THE EFFECTS OF TERT-BUTYLHYDROQUINONE ON THE MURINE NATURAL KILLER CELL ACTIVATION, EFFECTOR FUNCTION, AND PRIMARY RESPONSE TO INFLUENZA By Allison Paige Boss A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Human Nutrition – Doctor of Philosophy Pharmacology and Toxicology – Dual Major 2023 ABSTRACT Seasonal influenza poses a significant threat to the public due to its ability to cause illness that ranges in severity and can lead to hospitalization and death. Research to identify potential contributing factors to the susceptibility and severity of influenza infection is necessary to decrease the burden of disease. Natural killer (NK) cells provide early protection during infection by providing an early source of cytokines and cytotoxicity of virally infected cells. Recently, an immunomodulatory role for the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), has been shown in several immune cell types and is activated by oxidative stress and various exogenous compounds, such as the food additive, tert-butylhydroquinone (tBHQ). However, the role of Nrf2 and tBHQ in NK cells remains largely unknown. The overall goal of these studies is to understand the impact of tBHQ and Nrf2 on NK cell activity and to determine the effect of tBHQ on NK cell response to influenza. To assess this, we determined the effects of tBHQ on NK cell activation and effector function and whether these effects were dependent on Nrf2. Activation of NK cells was significantly decreased as shown by a reduction in early activation markers, CD25 and CD69, with 1 and 5 µM tBHQ compared to the control vehicle group (VEH). Additionally, tBHQ induced maturation as demonstrated by a decrease in the percentage of intermediate stage NK cells, CD27+CD11b+, and an increase in terminally differentiated NK cells (CD27-CD11b+). tBHQ also inhibited production of the antiviral cytokine, IFNg, at 1 and 5 µM tBHQ compared to the VEH control. Furthermore, cytotoxic molecules, perforin and granzyme B, were also reduced with tBHQ in a concentration-dependent manner. To investigate the role of Nrf2, Nrf2-deficient mice were utilized to assess whether these effects were mediated by Nrf2. Maturation of NK cells had a pronounced genotype and tBHQ effect with a significant increase in the percentage of immature NK cells (CD27-CD11b-) in activated splenocytes from Nrf2-null mice. In addition, tBHQ caused a Nrf2-dependent decrease in the expression of the activation markers, CD25 and CD69, in PMA/ionomycin-activated NK cells. Likewise, tBHQ decreased expression of fas ligand in IL-12/IL-18-activated NK cells in a Nrf2-dependent manner. In contrast, inhibition of IFNg induction by tBHQ was only partially Nrf2-dependent. These findings demonstrate tBHQ negatively impacts NK cell activation and effector function in vitro. However, little is known regarding the effect of dietary tBHQ on NK cells in vivo, specifically in response to influenza infection. Mice were fed a diet containing either 0.0014% tBHQ or control diet 10 days prior to being infected with influenza A/PR/8/34 (H1N1). NK cell responses were assessed at days two and three post-infection. Mice exhibited similar weight loss throughout infection regardless of their respective diet. At day three, tBHQ modestly decreased the percentage of NK cells in the lung, and NK cells adopted a more immature phenotype. tBHQ significantly reduced expression of fas ligand and production of IFNg in NK cells compared to those on a control diet at day two. NK cell cytotoxicity decreased with tBHQ at day three, as demonstrated by reduced CD107a expression. Induction of effector genes was decreased in the lungs of mice on a tBHQ diet at days two and three of infection. Collectively, these are the first studies show that the food additive, tBHQ, negatively impacts NK cell activation, effector function and early responses to influenza infection. ACKNOWLEDGEMENTS I would like to thank Dr. Cheryl Rockwell and Dr. Elizabeth Gardner for their constant encouragement and guidance during my PhD research. They always kept my goals in mind and challenged me to grow as an independent scientist. My sincere thanks must also go to the members of my committee: Drs. Jenifer Fenton, Wei Li, and Colleen Hegg. They generously gave their time and provided me valuable comments toward improving my scientific research and professional development. I am grateful to all of those whom I have had the pleasure to work with during my time in lab. Drs. Robert Freeborn, Yining Jin, and Luca Kaiser have all assisted in work within this dissertation, which would not have been possible without their contributions. Most importantly, I have my family to thank for their unwavering love and support in whatever I pursue. My parents are the greatest role models and continue to inspire me every day. A special thanks to brother, Jacob, and sister, Megan, for always being my biggest fans throughout life. Finally, to my boyfriend, Gavin: your love, patience, and understanding has meant everything to me. Thank you for supporting and encouraging me throughout this experience. iv TABLE OF CONTENTS LIST OF ABBREVIATIONS ................................................................................................................ vi CHAPTER 1: Literature review ....................................................................................................... 1 CHAPTER 2: The Nrf2 activator tBHQ inhibits the activation of primary murine natural killer cells ............................................................................................................................................... 28 CHAPTER 3: Activation of Nrf2 by tBHQ promotes maturation, while concurrently inhibiting activation and expression of effector molecules by activated NK cells........................................ 45 CHAPTER 4: The Nrf2 activator, tBHQ, inhibits early NK cell responses to influenza .................. 63 CHAPTER 5: Summary, Significance, and Future Directions ........................................................ 86 WORKS CITED ............................................................................................................................... 93 APPENDIX ................................................................................................................................... 120 v LIST OF ABBREVIATIONS ADCC Antibody-dependent cellular cytotoxicity ADI Allowable daily intake AHCC Active hexose correlated compound AhR Aryl hydrocarbon receptor ARE Antioxidant response element bZIP Basic leucine zipper CCR Chemokine receptor CDDO-Im 2-Cyano-3,12 dioxooleana-1,9 diene-28-imidazolide CLP Common lymphoid progenitor CMV Cytomegalovirus CNC Cap’n’collar COPD Chronic obstructive pulmonary disease Cul3 Cullin 3 CXCR CXC-chemokine receptor DC Dendritic cell DEM Diethylmaleate DMF Dimethylfumarate DNA Deoxyribonucleic acid Eomes Eomesodermin ER Endoplasmic reticulum FasL Fas ligand vi GSH Glutathione GST Glutathione S-transferase HA Hemagglutinin HBV Hepatitis B virus HCV Hepatitis C virus HIV Human immunodeficiency virus HO-1 Heme oxygenase-1 HSC Hematopoietic stem cells HSV Herpes simplex virus IAV Influenza A virus ID2 Inhibitor of deoxyribonucleic acid (DNA) binding 2 IFN Interferon Ig Immunoglobulin IL Interleukin iNK Immature natural killer cell Keap1 Kelch ECH-associated protein 1 M1 Matrix 1 M2 Matrix 2 MACRO Macrophage receptor with collagenous structure MHC Major histone complex NA Neuraminidase NOAEL No observed adverse effect level vii NQO-1 NAD(P)H oxidoreductase NCR Natural cytotoxicity receptor Neh Nuclear factor erythroid 2-related factor 2 (Nrf2)-ECHhomology NFE212 Nuclear factor (erythroid-derived 2)-like 2 NFkB Nuclear factor kappa B NK Natural killer NKP Natural killer cell precursor NP Nucleoprotein Nrf2 Nuclear factor erythroid 2-related factor 2 NSP1 Non-structural protein 1 NSP2 Non-structural protein 2 OVA Ovalbumin PA Polymerase acidic protein PB1 Polymerase basic protein 1 PB1-F2 Polymerase basic protein 1- F2 PB2 Polymerase basic protein 2 PERK Protein kinase ribonucleic acid (RNA)-like endoplasmic reticulum kinase PKC Protein kinase C PMA Phorbol 12-myristate 13-acetate preNKP Pre natural killer cell progenitor RNA Ribonucleic acid ROS Reactive oxygen species viii RSV Respiratory syncytial virus SFN Sulforaphane SLE Systemic lupus erythematosus T-bet T-box expressed in T cells tBBQ tert-butyl benzoquinone tBHQ tert-butylhydroquinone TCR T cell receptor TGFb Transforming growth factor b Th T helper Tim-3 T cell immunoglobulin mucin-3 TLR Toll-like receptor TNFa Tumor necrosis factor-a vRNP Viral ribonucleoproteins WT Wild-type ZEB2 Zinc finger E-box- binding protein 2 ix CHAPTER 1 Literature Review 1 I. Influenza Virus A. General Influenza A virus (IAV) belongs to the Orthomyxoviridae family and is a negative- sense, single-stranded, enveloped ribonucleic acid (RNA) virus that contains a segmented genome, which encodes several proteins involved in viral entry and infection, replication, and viral release including: hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), matrix 2 (M2), nucleoprotein (NP), non-structural protein 1 (NSP1), non-structural protein 2 (NSP2), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase basic protein 1- F2 (PB1-F2). Entry into the host cell is initiated by HA binding to sialic acid residues on the host cell’s membrane. The virus enters the host via receptor-mediated endocytosis. The acidic environment of the endosome allows fusion of viral and host membranes and opens M2 channel, leading to acidification and release of viral ribonucleoproteins (vRNP) from M1, which is comprised of PB1, PB2, and PA, into the cytoplasm. vRNP enter the nucleus by nuclear localization signals. Replication occurs by synthesis of positive-sense complementary RNA (cRNA) followed by copying of cRNA into newly synthesized negative-sense viral RNA. Newly assembled viral RNA is transported to the plasma cell membrane and released by NA1,2. IAV can be categorized based on the virus structure of surface glycoproteins HA and NA. Currently, there are 18 HA and 11 NA subtypes, but the main subtypes that are infectious to humans include H1N1 and H3N22. Although there are 4 types of seasonal influenza viruses, types A, B, C, and D, that can cause seasonal epidemics, only influenza 2 A virus is known to have caused pandemics, such as the pandemic in 2009.3 Influenza continues to pose a significant threat to society with 8% of the United States population infected each year.4 Globally, influenza infections are estimated at 5-10% in adults and 20-30% in children.3,5 However, it should be noted that precise data on influenza morbidity and mortality are based on data from industrialized countries. Therefore, the impact of influenza on lower income countries are less known.6,7 Influenza virus burden of disease persists due to high mutation rates in antigenic sites. Influenza viral RNA polymerase lacks proofreading machinery which results in significant mutations during the viral replication at a rate of 10-3 to 10-4. To evade immune responses, IAV evolve by mechanisms that include antigenic drift, antigenic shift, and recombination. Specifically, mutations in HA or NA proteins can escape host immune responses that have been acquired through immunization or previous infection. Vaccination against influenza is the most effective way to prevent disease, and it has been estimated to prevent millions of illnesses, medical visits, hospitalization, and deaths each season.8 Despite the advancements in vaccine effectiveness and coverage, the burden of disease remains high, affecting millions in the United States alone.9 Individual factors, including demographic, genetic, and environmental, are known to influence immune responses, which provides an explanation for the wide variations in susceptibility and severity of infection.10 Even though everyone can be affected by influenza infection, certain groups are at higher risk than others. High risk groups are defined as pregnant persons, children, elderly, and people with chronic medical 3 conditions. Additionally, certain occupations can increase exposure to influenza, such as health care workers.3 B. Innate Immunity Against IAV Initial immune response to viral pathogens is essential to limit viral replication and bridges host immune responses from initial infection to adaptive immunity. Although adaptive immunity is vital in viral clearance and provides protection from reinfection, the variable nature of IAV limits long term protection. Additionally, innate immunity plays a key role in providing signals, accelerating adaptive immune responses, and augmenting vaccine responses. 1. Natural Killer Cell Overview NK cells are effector innate lymphocytes with cytotoxicity and cytokine- producing effector functions against infected or transformed cells. Mechanisms used to differentiate between ‘self’ cells and target cells include a vast array of activating and inhibitory receptors, and engagement of receptors determine NK cell activation and target cell killing.11,12 Ligands detected by NK cell activating receptors can include stress-induced self-ligands, infectious non self-ligands, and Toll-like receptor (TLR) ligands.13,14 NK cell inhibitory receptors recognize constitutively expressed self- molecules, such as major histocompatibility complex (MHC) class I and non-MHC self- molecules (Clr-b, LLT-1, and CD48), that protect cells from NK cell-mediated killing.15 However, loss of MHC class I allows for NK cell activation and cytotoxicity.16 Robust NK cell responses depend on interactions with other immune cells, including dendritic cells (DCs), macrophages, and T cells, and cytokine signals.11,17 Conversely, negative 4 regulation of NK cell function can be mediated by transforming growth factor (TGF)-b secreted from regulatory T cells.18 NK cells reach optimal functional status through the process of maturation from immature to mature NK cells. Defects in NK cell maturation have been shown to diminish effector capabilities, including decreased target killing, and reduce the overall NK cell population in the periphery.19,20 Therefore, it is important to identify NK cell maturation status and the factors involved in the maturation process, such as cytokines and transcription factors. NK cell maturation occurs in a stepwise cell differentiation process. In the bone marrow, NK cells develop from hematopoietic stem cells (HSCs) and differentiate into common lymphoid progenitors (CLPs). At this stage, CLPs can develop into either pro-B cells, pro-T cells, innate lymphoid cell precursors or preNK cell progenitors (preNKPs). PreNKPs are early NK-committed precursors that can differentiate to NK cell precursors (NKPs). The above developmental processes are similar in murine and human NK cells. However, differences in development occur following the NKP stage in NK cell maturation. In mice, final commitment of NK cells happens after the acquisition of NK1.1 and natural cytotoxicity receptor (NCR) and are classified as immature NK cells (iNK). Following this stage, NK cells continue to mature in the bone marrow and periphery where they acquire functional capacity and can be identified by changes in expression of the phenotype markers CD27 and CD11b. This late-stage maturation is defined by the upregulation of CD11b and downregulation of CD27. During this process, maturing NK cells acquire cytotoxic capacity and gradually lose proliferative potential towards reaching terminal differentiation. Late-stage 5 maturation progresses in the following order from immature to terminally mature NK cells: CD27-CD11b-, CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+.21 In humans, expression of CD122 on NKPs is necessary for NK cell lineage commitment. Additionally, final NK cell maturation in humans is defined by the appearance of CD56, which can be further divided into subsets CD56bright and CD56dim. NK cell maturation in humans has been demonstrated by the transition from a more immature CD56bright NK cells to terminally mature subset CD56dim NK cells with the acquisition of CD16.22,23 The majority of circulating NK cells (up to 90%) are cytotoxic CD56dimCD16+ cells, whereas CD56brightCD16- produce more proinflammatory cytokines, such as IFNg.24,25 Figure 1: Stages of NK cell maturation. A schematic illustrating stages of NK cell maturation and associated surface markers. Created with Biorender. Transcription factors required for NK cell maturation include ID2 (inhibitor of DNA binding 2), T-bet (T-box expressed in T cells), Eomes (eomesodermin), and ZEB2 (zinc finger E-box- binding protein 2). ID2 is expressed at lineage commitment of NK cells and throughout subsequent maturation stages. Experimental evidence has shown 6 ID2 deficiency to reduce NK cell numbers and decrease CD11b and granzyme expression.26 Additionally, ID2 is needed for IL-15 receptor signaling, which is essential for mature NK cell survival and expansion.27–29 T-bet and Eomes are highly homologous T-box transcription factors that have been shown to play a key role in maturation. In T- bet-/- mice, NK cell numbers were decreased in peripheral tissues but increased in the bone marrow, suggesting the presence of T-bet is essential for NK cell egression from bone marrow. Additionally, T-bet deficiency was shown to reduce expression of CD11b.19 In addition, a study had found temporal depletion of T-bet and Eomes in mature NK cells downregulates NK1.1 and NKp46. Loss of Eomes alone resulted in the loss of maturity markers, which ultimately caused the cells to express a more immature phenotype identified as TRAIL+ and DX5-.30 T-bet also synergizes with the transcription factor Zeb2, which regulates terminal maturation downstream of T-bet. Furthermore, induction of T-bet was found to upregulate expression of Zeb2. In addition, loss of ZEB2 in mice resulted in retention of NK cells in the bone marrow, whereas overexpression of ZEB2 increased NK cell numbers in peripheral tissues while decreasing the NK cell population in bone marrow, suggesting ZEB2 is essential for NK cell homeostasis.20 There is increasing evidence regarding the role of NK cells in negatively regulating cellular and humoral immune responses.31–34 For instance, NK cells can lyse immature dendritic cells that lack inhibitory receptors, KIRs (human)/Ly49 (mouse), whereas mature DCs are protected from lysis due to the upregulation of MHC class I.35– 37 Microglia cells have also been shown to be susceptible to NK cell-mediated cytotoxicity via NKG2D and NKp46 engagement. However, upregulation of MHC class I 7 molecules inhibited NK cell lysis of microglia cells.38 Studies have also shown NK cells to influence T helper (Th) cell polarization and B cell activation and isotype switching.39–41 NK cells rapidly secrete tumor necrosis factor-a (TNFa) upon direct contact with mature DCs, which promotes polarization of activated T cells towards the Th1 phenotype. However, activated T cells upregulate ligands for the NK cell activating receptor, NKG2D, making them susceptible to NK cell-mediated lysis. Conversely, simultaneous expression of ligands specific to NK cell inhibitory receptor, NKG2A, can inhibit NK cell activity.35,42 Although NK cells are potent producers of Th1-associated cytokines, such as IFNg, studies have shown prolonged activation leads to augmented production of the regulatory cytokine, IL-10.43 Furthermore, studies of autoimmunity have suggested a protective role for NK cells through the secretion of Th2-associated cytokines, IL-13 and IL-5.44–47 NK cells in mucosal tissues have been shown to produce IL-22, a cytokine involved in tissue repair.48,49 Together, NK cells can function to limit or exacerbate immune responses, which is dependent on cell interactions and the cytokine microenvironments. 2. Natural Killer Cell Responses to Primary Infection NK cells are large granular lymphocytes that can rapidly produce cytokines and directly kill virally infected cells without prior exposure to IAV. They play an essential role in limiting viral replication and potentiating the adaptive immune response.50 Unlike the adaptive immune response, NK cells elicit effector functions without priming or prior sensitization to pathogens. Instead, NK cells rapidly respond to transformed or infected cells via mechanisms involving a host of activating and inhibitory receptor 8 signals, cytokine stimulation, and antibody-dependent cellular cytotoxicity (ADCC).11 The importance of NK cell antiviral activity was first noted when a patient lacking NK cells was diagnosed with reoccurring viral infections.51 After this key finding, numerous studies were conducted to elucidate the role of NK cells in viral infection, further emphasizing the significance of NK cells in host immune responses. Resident NK cells in the lung make up approximately 10% of the lymphocyte population under homeostatic conditions.52 During initial hours to days of infection, NK cells are recruited to the lung by CXC-chemokine receptor 3 (CXCR3) and CC- chemokine receptor 5 (CCR5), which have been found to be the main contributor for increased NK cell accumulation in the lung while proliferation of resident NK cells is minimal53 NK cell responses during viral infection are regulated by a wide array of germline-encoded activating and inhibitory receptors. Normal healthy cells express MHC class I molecules that are recognized by NK cell inhibitory receptors. However, in virally infected cells, MHC class I is downregulated or lost thereby withholding inhibitory signals that would prevent target cell lysis.54 Virus-infected cells upregulate self-encoded molecules and stress ligands which bind to natural cytotoxicity receptors (NCRs), NKp30, NKp44, and NK46, and C-type lectin-like receptors, such as NKG2D.13,55,56 Of relevance to influenza infection, NK cell activating receptor, NKp46, can interact with influenza HA protein via the a-2,6-linked terminal sialic acid and become activated.57 NK cell receptor binding to corresponding ligands induces signaling cascades modulating NK cell function, which include release of cytotoxic granules containing granzymes and perforin, cytokine production, and Fas ligand (FasL)- mediated apoptosis.24,58 Additionally, NK cells can 9 execute cell-to-cell cytolysis of virus-infected cells coated with immunoglobulin (Ig)G antibodies via ADCC upon ligation of the CD16 receptor.59 Activation of NK cells through contact-dependent and -independent mechanisms has also been demonstrated with virus-infected macrophages.60 Contact- independent mechanisms include cytokine signals secreted by antigen-presenting cells and virus-infected cells which activate NK cells during infection. Cytokines, including, but not limited to IL-2, IL-12, IL-15, IL-18, and type 1 interferons, are important in NK cell survival, proliferation, and activation.61,62 Influenza is a respiratory virus that mainly infects lung epithelial cells. Like many viruses, infection may lead to severe inflammation and tissue damage. The role of NK cells in response to influenza virus remains complex as studies have shown differing outcomes in relation to the magnitude of NK cell response. Defects in activation or depletion of NK cells have been shown to impair viral clearance and increase morbidity and mortality.63–68 Conversely, studies have found NK cells to cause immunopathology in mice when infected with influenza virus. For example, mice depleted of NK cells infected with a high viral dose of influenza A/PR/8/34 virus had increased survival rates compared to mice depleted of NK cells receiving a low dose of virus.69 Additionally, NK cells adoptively transferred from mice infected with a high dose of virus resulted in increased morbidity and mortality. Whereas NK1.1+ cell-depleted mice found to have minimal morbidity and no effect on viral clearance when infected with a lethal dose of virus.70 10 3. Nutritional Interventions and IAV Outcomes Alterations in NK cell development, activation, and effector function have resulted in dampened responses to influenza infection. Our group has shown differences in NK cell activity in both aged and calorie-restricted mice following primary influenza infection. Compared to young mice, aged mice exhibited a reduction in the percentage of NK cells in the lungs and decreased NK cell production of IFNg after infection.63,71 Additionally, calorie restriction in mice increased mortality and decreased NK cell cytotoxicity following influenza infection.63 Furthermore, in aged mice, calorie restriction further diminished the response to influenza resulting in decreased survival, increased virus titers, and reduced NK cell activity compared to aged mice fed ad libitum and calorie restricted, young mice.72,73 Others have found diet-induced obesity in mice decreased NK cell cytotoxicity and the expression anti-viral cytokines.74 In contrast, supplementation of active hexose correlated compound (AHCC) increased NK cell activity and NK cell number in the lungs following influenza infection.75 Other interventions have been shown to alter NK cell activity and effector function in the absence of infection, including vitamin supplementation and alcohol consumption.76,77 The following studies suggest that NK cells are highly influenced by a variety of environmental factors, and impairment of NK cells during influenza infection may lead to decreased viral clearance and survival. 11 II. Nrf2 A. Discovery Nuclear factor erythroid 2-related factor 2 (Nrf2) was discovered in 1994 as a stress-induced transcription factor with a basic leucine zipper (bZIP) DNA binding domain that belongs to the cap’n’collar (CNC) family and is encoded by the gene NFE212 (nuclear factor (erythroid-derived 2)-like 2).78 Nrf2 is highly expressed among various tissues and is structurally and functionally conserved across species.78–80 Canonically, Nrf2 binds to the cis-acting DNA sequence termed antioxidant response element (ARE) as a heterodimer with small Maf proteins and regulates gene transcription of phase II detoxifying enzymes, such as glutathione S-transferases, NAD(P)H oxidoreductase (NQO-1), and g-glutamyl cysteine synthase.81–85 Nrf2 has also been found to associate with c-Jun to upregulate ARE-mediated expression of NQO-1 gene.86 To determine the function of Nfe2l2 (Nrf2) gene, Nrf2-deficient mice were generated. Although Nrf2 is constitutively expressed, the Nfe2l2 gene function was found not necessary for mouse development, growth, and fertility.87 The development of Nrf2-deficient mice were found to have increased susceptibility to oxidative and xenobiotic stressors, which resulted in various pathological characteristics.88–91 In humans, Nrf2 polymorphisms have been identified in the regulatory region of the gene. However, Nrf2 polymorphisms was not associated with increased risk for systemic lupus erythematosus and chronic obstructive pulmonary disease.92 12 B. Structure and Regulation Nrf2 is divided into six highly conserved domains, Neh1 to Neh6 (Nrf2- ECHhomology). A considerable number of studies identifying functional activity of Nrf2 have been implemented, including in-depth analysis of the Nrf2 domains. Nrf2 is regulated by its repressor protein, Kelch ECH-associated protein 1 (Keap1), which is localized in the cytoplasm. Keap1 mediates Nrf2 repression by binding specifically to Neh2 domain, which is one of the six highly conserved regions of Nrf2. Keap1 regulates the cellular level of Nrf2 protein through proteasomal-mediated degradation via the 26S proteasome.93 This process involves the direct interaction between the intervening- region (IVR) domain of Keap1 with the N-terminal region of Cullin 3 (Cul3), a subunit of the E3 ligase complex .93–98 In the presence of oxidative stress or electrophiles, Nrf2/Keap1 complex is disrupted allowing Nrf2 translocation to the nucleus, heterodimerization with small maf proteins, and transcriptionally activation of ARE- dependent genes.99 In addition, several exogenous activators of Nrf2 interact with highly reactive cysteine residues of Keap1, which act as primary cellular sensors.100 Modifications can alter the configuration of the Nrf2/Keap1 complex inhibiting Nrf2 release and degradation, which allows Nrf2 accumulation via de novo synthesis. Another mechanism involves Nr2 dissociation from Keap1 resulting in Nrf2 translocation to the nucleus.101,102 Additionally, dissociation of the E3 ligase complex from the Nrf2/Keap1 complex has been proposed, which allows newly synthesized Nrf2 to evade degradation.103 A noncanonical pathway for Nrf2 activation has also been demonstrated through the upregulation of p62 by autophagy deficiency in which p62 13 sequesters Keap1 into aggregates. Aggregation of Keap1 hinders Nrf2 ubiquitination by E3 ligase complex thereby inhibiting Nrf2 degradation.104–106 Post-translational modifications involving phosphorylation of Nrf2 are known to regulate Nrf2 activation and induction of ARE-mediated gene expression. Several cytosolic kinases have been shown to play a role in the activation of Nrf2 signaling. Phosphorylation at serine 40 residue (S40) of Nrf2 by protein kinase C (PKC) induced nuclear translocation and upregulation of ARE-mediated genes.107,108 Additionally, casein kinase 2 (CK2) was found to phosphorylate transcription activation domains Neh4 and Neh5 of Nrf2, and in the presence of tBHQ, phosphorylation increased nuclear 109,110 translocation. Others have shown Nrf2 phosphorylation at the Neh2 domain by protein kinase RNA-like endoplasmic reticulum kinase (PERK) after exposure to endoplasmic reticulum (ER) stress.111,112 Acetylation of Neh1 domain of Nrf2 has been found to enhance Nrf2 binding to ARE.111 C. Nrf2 Activators Nrf2/Keap1 responds to a wide range of chemical compounds using various distinct mechanisms including Nrf2 stabilization, oxidative stress, and Keap1 inhibition. These chemical compounds include endogenous compounds, such as reactive oxygen species, reactive nitrogen species, lipid aldehydes, 15-deoxy-D12,14- prostaglandin J2, and exogenous compounds, including heavy metals and electrophilic compounds.113,114 Several studies have established specific mechanisms for the activation of Nrf2, which include electrophilic reactions with cysteines of Keap1 and phosphorylation of Nrf2. For example, tert-butylhydroquinone (tBHQ), dimethylfumarate (DMF), sulforaphane (SFN), 14 ebselen, diethylmaleate (DEM), and 2-cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im) are Nrf2 inducers that react with cysteine residue, Cys151, of Keap1 to trigger the Keap1-Nrf2 response. Cys151 was found to be critical for tBHQ, DEM, and DEF.115 However, Cys151-independent Nrf2 inducers have been recognized. Cadmium chloride (CdCl2) has been shown to modulate zinc binding capacity of Keap1, and studies have found Cys151 is not required for activation in this case.114,116 tBHQ and SFN have been shown to stabilize Nrf2 by inhibiting Keap1-mediated ubiquitination. Conversely, mechanism for activation of Nrf2 by arsenic trioxide (As(III)) is through augmented association of Keap1 and Cul3, which ultimately prevents continual degradation of Nrf2 by Keap1.117 However, the studies on inorganic arsenic compounds overall demonstrate that multiple mechanisms are involved in Nrf2 activation. In contrast to arsenic trioxide, sodium arsenite increased expression of Nrf2 and accumulation of Nrf2 in the nucleus, which was enhanced by production of H2O2, while simultaneously increasing Keap1 expression.118 Other studies found Cys151, Cys273, and Cys288 in Keap1 were required for Nrf2 activation by inorganic arsenic.119 Interestingly, copper-mediated oxidation of para- or ortho- hydroquinone compounds was necessary for subsequent interaction with cysteine residues of Keap1.120 In contrast, tBHQ-mediated Nrf2 activation has been shown to occur through the induction of mitochondrial oxidative stress, at least in some model systems.121 15 Figure 2: Nrf2 activation. A representation illustrating A) unactivated Nrf2 and B) activated Nrf2 by tBHQ interaction with Keap1. Created with Biorender. D. Immune Cells 1. Macrophages Nrf2 responses are often recognized as anti-inflammatory. Studies have shown Nrf2 directly limits induction of inflammatory cytokine genes, IL-6, and IL-1b, in M1 macrophages.122 Additionally, activation of Nrf2 in macrophages skewed polarization towards an M2-phenotype while downregulating IL-1 and IL-6. Knocking down Keap1 in macrophages increased M2- macrophages polarization by cancer cells. Consistently, the Nrf2 activator, DEM, upregulated Nrf2 activation in macrophages therefore promoting cancer cell-induced M2 macrophages.123 Several studies have also identified heme oxygenase-1 (HO-1), a cytoprotective gene induced by Nrf2 activation, to influence M2 macrophage polarization.124–126 In patients with chronic obstructive pulmonary disease (COPD), alveolar macrophages treated with SFN enhanced bacterial phagocytosis by increasing expression of scavenger receptor, macrophage receptor with collagenous 16 structure (MACRO), with Nrf2 activation.127 In Nrf2-deficient mice, hyperoxia induced oxidative stress, which was accompanied by an increase in inflammatory cytokine gene expression of IL-6 and IL-1b in alveolar macrophages. Additionally, macrophages from Nrf2-deficient mice were defective in bacterial killing compared to wild-type macrophages.128 Others have found macrophage-mediated phagocytosis and clearance of Listeria monocytogenes to be inhibited by Nrf2-regulated expression of CD36 and HO- 1 and T cell immunoglobulin mucin-3 (Tim-3).129 2. Dendritic Cells Bone marrow derived DCs from Nrf2-deficient mice show increased oxidative stress. Additionally, Nrf2-deficient DCs have higher cell surface expression of MHC class II and costimulatory molecules suggesting a heightened proinflammatory state. 130 Similarly, bone marrow-derived Nrf2-deficient DCs were found to have impaired phagocytosis, increased DC maturation, and increased capacity to induce CD8 T cell proliferation and activation.131 Another study assessing the adjuvant effects of ultrafine particles (UFP) on allergic sensitization to ovalbumin (OVA) in mice found Nrf2-deficient DCs to possess a cytokine profile favoring Th2 polarization with low expression of IL- 12p70 and increased expression of IL-6 when compared to wild-type DCs.132 In bone marrow derived DCs from Nrf2-deficient mice, increased amounts of IL-6 and TNF-a were secreted compared to wild-type counterparts.133 In activated DCs, treatment with either Nrf2 activators, arsenic or tBHQ, significantly inhibited IL-12 expression at the transcriptional and protein level. However, arsenic-induced reduction of IL-12 was only exhibited in DCs from wild-type mice and not Nrf2-deficient mice.134 17 3. Natural Killer Cells and Natural Killer T Cells The role of Nrf2 in NK cells and NKT cells remains largely unknown. In tumor- bearing mice, mice exposed to topical tBHQ delayed tumor growth in Rag2-/- mice but not Rag2-/- γc-/- suggesting NK cell involvement in the mediation of tumor rejection. Nrf2 was found to directly induce IL-17D expression, whereas knockdown of IL-17D or Nrf2 within tumor cells abolished any NK cell increase that was mediated by tBHQ.135 In mice infected with influenza A/PR/8/34 H1N1 virus, IL-27-induced increase in NK cell function was mediated by activating receptor, NKG2D. Additionally, Mafs and Nrf2 transcriptional pathways were partly dependent in mediating IL-27 signaling.136 In contrast, our lab has demonstrated impaired NK cell activation and effector function with tBHQ in murine splenocytes.137 In Keap1-deficient mice, NKT cells had elevated apoptosis and reduced peripheral NKT cell numbers due to defects in development. However, Nrf2 deletion restored NKT cell defects observed in Keap1-deficient mice.138 4. B Cells While many differences in immunoglobulin levels have been described between wild-type and Nrf2-null mice, there is limited evidence regarding a direct role for Nrf2 in modulating B cell response. Our group has demonstrated a Nrf2-dependent decrease in B cell activation and increase in IgM production with tBHQ in activated murine B cells.139 Additionally, in mice infected with Haemophilus influenzae, Nrf2-deficient mice had increased lung infiltration and higher immunoglobulin-secreting cells compared to wild- type mice.140 18 5. T Cells There has been increasing evidence regarding the immunomodulatory role of Nrf2 in T cells. In the development of pulmonary fibrosis, Nrf2 was found to regulate Th1 and Th2 immune responses. Specifically, Nrf2-deficient mice were more susceptible to bleomycin-induced pulmonary fibrosis and had increased levels of TNF-a and nuclear factor Kappa B (NFkB) activation compared to wild-type mice. Regarding Th1/Th2 balance, Nrf2-deficient mice exhibited, IFNg-producing CD4+ and CD8+ T cells was significantly lower while IL-4-producing CD4+ and CD8+ T cells as enhanced when compared to wild-type mice. Furthermore, assessment of Th1 and Th2 master regulators, T-bet and GATA3 respectively, showed an increase in GATA3 and decrease in T-bet levels in Nrf2-deficient mice suggesting absence of Nrf2 skews Th1/Th2 balance toward Th2 phenotype in the pulmonary fibrosis disease model.141 Conversely, our group has shown Nrf2 activation skews CD4+ T cells toward Th2 differentiation by demonstrating increased IFNg-expression by anti-CD3/anti-CD28-activated CD4 T cells derived from Nrf2-deficient mice relative to wild-type. Likewise, tBHQ inhibited IFNg induction by activated wild-type, but not Nrf2-null, CD4 T cells. Additionally, Nrf2- deficient mice had decreased secretion of the Th2 cytokines, IL-4, IL-5, and IL-13, by activated CD4+ T cells. The transcription factors, GATA-3 and T-bet, DNA-binding was also assessed, which showed enhanced binding of T-bet, which promotes Th1 polarization, and diminished binding of GATA-3, which promotes Th2 polarization, in CD4 T cells derived from Nrf2-null mice.142 Consistent with these effects in primary murine CD4 T cells, in primary human CD4+ T cells, tBHQ treatment induced expression 19 of Nrf2-related genes, while decreasing production of IL-2 and IFNg and early CD4+ T cell activation markers.143 In mice genetically modified to express high levels of Nrf2 in T cells, mice induced with ischemia reperfusion (IR) exhibited increased regulatory T cells and decreased levels of CD4+ T cell cytokines, TNFa, IFNg, and IL-17. Additionally, high expression of Nrf2 protected mice from IR injury and increased survival.144 The Nrf2- activator, CDDO- Im, was shown to inhibit IFNg-secretion by activated T cells derived from wild-type mice in a Nrf2-dependent manner.145 In Jurkat T cells, treatment of tBHQ demonstrated decreased IL-2 production and CD25 expression, which was associated with reduced NFkB transcriptional activity.146 To assess the role of Nrf2 in Jurkat T cells, Nrf2 was deleted using CRISPR/Cas9 gene editing. These studies showed tBHQ-mediated suppression of IL-2 was largely independent of Nrf2 while inhibition of CD25 (IL-2 receptor) was partially Nrf2- dependent in Jurkat T cells. In contrast to tBHQ, IL-2 suppression by CDDO-Im in activated Jurkat T cells was Nrf2-dependent.147 In addition to effects on cytokines and cytokine receptors, another study showed that Keap1 deletion in T cells increased antioxidant potential which was dependent on Nrf2 expression. Additionally, primary human T cells with Keap1 deletion in vitro had higher frequency of CD4+ T cells and lower frequency of CD8+ T cells relative to controls. CD25 expression was also significantly increased while production of IL-17 was reduced with Keap1 deletion. More specifically, Keap1-edited regulatory T cells had significantly increased CD69 and IL-10 expression, suggesting Nrf2 involvement in promoting immunosuppressive T cells in this model.148 Interestingly, and in contrast to 20 this study, in a cre-flox mouse model, mice heterozygous mice with Keap1 deletion in FoxP3+ cells had increased CD4+ T cell activation and effector memory T cells compared to wild-type mice. Homozygous mice for Keap1 deletion in FoxP3+ cells had increase IFNg- producing T cells and liver and lung inflammation, suggesting constitutive Nrf2 activation results in diminished regulatory T cell-mediated immune tolerance.149 E. Nrf2 Autoimmunity Characterization of Nrf2-deficient mice lead to the discovery of Nrf2 as a potential candidate in determining susceptibility to autoimmune disease. In aged, Nrf2- null female mice, characteristics similar to human lupus nephritis were observed, which lead to the utilization of Nrf2-deficient mice as a model for lupus-like autoimmune nephritis.92 Similarly, Nrf2-deficient female mice on a different genetic background, developed an autoimmune disease resembling systemic lupus erythematosus (SLE) as demonstrated by splenomegaly, vasculitis, glomerulonephritis, hepatitis, and myocarditis. In addition, increased oxidative stress and apoptosis in the absence of Nrf2 expression strongly suggests Nrf2 involvement in the prevention of SLE.150 In addition to multiorgan disease manifestation, CD4+ T cells from Nrf2-deficient mice exhibited hyperproliferation upon T cell receptor (TCR) activation resulting in a potential mechanism for Nrf2 mediation of oxidative tissue damage and T proliferation.151 Likewise, the Nrf2 activator, SFN, ameliorated renal injury in pristine-induced lupus nephritis in wild-type, but not Nrf2-null mice, by negatively regulating NFkB and TGFb1 signaling pathways. In the same study, elevated expression of Nrf2-target genes was observed in human lupus nephritis patients, but not in normal kidney tissue.152 Nrf2 has 21 also been implicated in psoriasis, multiple sclerosis, rheumatoid arthritis, Type 1 diabetes, graft versus host disease, among other autoimmune diseases.153–162 F. Nrf2 in Infectious Disease Infectious agents include bacteria, fungi, viruses, and parasites that can produce infection and/or infectious disease. Infection by these organisms often leads to ROS production which induces activation of the Nrf2/Keap1 signaling pathway. In addition, an alternate ROS-independent mechanism for Nrf2 activation involves Toll-like receptor (TLR) signaling. Emerging evidence suggest an immunomodulatory role for Nrf2 in host immune responses in the context of various pathogens, including viruses. Respiratory syncytial virus (RSV) causes acute viral respiratory tract infections and hospitalizations, particularly in children and infants. In Nrf2-null mice, RSV infection resulted in increased viral titers, inflammation, and lung injury compared to wild-type mice.163 Additionally, there was significant induction of Nrf2-target genes in RSV- infected wild-type mice, which was not observed in Nrf2-null mice.164 Conversely, RSV was also shown to induce Nrf2 degradation via Keap1-independent mechanisms. However, treatment with Nrf2 inducers was shown to rescue Nrf2-dependent gene expression in the context of RSV infection in epithelial cells and in mice.165,166 Influenza virus is also a persistent threat to society as it causes millions of infections and hundreds of thousands of hospitalizations and deaths yearly.7 It is well established that Nrf2 modulates virus-associated inflammation. However, the relationship between Nrf2 activation and virus susceptibility and clearance has yet to be explained. Notably, suppression of Nrf2 was shown to increase viral entry of influenza 22 in epithelial cells in vitro, while overexpression of Nrf2 blocked entry. Additionally, pre- treatment of epithelial cells with Nrf2 activators significantly decreased viral entry.167 In humans, nasal lavage fluids and biopsies were collected from smokers after receiving live attenuated influenza virus vaccine to assess Nrf2-dependent enzyme expression in the context of influenza A infection. Short term ingestion of broccoli sprout homogenates, which are a source for the Nrf2 activator sulforaphane, increased NAD(P)H.168 In Nrf2-deficient mice exposed to cigarette smoke and infected with influenza, macrophages showed increased NFkB activation and inflammatory gene expression in macrophages when compared to macrophages from wild-type mice. Moreover, cigarette smoke exposure in Nrf2-deficient mice caused greater lung pathology and mortality rates following influenza infection compared to virus-infected wild-type mice.169 When the Nrf2 activator, carbocisteine, was given to cigarette smoke-exposed wild-type mice during infection, neutrophil counts in the BALF were significantly reduced compared to controls. Treatment with carbocisteine also decreased inflammation and mucus hypersecretion in infected wild-type mice exposed to cigarette smoke.170 A role for Nrf2 activation was also discovered in mosquito-borne viral diseases. With Zika virus infection, Nrf2 activation and upregulation of HO-1 by its natural physiological substrate hemin impaired viral replication.171 In dengue virus-exposed macrophages, genome-wide transcriptome studies found Nrf2 to upregulate antioxidant pathways in response to ROS production. Silencing of Nrf2 with Nrf2-specific siRNA decreased antioxidant genes and increased ROS production in human monocyte-derived 23 dendritic cells infected with dengue virus.172 Dengue virus was shown to induce ER stress-PERK activation of Nrf2 leading to the upregulation of C-type lectin domain family 5, member A and enhanced TNFa production.173 In dengue-infected patients, Nox-2 (NADPH oxidase) and Nrf2 expression were down regulated upon hospital admittance in comparison to other febrile illness. Interestingly, between time of hospital admittance to release, Nox-2 expression increased in non-severe and severe dengue cases, whereas increased Nrf2 expression was only observed in non-severe dengue cases.174 Human immunodeficiency virus-1 infection (HIV) is enhanced with NFkB activation, whereas activated Nrf2 has been shown to inhibit NFkB activation. Consistent with this, treatment of HIV-infected monocyte-derived macrophages with the Nrf2-activator, dimethyl fumarate, diminished HIV replication, inhibited NFkB nuclear entry, and suppressed TNFa production while enhancing Nrf2 and NQO-1 expression.175 Similarly, sulforaphane inhibited HIV infection in primary macrophages, and siRNA-mediated depletion of Nrf2 increased susceptibility to HIV.176 HIV-1 Tat, a protein necessary for viral replication, was shown to enhance Nrf2 at both transcriptional and protein levels.177 In hepatitis B virus (HBV), ARE-gene expression was upregulated, and specific viral proteins for hepatitis C virus were shown to activate the Nrf2/ARE pathway by various mechanisms, including ROS-dependent and -independent phosphorylation.178 Nrf2 knockdown in a hepatitis C virus (HCV)-persistently-infected cell line demonstrated a critical role for Nrf2 in lipid droplet formation.179 Others have shown treatment with 24 the Nrf2 activator bardoxolone methyl (BARD) inhibited replication of HBV and HCV through both Nrf2-dependent and -independent mechanisms.180 Nrf2 activation in Herpes simplex virus (HSV)-1 was implicated in resistance to infection, and addition of the Nrf2 activators, sulforaphane or bardoxolone methyl, impaired virus replication.181 In Nrf2-deficient mice, type I IFNs were significantly increased and HSV replication decreased compared to wild-type mice following HSV challenge. Furthermore, Keap1 deletion promoted HSV replication, suggesting Nrf2 dampens innate antiviral immune responses and increased susceptibility to HSV.182 Cytomegalovirus (CMV) infection in primary human fibroblasts was shown to activate Nrf2, and upregulation of HO-1 gene expression in infected cells was dependent on Nrf2.183 Nrf2-deficient mice infected with mouse CMV had decreased survival, increased viral burden, and impaired immune cell recruitment to the peritoneum compared to wild-type mice. Also, injection of the Nrf2 activator, tBHQ, decreased weight loss and viral burden against mouse CMV infection in Nrf2-deficient mice. However, the protective effects of tBHQ were greater in CMV-infected wild-type mice as compared to Nrf2-null mice.184 G. Nrf2 Activator, tert-Butylhydroquinone tBHQ is a widely used food additive used in a variety of foods to prevent oxidation and rancidification of fats. Foods containing tBHQ include cooking oils, cereals, and crackers, among others. Initial toxicology studies in rats, mice and dogs concluded that TBHQ was not carcinogenic.185 The World Health Organization’s and United Nations’ Food and Agriculture Organization’s Joint Expert Committee of Food 25 Additives (JECFA) has set the allowable daily intake (ADI) of tBHQ at 0.7 mg/kg/day, which was based on a no-observed-adverse-effect-level (NOAEL) of 72 mg/kg/bw per day in dogs with a 100-fold safety factor.186 Dietary tBHQ exposure in humans is difficult to quantify. Therefore, experts have estimated model diets based on countries, such as United States, Australia, and China, and found high consumers of fats, oils, fat emulsions, and frozen fish products are capable of exceeding the recommended ADI for tBHQ.186 In addition to its commercial use in the food industry, tBHQ is also used experimentally as a well-characterized Nrf2 activator.187 The metabolic fate of tBHQ has been evaluated in several species.188–192 In rats, dogs, and humans, more than 90% of tBHQ is absorbed in the gastrointestinal tract and distributed via serum albumin proteins. During phase I metabolism in the liver, tBHQ is oxidized to tert-butyl benzoquinone (tBBQ) by either cytochrome P450 enzymes or autoxidation and/or Cu2+ ion-catalyzed redox cycling. In phase II, glutathione (GSH) conjugation of tBBQ is facilitated by metabolizing enzymes, such as NQO-1, glutathione S-transferase (GST), UDP-glucuronosyltransferase, and epoxide hydrolase.193 Furthermore, studies in rats and dogs have shown tBHQ to be primarily excreted as O- sulphate and O-glucuronide conjugates in the urine within the first 24 hr.188,194 However, an increase in tBHQ intake (from 100 mg/kg to 400 mg/kg) was found to decrease urinary elimination with the remaining amounts recovered in feces.188 In addition, tBHQ accumulation in tissues was assessed in short-term and long-term exposure studies in rats, which found insignificant levels of tBHQ residues in liver, brain, and kidney. In humans, tBHQ in the serum and metabolites of tBHQ in the urine have 26 been examined following the ingestion of 100- 150 mg of tBHQ. Similar to rats and dogs, tBHQ was excreted as O-sulphate and O-glucuronide conjugates at a ratio of approximately 3:1 in the urine within the first 24 h. Unchanged tBHQ was not detected in the urine of humans, while minor amounts of tBHQ were excreted in feces (estimated between 2.4- 3.7 %). It should be noted, the urinary recoveries of tBHQ were shown to be dependent on the vehicle. For example, a vehicle containing 30% corn oil had a greater recovery of tBHQ in the urine compared to a vehicle containing 10% corn oil, suggesting tBHQ consumed in a high fat vehicle increases absorption compared to a low-fat vehicle.188 III. Rationale for the Presented Studies Evidence regarding the immunomodulatory role of Nrf2 in disease and on T and B cell immune responses has increased, with previous studies from our lab demonstrating Nrf2 activation by tBHQ modulates both T and B cell activity.139,142,143,145–147 In particular, tBHQ significantly reduced IFNg secretion in activated primary human and primary murine T cells.142,143 Additional studies from our lab have shown impaired NK cell response to primary influenza infection and increased influenza severity in calorie- restricted mice.63,71 Given the importance of NK cell responses during primary influenza infection, the widespread use of tBHQ in food and the unknown effects of tBHQ on NK cells, the following studies described aim to determine whether tBHQ impairs NK cell activation in a Nrf2-dependent manner and whether dietary tBHQ at concentrations relevant to human exposure diminishes NK cell responses to influenza infection in vivo. 27 CHAPTER 2 The Nrf2 activator tBHQ inhibits the activation of primary murine natural killer cells 28 Abstract Tert-butylhydroquinone (tBHQ) is a commonly used food preservative with known immunomodulatory activity; however, there is little information regarding its role on natural killer (NK) cell activation and function. tBHQ is a known activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which results in induction of cytoprotective genes. Activation of Nrf2 has been shown to modulate immune responses in a number of different models. In addition, studies in our laboratory have shown that tBHQ inhibits numerous early events following T cell activation. In the current study, we investigated whether activated NK cells are impacted by tBHQ, since many signaling cascades that control NK cell effector function also contribute to T cell function. Splenocytes were isolated from female, wild-type C57Bl/6J mice and treated with 1 µM or 5 µM tBHQ. NK cell function was assessed after activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 24 h. Activation of NK cells in the presence of tBHQ decreased total NK cell percentage, production of intracellular interferon gamma (IFNg), granzyme B, and perforin, and induction of the cell surface proteins CD25 and CD69, which are markers of NK cell activation. In addition to NK cell effector function, NK cell maturation was also altered in response to tBHQ. Notably, this is the first study to demonstrate that the Nrf2 activator, tBHQ, negatively impacts effector function and maturation of NK cells. 29 Introduction Natural killer (NK) cells play a vital role in innate immunity and are necessary for a rapid response against infected and cancerous cells. Activating and inhibitory receptors allow NK cells to effectively monitor tissues for abnormal cells.195 Upon infection, NK cells are able to recognize and directly kill infected cells, in part through secretion of perforin, granzyme, and proinflammatory cytokines, such as interferon gamma (IFNg).196,197 Mechanisms for activation not only occur with recognition through activating receptors or lack of ligand binding inhibitory receptors, but also by cytokine stimulation. Effector functions are acquired as NK cells mature and can be observed at distinct stages. NK cells are produced from lymphoid progenitors in the bone marrow and circulate in the periphery where they can continue to mature and gain function 198. Maturation in mice can be classified using surface markers, CD27 and CD11b, in four stages: CD27-CD11b-, CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+.199 CD27 is the first marker expressed followed by the appearance of CD11b, which is an indicator of cell effector function.199 Double positive NK cells, CD27+CD11b+, have a lower activation threshold and therefore, are the most responsive phenotype with high cytolytic capacity and rapid cytokine production.200 NK cells that are terminally differentiated are identified by the loss of CD27 expression and are also capable of inducing effector functions. However, these cells are more tightly regulated and have a higher activation threshold due to increased expression of Ly-49, an inhibitory receptor.198,200,201 Studies from our group and others suggest that NK cell cytotoxicity and development are greatly influenced by dietary interventions. Our laboratory previously demonstrated alterations in NK cell maturation and impaired function in calorie-restricted mice.202–204 Other 30 studies involving vitamin supplementation, alcohol consumption, and excessive energy intake have also been shown to impact NK cell function and development 205–208. Tert-butylhydroquinone (tBHQ) is widely used as a food additive to delay rancidification of fats, and it can be found in many foods including oils, crackers, and cereals, among others.209 The U.S. Food and Drug Administration regulates the amount of tBHQ in foods with a limit of 0.02% of the oil or fat content of the food.210 Additionally, the acceptable daily intake (ADI) of tBHQ is 0.7 mg/kg body weight per day. Regardless of the regulations and recommendations, the amount of tBHQ consumed by an individual is difficult to determine. Estimates of tBHQ intake have been performed using several methods. Based on poundage, the estimates of tBHQ intake were below the ADI. Consistently, model diets measuring the intake of tBHQ found the average consumer below the ADI, yet individuals with high tBHQ consumption exceeded the ADI, up to 1100% of the ADI.186 Previous studies in humans have found serum concentrations in the high micromolar range after consuming 100-150 mg tBHQ, suggesting that the compound is readily absorbed following consumption.191 tBHQ is a well characterized nuclear factor erythroid 2-related factor 2 (Nrf2) activator. Nrf2 is a transcription factor that acts as a sensor for cell stress by inducing expression of cytoprotective genes when activated. Under basal conditions, Nrf2 is tethered to Kelch ECH associating protein 1 (Keap1), a cytosolic repressor protein, and is polyubiquitinated for proteasomal degradation. Activation by cellular and environmental stresses leads to Nrf2 translocation to the nucleus where it binds to antioxidant response elements, causing upregulation of cytoprotective genes.211 tBHQ is a potent activator of Nrf2 through 31 modification of thiol groups of cysteines on the Keap1 protein, which prevents Nrf2 repression by Keap1, therefore allowing induction of antioxidant responses by Nrf2.212 Much of the toxicology research on tBHQ has primarily focused on potential carcinogenic effects in humans, with the research indicating that tBHQ is not carcinogenic.209 However, research from our laboratory has shown that Nrf2 has numerous immunomodulatory effects in T cells. We previously showed tBHQ significantly reduces IFNg secretion and skews differentiation of primary murine CD4+ T cells from T helper type 1 (Th1) to T helper type 2 (Th2) in a Nrf2-dependent manner.213 Additionally, in human Jurkat T cells, tBHQ inhibits interleukin (IL)-2 production and decreases expression of CD25.214,215 More recently, treatment of primary human T cells with tBHQ causes decreased expression of CD25 and CD69 surface proteins and reduced production of IL-2 and IFNg, suggesting that overall T cell activation may be inhibited.216 Based on these findings, we hypothesized that NK cells could also be impacted by tBHQ because NK cells and T cells possess many similarities, including robust IFNg production and cytotoxic capabilities, and shared transcription factors and cell surface molecules.217 To our knowledge, the role of the food additive tBHQ has not been studied in isolated NK cells. The goal of the present study was to determine the effects of the Nrf2 activator, tBHQ, on NK cells in a mixed murine splenocyte preparation using flow cytometric analysis. Notably, this is the first study to demonstrate that the Nrf2 activator, tBHQ, inhibits activation, alters maturation, and diminishes effector function in NK cells. 32 Materials and Methods Materials All materials were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise specified. Mice Female, wild-type C57Bl/6J mice (13 weeks of age) were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were acclimated for a week and given food and water ad libitum. All animal studies were conducted in accordance with the Guide for the Care and Use of Animals as adopted by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) at Michigan State University. Cell Preparation Spleens were aseptically removed for lymphocyte isolation by grinding and filtering tissues through a 40 µm strainer. Cells were subsequently washed, counted, and adjusted to a cell density of 5 x 106 c/mL. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid solution (HEPES), 10% fetal bovine serum (FBS), non-essential amino acid (1X final concentration from 100X), 100 U/mL penicillin, 100 U/mL streptomycin, and 1000 U/mL 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA). Splenocytes were either treated with tBHQ or vehicle (VEH) at concentrations indicated in the figure legends 30 min prior to activation with 40 nM phorbol 12-myristate 13-acetate (PMA) and 0.5 µM ionomycin. Up to 5µM tBHQ was used in the present studies; the concentrations were selected in part because they are known not to affect viability based on broad concentration responses previously conducted in our lab. Further, 33 while information on the blood concentrations of tBHQ within the general public is not readily available, research has shown that a bolus dose of tBHQ provided orally to humans translates into a blood concentration reaching around 200µM, suggesting the concentrations used in this study are well within the range of potential human exposure. 191 Immediately following the addition of PMA and ionomycin, 15 µM monensin solution was used to block protein secretion for intracellular labeling (eBioscience, San Diego, CA). Splenocytes were then incubated for 24 h. Flow Cytometry After 24 h of incubation, splenocytes were transferred to a 96-well V-bottom plate and stained with Zombie Aqua Fixable Viability Dye following the manufacturer protocol (Biolegend, San Diego, CA). Samples were treated with Fc-Block (antibody to CD16/32 [2.4G2], BD Bioscience, San Jose, CA) and labeled for surface markers using appropriate antibodies for 30 min. Subsequently, samples were fixed using BD Cytofix Fixation Buffer (BD Biosciences). For intracellular staining, splenocytes were permeabilized, labeled for intracellular markers, and fixed following previously published procedures with minor modifications.218 Combinations of the following fluorochrome-conjugated antibodies (Biolegend, eBioscience or BD Biosciences) were used: CD3ε (AlexaFluor488 [17A2] or AlexaFluor700 [eBio500A2]), NK1.1 (BrilliantViolet711 [PK136]), CD27 (PerCP/eFluor710 [LG.7F9]), CD11b (AlexaFluor647 [M1/70]), CD69 (AlexaFluor700 [H1.2F3]), CD25 (PE [PC61] or [3C7]), IFNg (APC [XMG1.2]), granzyme B (FITC [GB11]), and perforin (PE [eBio0MAK-D]). Samples were acquired and analyzed on an Attune NxT Acoustic Focusing Cytometer from Life Technologies (Thermo Fisher Scientific, Waltham, MA). 34 Statistical Analysis Treatment groups are presented as mean ± standard error (SE). SigmaPlot 12.3 (Systat, Chicago, IL) was used to statistically analyze data. The Shapiro-Wilk Normality Test was performed to determine normal distribution. Normally distributed data were analyzed by one- way ANOVA followed by a Dunnett’s post-hoc test to determine statistical significance of p < 0.05 comparing all groups to the VEH treatment. Data not normally distributed were analyzed using Kruskal-Wallis one-way analysis of variance on ranks. A sample size of 5 or 10 mice was used for each experiment and is noted in the figure legends. Results The Nrf2 Activator, tBHQ, Modestly Decreases the Percentage of NK Cells in Spleen Our laboratory has previously shown the immunomodulatory effects of Nrf2 activation by tBHQ in primary murine CD4+ T cells, Jurkat T cells, and primary human CD4+ T cells; however, the effects of Nrf2 activation by tBHQ in NK cells has yet to be characterized. 213–216 To determine the effect of tBHQ on NK cells, we quantified the NK cell population by gating CD3-NK1.1+. tBHQ concentrations were selected based on concentrations used previously in our lab that did not demonstrate cytotoxicity in these experiments. Consistent with these previous studies, tBHQ did not decrease viability in the current studies (data not shown). Compared to the VEH group, splenocytes treated with 5 µM tBHQ displayed a modest, though statistically significant, reduction in the percentage of NK cells within the splenocyte population (Figure 3, VEH, 2.19 ± 0.08%; 5 µM tBHQ, 1.79 ± 0.1%). 35 Figure 3: tBHQ treatment modestly decreases the percentage of NK cells in spleen. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were then labeled with fluorescently conjugated antibodies against CD3ε and NK1.1 for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive. n = 10 *Indicates p < 0.05 compared to VEH group. The Nrf2 Activator, tBHQ, Decreases NK Cell Expression of CD25 and CD69 The activation of NK cells by PMA and ionomycin can be assessed in part via the induction of the cell surface molecules CD25 and CD69, which serve as markers of activation of lymphocytes. Previously, our laboratory demonstrated reduced induction of CD25 and CD69 in activated T cells with tBHQ treatment.216 This prompted us to quantify CD25 and CD69 expression in activated NK cells. The percentage of NK cells expressing CD25 was significantly reduced in groups treated with 1 µM and 5 µM tBHQ compared to the VEH group (Figure 4A, VEH, 1.4 ± 0.15%; 1 µM tBHQ, 0.85 ± 0.07%; 5 µM tBHQ, 0.58 ± 0.13%). Likewise, NK cell expression of CD69 followed the same trend with significant decline in expression for both tBHQ-treated groups (Figure 4B, VEH, 63.57 ± 1.39%; 1 µM tBHQ, 53.39 ± 0.88%; 5 µM tBHQ, 46.08 ± 1.01%). Activated NK cells in the presence of tBHQ markedly decreased induction of CD25 and CD69 to levels lower than that of unactivated NK cells (BKG) (Figure 4, CD25 BKG, 1.1 36 ± 0.12%; CD69 BKG, 52.46 ± 1.07%). Taken together, the food preservative tBHQ inhibits expression of activation markers CD25 and CD69 in activated NK cells, which is consistent with the effects of tBHQ in T cells. Figure 4: tBHQ treatment inhibits expression of cell surface molecules CD25 and CD69 in NK cells. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε, NK1.1, CD25, and CD69 for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to analysis of (A) CD25 and (B) CD69 expression. n = 5 * Indicates p < 0.05 compared to VEH group. Treatment with the Nrf2 Activator, tBHQ, Alters NK Cell Maturation In Vitro NK cell effector function is acquired through maturation and is defined by distinct stages.217 Therefore, we examined whether the Nrf2 activator, tBHQ, altered NK cell maturation within the total NK cell population. As expected, NK cells expressing an immature phenotype, CD27-CD11b-, were significantly greater in the BKG group likely due to the absence of cell activation (Figure 5A, VEH, 27.87 ± 2.73%; BKG, 37.91 ± 3.48%). A more mature, yet not fully functional, subset of NK cell maturation is defined by the expression of CD27. We found this NK cell subset, CD27+CD11b-, at significantly lower percentages in groups treated with 1 µM (5.23 ± 0.41%) and 5 µM tBHQ (3.21 ± 0.32%) in relation to the VEH group (7.19 ± 0.61%) 37 (Figure 5B). Following the progression of maturation, the most responsive NK cell subset, CD27+CD11b+, was highest in the VEH group, while the percentage of this subset was decreased in the tBHQ-treated groups. Though these results were not significant (p = 0.052), treatment with 5 µM tBHQ (2.09 ± 0.37%) exhibited the greatest difference compared to the VEH group (4.6 ± 0.71%) in which 5 µM tBHQ decreased the CD27+CD11b+ subset to a percentage lower than that of the (unactivated) BKG group (Figure 5C). Interestingly, the most mature and terminally differentiated NK cell phenotype, CD27-CD11b+, was significantly increased with treatment of 5 µM tBHQ (67.83 ± 2.67%) compared to the VEH group (60.33 ± 2.34%) (Figure 5D). Collectively, these results indicate that the food preservative, tBHQ, has significant effects on NK cell maturation. 38 Figure 5: tBHQ treatment alters NK cell maturation. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε, NK1.1, CD27, and CD11b for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to FACS analysis of maturation markers, CD27 and CD11b. Distinct stages of NK cell maturation occur in the following order: (A) CD27-CD11b-, (B) CD27 +CD11b-, (C) CD27 +CD11b+, (D) CD27-CD11b+. n = 10 *Indicates p < 0.05 compared to VEH group. Treatment with the Nrf2 Activator, tBHQ, Inhibits IFNg Production in NK Cells As NK cells mature and express CD11b, they acquire effector function and have increased capacity for producing cytokines such as IFNg. A previous study from our laboratory showed tBHQ decreased production of IFNg in CD4+ T cells.216 Because tBHQ altered NK cell maturation, we investigated the effects of tBHQ on IFNg production by NK cells. The 39 percentage of NK cells producing IFNg was significantly reduced in groups treated with 1 µM and 5 µM tBHQ in a concentration-dependent manner (Figure 6A, VEH, 53.38 ± 4.64%; 1 µM tBHQ, 29.35 ± 2.28%; 5 µM tBHQ, 12.62 ± 1.25%). In addition to a decrease in the percentage of IFNg-producing NK cells, tBHQ also caused a reduction in mean fluorescence intensity (MFI) (Figure 6B, VEH, 14567.2 ± 1876.06; 5 µM tBHQ, 2261.2 ± 128.83), suggesting a decrease in IFNg expression in individual NK cells. Figure 6: tBHQ treatment inhibits production of the cytokine IFNg in NK cells. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε and NK1.1 followed by intracellular labeling of IFNg with fluorescently conjugated antibodies for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to analysis of IFNg by (A) percentage and (B) mean fluorescence intensity (MFI). n = 5 *Indicates p < 0.05 compared to VEH group. The Nrf2 Activator, tBHQ, Decreases Production of Perforin and Granzyme B in NK Cells Due to a decreased production of IFNg in NK cells treated with tBHQ, we decided to further investigate fundamental NK cell effector functions, including production of perforin and granzyme B. As concentrations of tBHQ increased, the percentage of NK cells producing perforin was reduced in a concentration-dependent manner with significant reduction 40 observed by 5 µM tBHQ (Figure 7A, VEH, 3.98 ± 0.29%; 5 µM tBHQ, 1.8 ± 0.09%). Similarly, granzyme B production by activated NK cells was significantly decreased in both groups treated with tBHQ compared to the VEH group (Figure 7B, VEH, 31.74 ± 2.21%; 1 µM tBHQ; 20.37 ± 2.46%; 5 µM tBHQ, 14.9 ± 1.67%). Collectively, these results demonstrate inhibitory effects of the food preservative, tBHQ, on vital NK cell cytolytic events. Figure 7: tBHQ treatment inhibits the production of perforin and granzyme B in NK cells. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε and NK1.1 followed by intracellular labeling of perforin and granzyme B with fluorescently conjugated antibodies for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to analysis of (A) perforin and (B) granzyme B. n = 5 *Indicates p < 0.05 compared to VEH group. Discussion The current study is the first to show inhibition of NK cell activation and effector functions in murine splenocytes with increasing concentrations of tBHQ. tBHQ at low micromolar concentrations diminished NK cell activation-induced CD25 and CD69 expression in accord with previous studies using primary human CD4+ T cells treated with tBHQ.216 Additionally, tBHQ induced NK cell maturation as indicated by the decreased percentage of the 41 intermediate NK cell subsets, CD27+CD11b- and CD27+CD11b+, and increased percentage of terminally differentiated NK cells, CD27-CD11b+. NK cell effector function was also found to be markedly reduced after supplementation of tBHQ. Notably, tBHQ significantly inhibited the production of IFNg in NK cells in a concentration-dependent manner. Likewise, significant reductions in perforin and granzyme B production were observed with increasing concentrations of tBHQ, suggesting impairment of NK cell effector function after supplementation with tBHQ. The cell surface proteins CD27 and CD11b are commonly used to identify specific stages of murine NK cell development.199 NK cell function has correlated with developmental stages based on expression of CD27 and CD11b.200 In the spleen, NK cells represent a small fraction (< 2.5%) of the lymphocyte population with the majority of NK cells expressing CD27- CD11b+.200,219 Consistent with our results, less than 2.5% of the splenic lymphocytes were identified as NK cells, and of those NK cells, CD27-CD11b+ was the predominant phenotype within all treatment groups (Figure 1 and Figure 5D). Interestingly, the percentage of CD27- CD11b+ NK cells was significantly greater when treated with 5 µM tBHQ, while CD27+CD11b+ NK cells were reduced in both tBHQ-treated groups (Figure 5C and 5D). Prior studies have also demonstrated decreased cytolytic capacity and cytokine production in mature CD27-CD11b+ NK cells compared to CD27+CD11b+ NK cells.198,200 The present study found a decreased percentage of CD27+CD11b+ NK cells to be reflective of the diminished production of IFNg, perforin, and granzyme B with tBHQ treatments (Figure 5C, Figure 6 and Figure 7). The increase in CD27-CD11b+ NK cells treated with 5 µM tBHQ (Figure 5D) may provide relatively small amounts of IFNg, perforin, and granzyme B. Still, the majority of effector functions are 42 elicited by the CD27+CD11b+ NK cell subset. NK cells identified as CD27+CD11b- were significantly reduced in the 1 µM and 5 µM tBHQ-treated groups (Figure 5B). However, this subpopulation does not exhibit substantial cytolytic function or cytokine production, therefore does not contribute to the reduced effector functions seen with tBHQ treatment. Taken together, our results suggest that although tBHQ may cause an increase in the proportion of mature, fully differentiated NK cells, it may also diminish the effector function of these cells. Several studies have noted immunomodulatory effects of Nrf2 when activated by tBHQ. As mentioned earlier, our laboratory has demonstrated that tBHQ strongly activates Nrf2 and modulates T cells in primary mouse CD4+ T cells, Jurkat T cells, and primary human CD4+ T cells.213–216 Furthermore, dendritic cells were found to be affected by tBHQ through Nrf2 activation. Treatment of tBHQ in lipopolysaccharide-activated human dendritic cells potently inhibited the secretion of the cytokine IL-12, which is involved in the activation NK cells and the differentiation of CD4+ T cells.220,221 Studies have also shown macrophages to be affected by Nrf2 activation. In particular, macrophages producing proinflammatory cytokines, tumor necrosis factor alpha (TNFa) and IL-6, were inhibited with tBHQ treatment in a concentration- dependent manner after lipopolysaccharide stimulation.222,223 Additionally, activation of Nrf2 by tBHQ was found to regulate macrophage polarization by promoting the anti-inflammatory M2 macrophages.223 Consistent with other immune cell types, our results also demonstrate the immunomodulatory effects of the food additive, tBHQ, on splenic murine NK cells, including reduced expression of activation markers CD25 and CD69, diminished production of IFNg, perforin, and granzyme B, and altered maturation. 43 Collectively, these results suggest treatment of murine splenocytes with the Nrf2 activator, tBHQ, greatly impact activated NK cells. tBHQ inhibited the upregulation of CD25 and CD69 in NK cells upon activation with PMA and ionomycin in a concentration-dependent manner. Additionally, tBHQ treatment increased the percentage of NK cells displaying a more mature phenotype, CD27-CD11b+. Interestingly, markers that measure NK cell function, such as IFNg, perforin, and granzyme B, were significantly decreased in groups treated with tBHQ. In conclusion, this study has shown that doses of tBHQ that are relevant to human exposure can significantly impair NK cell function in vitro. Therefore, future studies should determine if the effects of tBHQ on NK cell maturation and function will significantly impact human health and disease, particularly in the context of host defense and cancer. 44 CHAPTER 3 Activation of Nrf2 by tBHQ promotes maturation, while concurrently inhibiting activation and expression of effector molecules by activated NK cells 45 Abstract The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) upregulates antioxidant, detoxification, and cell stress genes when activated by oxidative stress or exogenous compounds. tert-butylhydroquinone (tBHQ) is a potent activator of Nrf2 and a food preservative. Previously, we have found tBHQ negatively impacted NK cell activation and effector function and altered NK cell maturation. In the current study, we examined the role of Nrf2 in these effects. Splenocytes were isolated from wild-type (WT) C57Bl/6J mice or Nrf2- deficient mice and treated with varying concentrations of tBHQ (0.1 µM- 5 µM tBHQ). Following treatment, NK cells were activated for 24 hours with either phorbol 12-myristate 13- acetate (PMA) and ionomycin or a specific NK cell activator, IL-12/IL-18 cytokines prior to flow cytometry analysis. There were pronounced genotype and tBHQ effects on NK cell maturation. Specifically, there was a significant increase in the percentage of immature NK cells (CD27- CD11b-) in activated splenocytes derived from Nrf2-knockout mice. tBHQ increased the percentage of terminally differentiated NK cells derived from wild-type, but not Nrf2-null, mice. In addition, tBHQ caused a Nrf2-dependent decrease in expression of the activation markers, CD25 and CD69, in PMA/ionomycin-activated NK cells. Likewise, tBHQ decreased expression of Fas ligand in IL-12/IL-18-activated NK cells in a Nrf2-dependent manner. In contrast, inhibition of IFNg induction by tBHQ appeared to be only partially Nrf2-dependent. Taken together, the data suggest that activation of Nrf2 by tBHQ promotes NK cell terminal differentiation, while concurrently inhibiting activation and effector function, which could negatively impact NK cell- mediated host defense. 46 Introduction The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of cytoprotective genes in response to endogenous and exogenous cellular stresses. Under quiescent conditions, Nrf2 is retained in the cytoplasm by its repressor protein, Kelch ECH associating protein 1 (Keap1), and is rapidly degraded through the ubiquitin- proteasome pathway.224 However, in the presence of oxidative and electrophilic stimuli, critical cysteine residues in Keap1 that serve as stress sensors undergo modification resulting in conformational changes, thereby disrupting the ubiquitination and subsequent proteasomal degradation of Nrf2. Nrf2 accumulates in the cytoplasm and translocates to the nucleus where it binds to antioxidant response elements, leading to the upregulation of cytoprotective genes that enhance cell survival.81,225–227 Tert-butylhydroquinone (tBHQ) is a well-characterized inducer of Nrf2 and is commonly used as a food preservative to prevent rancidification and oxidation of lipids.185,187 Previous studies in humans have shown serum levels of tBHQ reaching the high micromolar range following ingestion of 150 mg tBHQ.191 Additionally, assessments of tBHQ intake based on model diets suggest high consumers of fats, oils, and frozen fish products are likely to exceed the recommended acceptable daily intake (ADI) of tBHQ, which is set at 0.7 mg/kg body weight.186 The current study used concentrations of tBHQ in the low micromolar range of 0.1- 5 µM tBHQ, which our lab has previously shown to activate Nrf2 without causing cytotoxicity in T cells.142 Increasing evidence shows involvement of Nrf2 in mediating anti-inflammatory responses in various disease models, including allergy, autoimmune diseases, and inflammatory 47 diseases.150,151,228–234 Studies have also identified Nrf2-mediated effects in several immune cell types when treated with tBHQ. Our lab demonstrated Nrf2 activation by tBHQ promotes Th2 differentiation, whereas absence of Nrf2 skews CD4+ T cells toward Th1 differentiation.142 Additional studies found the Nrf2 activator, tBHQ, inhibited early events following T cell activation in human Jurkat T cells, primary human CD4+ T cells, and primary murine splenocytes.142,143,145–147 In B cells, we showed a Nrf2-dependent increase in IgM production by tBHQ. Others showed tBHQ potently inhibited IL-12 expression in LPS-stimulated dendritic cells.235 In macrophages, tBHQ attenuated LPS-induced production of pro-inflammatory markers and promoted M2 polarization, which was further confirmed by the finding that Nrf2 interferes with the transcriptional upregulation of proinflammatory cytokines induced by LPS.236–238 Importantly, the role of Nrf2 in NK cells remains largely unknown. Previously, our lab investigated the effects of tBHQ on activated NK cells and found tBHQ induced maturation and inhibited activation and effector function.137 Since tBHQ is a potent activator of Nrf2, the current study examines whether these effects are mediated by Nrf2. Materials and Methods Materials tBHQ and all other materials were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise stated. Animals and Housing Nrf2-null mice on a mixed C57BL/6 and background were generated and received from Dr. Jefferson Chan at the University of California San Francisco, San Francisco, CA.239 Mice were 48 backcrossed on the C57BL/6 background for eight generations and were 99% congenic (analysis performed by Jackson Laboratory, Bay Harbor, ME). Wild-type (Jackson Laboratories, Bar Harbor, ME) and Nrf2-null female mice, ages 9-22 weeks old, were used for this study. Age- matched mice were given food and water ad libitum. Animal studies were performed following the Guide for Care and Use of Animals adopted by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) at Michigan State University. Cell Preparation Single-cell suspensions from spleens were obtained via grinding and filtering of tissue through a 40 µm strainer. Subsequently, cells were washed and depleted of red blood cells by using ACK lysis buffer following manufacturer’s protocol. Cells adjusted to a concentration of 5x106 cells/mL and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid solution (HEPES), 10% fetal bovine serum (FBS), non-essential amino acid (1X final concentration from 100X), 100 U/mL penicillin, 100 U/mL Streptomycin, and 100 U/mL 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA). Cells were left alone (background, BKG) or treated with tBHQ at various concentrations ranging from 0.1- 5 µM tBHQ or 0.005% ethanol (vehicle, VEH). Concentrations of tBHQ are consistent with previous studies conducted in our lab and are relevant to human exposure, as research has shown a bolus dose of tBHQ taken orally equates to an estimated blood concentration of 200 µM.191 After 30 min, cells were activated with either 10 ng/mL of IL-12/IL- 18 or 40 nM phorbol 12-myristate 13-acetate (PMA)/0.5 µM Ionomycin for 24 h. Cells in the BKG treatment group did not receive either activator. Monensin (1X) was added to cells 5 h 49 prior to cell collection (19 h post treatment) to inhibit cytokine secretion for intracellular labeling (eBioscience, San Diego, CA). Flow Cytometry After 24 h of incubation, cells were transferred to a 96-well V-bottom plate and washed with dPBS prior to staining with Zombie Aqua Fixable Viability Dye following manufacturer’s protocol (Biolegend, San Diego, CA). Cells were washed with FACS buffer (1% FBS in dPBS) and treated with Fc-Block (CD16/32 [2.4G2], BD Bioscience, San Jose, CA) prior to surface marker staining with fluorescently conjugated antibodies (Table 1). After 30 min, samples were permeabilized using FoxP3/Transcription Buffer Staining Kit (Invitrogen, Waltham, MA) following the manufacturer’s protocol and stained with the intracellular antibodies listed in Table 1 for 30 minutes. Cells were subsequently washed and immediately analyzed by flow cytometry using an Attune NxT Flow Cytometer (Thermo Fisher, Waltham, MA). Following the manufacturer’s protocol, compensation was performed using Ultra-Comp eBeads (Invitrogen, Waltham, MA). Table 1: List of Antibodies Antibody Fluorochrome Clone Supplier Live/Dead Zombie Aqua Dye Biolegend CD3 Alexa Fluor 488 17A2 Biolegend NK1.1 Brilliant Violet 711 PK136 Biolegend CD27 PerCP-eFluor 710 LG.7F9 eBioscience CD11b Alexa Fluor 647 M1/70 Biolegend CD25 Pacific Blue PC61 Invitrogen 50 Table 1 (cont’d): List of Antibodies CD69 Alexa Fluor 700 H1.2F3 Biolegend CD107a PE-Cy7 1D4B BD Biosciences CD178 (FasL) PE MFL3 Biolegend Granzyme B Pacific Blue QA16A02 Biolegend IFN-𝛾 APC XMG1.2 Biolegend Perforin PE S16009B Biolegend IFNg ELISA Supernatants from samples activated with IL-12/IL-18 were collected 24 h after treatment for the analysis of IFNg protein by ELISA. Mouse IFNg ELISA MAX Standard Kit (Biolegend, San Diego, CA) was used to detect IFNg and quantified at an absorbance of 450 nm by Tecan Infinite M1000 Pro Microplate Reader (Tecan; San Jose, CA). Statistical Analysis The mean ± standard error (SE) was determined for each treatment group. Normally distributed data were analyzed by two-way ANOVA followed by a Dunnett’s post hoc test to compare treatment groups to vehicle control. Analyses were performed using GraphPad PRISM software version 8.4.2 (GraphPad Software, La Jolla, CA). Results Nrf2 Modestly Increases NK Cells with IL-12/IL-18 Activation. Our previous studies showed a potent effect of the food additive tBHQ on PMA/ionomycin-activated NK cells in vitro.137 To determine the role of Nrf2 in these effects, we 51 used splenocytes from wild-type and Nrf2-null mice. We found no difference in the percent of NK cells between Nrf2-null and wild-type mice, which is consistent with a previous report that found no significant difference in the numbers of splenic NK cells between wild-type and Nrf2- null mice.240 We next wanted to identify the effects of tBHQ on NK cells following activation with IL-12/IL-18 or PMA/Ionomycin (where PMA/ionomycin was the NK cell activator we used in our previous study). We did not observe a difference between treatment groups in the percentage of NK cells regardless of activation, however we observed a trend toward a modest increase in percentage of NK cells in the 5 µM tBHQ + IL-12/IL-18 group that was not statistically significant (Figure 8A). Overall, the results suggest that neither tBHQ nor Nrf2 affects the percentage of splenic NK cells following activation with either PMA/Ionomycin or IL- 12/IL-18. A) B) NK cells NK cells 5 5 WIld-type WIld-type 4 4 Nrf2-null Nrf2-null % NK cells % NK cells 3 3 2 2 1 1 0 0 H Q Q Q Q H Q Q Q Q VE H H H H VE H H H H tB tB tB tB tB tB tB tB µM µM µM µM µM µM µM µM 0.1 0.5 1 5 0.1 0.5 1 5 IL12/IL18 Activation PMA/Ionomycin Activation Figure 8: Nrf2 modestly increases NK cells with IL-12/IL-18 activation. Splenocytes were isolated from wild-type or Nrf2-null C57Bl/6 mice. Cells were either left untreated (BKG) or treated with tBHQ (0.1- 5 µM) or 0.005% ethanol (VEH) for 30 min prior to addition of activators (A) IL-12/IL- 18 or (B) PMA/Ionomycin. After 24 h, cells were stained with viability dye and fluorescently conjugated antibodies against CD3ε and NK1.1 for analysis by flow cytometry. NK cells were identified as viable CD3-NK1.1+ cells. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared to vehicle of respective genotype 52 Figure 8 (cont’d): # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to wild-type vs Nrf2-null of respective treatment n= 5 Nrf2 Increases Maturation of NK Cells. NK cell maturation is defined by differential surface expression of CD27 and CD11b with four distinct stages of maturation which progress in the following order: CD27-CD11b-, CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+. During the maturation process, NK cells gradually acquire cytotoxic capabilities.21 Unexpectedly, we found significant genotype differences in NK cell maturation in the untreated groups with an increase in percentage of immature NK cells in the spleens from Nrf2-null mice, whereas wild-type splenic NK cells displayed a more mature phenotype (Figure 9A-D). tBHQ treatment did not affect maturation with IL-12/IL-18 activation. However, in PMA/Ionomycin activated cells, 5 µM tBHQ increased the percentage of terminally mature NK cells from wild-type, but not Nrf2-null, mice (Figure 2H). Overall, these data indicate that Nrf2 plays a role in the progression of maturation of NK cells. 53 Figure 9: Nrf2 increases maturation of NK cells. Splenocytes were isolated from wild-type or Nrf2-null C57Bl/6 mice. Cells were either left untreated (BKG) or treated with tBHQ (0.1- 5 µM) or 0.005% ethanol (VEH) for 30 min prior to addition of activators (A-D) IL-12/IL-18 or (E-H) PMA/Ionomycin. After 24 h, cells were stained with viability dye and fluorescently conjugated antibodies against CD3ε, NK1.1, CD27, and CD11b for analysis by flow cytometry. NK cells were identified as viable CD3-NK1.1+ cells prior to FACS analysis of maturation markers, CD27 and CD11b. Maturation occurs in the following order from Immature to mature NK cells: (A, E) CD27-CD11b-, (B, F) CD27+CD11b-, (C, G) CD27+CD11b+, (D, H) CD27-CD11b+. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared to vehicle of respective genotype # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to wild-type vs Nrf2-null of respective treatment n= 5 tBHQ Treatment Decreases Activation of NK Cells Independently of Nrf2. To evaluate activation by IL-12/IL-18 and PMA/ionomycin, surface expression of CD69 and CD25 were assessed on NK cells. Interestingly, early activation markers significantly decreased with 5 µM tBHQ in IL-12/IL-18- activated NK cells. This decrease was not dependent on Nrf2, as a reduction in CD69+CD25+ expression occurred in both wildtype- and Nrf2-null NK cells (Figure 10A). However, in PMA/ionomycin-activated NK cells, CD69+CD25+ expression 54 was inhibited with 5 µM tBHQ solely in wildtype cells (Figure 10B). Overall, the data suggest that tBHQ inhibits early activation markers in NK cells regardless of the mode of activation. The inhibition is largely Nrf2-dependent when the cells are activated with PMA/ionomycin, but interestingly, independent of Nrf2 when activated with IL-12/IL-18. Figure 10: tBHQ decreases activation of NK cells independently of Nrf2. Splenocytes were isolated from wild-type or Nrf2-null C57Bl/6 mice. Cells were either left untreated (BKG) or treated with tBHQ (0.1- 5 µM) or 0.005% ethanol (VEH) for 30 min prior to addition of activators (A) IL-12/IL-18 or (B) PMA/Ionomycin. After 24 h, cells were stained with viability dye and fluorescently conjugated antibodies against CD3ε, NK1.1, CD69, and CD25 for analysis by flow cytometry. NK cells were identified as viable CD3-NK1.1+ cells prior to FACS analysis of activation markers, CD69 and CD25. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared to vehicle of respective genotype # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to wild-type vs Nrf2-null of respective treatment n= 5 5 µM tBHQ Decreases FasL Expression in NK Cells in a Nrf2-Dependent Manner with IL-12/IL-18 Activation. FasL is expressed on activated NK cells, and upon ligation to target cells, the apoptotic signaling cascade is activated which leads to the activation of caspase 8 and 10 and ultimately induction of apoptosis.241 Here we show a Nrf2-dependent decrease in FasL expression in NK 55 cells activated with IL-12/IL-18, as demonstrated by a decrease in FasL at 5 µM tBHQ in NK cells from wild-type, but not Nrf2-null, mice (Figure 11A). Conversely, FasL was decreased in both genotypes at 5 µM tBHQ when activated with PMA/Ionomycin. However, there is a non- statistically significant trend toward greater inhibition in the WT group, suggesting both Nrf2- dependent and Nrf2-independent mechanisms may play a role. Figure 11: tBHQ decreases FasL expression in a Nrf2-dependent manner with IL-12/IL-18 activation. Splenocytes were isolated from wild-type or Nrf2-null C57Bl/6 mice. Cells were either left untreated (BKG) or treated with tBHQ (0.1- 5 µM) or 0.005% ethanol (VEH) for 30 min prior to addition of activators (A) IL-12/IL-18 or (B) PMA/Ionomycin. After 24 h, cells were stained with viability dye and fluorescently conjugated antibodies against CD3ε, NK1.1 and FasL for analysis by flow cytometry. NK cells were identified as viable CD3-NK1.1+ cells prior to FACS analysis of FasL. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared to vehicle of respective genotype # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to wild-type vs Nrf2-null of respective treatment n= 5 Nrf2 Promotes NK Cell Cytotoxicity. Previously, we found tBHQ decreases NK cell production of perforin and granzyme following activation with PMA/Ionomycin.137 Similarly, we found 5 µM of tBHQ to significantly decrease granzyme B in wild-type NK cells with PMA/Ionomycin (Figure 12C). However, while we did not see statistically significant differences with tBHQ treatments in Nrf2-null NK cells, 56 there was a trend toward decreased granzyme B in the Nrf2-null group, suggesting that a Nrf2- independent mechanism may be involved. While there was considerable variability in the granzyme B data sets, there was a consistent trend toward increased granzyme B induction in the NK cells derived from Nrf2-null mice. Likewise, perforin displayed significant differences between genotypes with wild-type NK cells producing significantly more perforin compared to Nrf2-null NK cells with both activators (Figure 12B and 12D). Additionally, treatment with 5 µM tBHQ significantly decreased perforin in wild-type NK cells but not Nrf2-null NK cells (Figure 12B and 12D). Taken together, the data suggest Nrf2 promotes perforin, and possibly granzyme B, induction and this effect does not require exogenous Nrf2 activators. Figure 12: Nrf2 promotes perforin induction in NK cells. Splenocytes were isolated from wild- type or Nrf2-null C57Bl/6 mice. Cells were either left untreated (BKG) or treated with tBHQ 57 Figure 12 (cont’d): (0.1- 5 µM) or 0.005% ethanol (VEH) for 30 min prior to addition of activators (A-B) IL-12/IL-18 or (C-D) PMA/Ionomycin. Monensin was added to samples 5 h prior to flow cytometry analysis (19 h post treatment). After 24 h, cells were stained with viability dye and fluorescently conjugated antibodies against CD3ε, NK1.1, granzyme B, and perforin for analysis by flow cytometry. NK cells were identified as viable CD3-NK1.1+ cells prior to FACS analysis of of intracellular markers, (A, C) granzyme B and (B, D) perforin. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared to vehicle of respective genotype # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to wild-type vs Nrf2-null of respective treatment n= 5 Reduction of IFNg by Nrf2 Activator tBHQ. IFNg is an important antiviral cytokine that is rapidly secreted upon activation. Our lab has shown tBHQ to significantly reduce NK cell expression of IFNg with PMA/Ionomycin activation.137 Consistently, IFNg significantly decreased with 1 and 5 µM tBHQ in wild-type NK cells with PMA/Ionomycin activation (Figure 13B). However, in Nrf2-null NK cells, IFNg reduction was only demonstrated with 5 µM tBHQ (Figure 13B). With IL-12/IL-18 activation, 5 µM tBHQ-treated cells decreased NK cell expression of IFNg in both genotypes (Figure 13A), suggesting a Nrf2-independent decrease in NK cell expression of IFNg with tBHQ. Unexpectedly, however, when we measured secreted IFNg in supernatants of cells activated with IL-12/IL-18, 5 µM tBHQ significantly decreased IFNg in wild-type splenocytes, while tBHQ treatment did not affect IFNg secretion in Nrf2-null splenocytes, which is not consistent with a fully Nrf2-independent mechanism (Figure 14). The contradiction between the two data sets may be due to kinetics as MFI is a “snapshot” of IFNg expression, whereas the ELISA values indicate IFNg accumulation over time. Collectively, these data indicate that tBHQ inhibits IFNg through a partially Nrf2-dependent mechanism at 1, but not 5 µM, tBHQ, when looking at 58 expression at 24 h via MFI. In addition, the tBHQ-mediated decrease in IFNg accumulation over 24 h appears to occur through a partially Nrf2-dependent mechanism. Figure 13: Reduction of IFNg expression with tBHQ is independent of Nrf2 in NK cells. Splenocytes were isolated from wild-type or Nrf2-null C57Bl/6 mice. Cells were either left untreated (BKG) or treated with tBHQ (0.1- 5 µM) or 0.005% ethanol (VEH) for 30 min prior to addition of activators (A) IL-12/IL-18 or (B) PMA/Ionomycin. Monensin was added to samples 5 h prior to flow cytometry analysis (19 h post treatment). After 24 h, cells were stained with viability dye and fluorescently conjugated antibodies against CD3ε, NK1.1, and IFN𝛾 for analysis by flow cytometry. NK cells were identified as viable CD3-NK1.1+ cells prior to FACS analysis of intracellular marker, IFN𝛾. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared to vehicle of respective genotype # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to wild-type vs Nrf2-null of respective treatment n= 5 59 Figure 14: Secreted IFNg protein accumulation is inhibited by tBHQ in a partially Nrf2- dependent manner. Splenocytes were isolated from wild-type or Nrf2-null C57Bl/6 mice. Cells were either left untreated (BKG) or treated with tBHQ (0.1- 5 µM) or 0.005% ethanol (VEH) for 30 min prior to addition of activator, IL-12/IL-18; BKG did not receive IL-12/IL-18. After 24 h, supernatants were collected for analysis of secreted IFN𝛾 protein by ELISA. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 compared to vehicle of respective genotype # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to wild-type vs Nrf2-null of respective treatment n= 5 Discussion Collectively, these results suggest that activation of Nrf2 by tBHQ alters NK cell maturation, decreases NK cell activation, and negatively impacts effector function. Notably, the presence of Nrf2 promoted terminal differentiation in NK cells, while tBHQ further increased the percent of mature NK cells with PMA/ionomycin activation. tBHQ treatment inhibited the upregulation of CD25 and CD69 through a partially Nrf2-dependent mechanism when the NK cells were activated with PMA/ionomycin, but not IL-12/IL-18. Similarly, tBHQ decreased FasL expression in NK cells activated with IL-12/IL-18 in a Nrf2-dependent manner. IFNg expression in NK cells was also inhibited by tBHQ through a partially Nrf2-dependent mechanism at 1 µM, but not 5 µM, tBHQ. However, secreted IFNg protein accumulation was reduced by tBHQ in a 60 partially Nrf2-dependent manner in the 5 µM tBHQ group. This study demonstrates a potential role of Nrf2 activation by tBHQ on NK cell maturation, activation, and effector function. In the current study, we used two different NK cell activators, including PMA/ionomycin and the cytokines IL-12 and IL-18. IL-12 and IL-18 are known NK cell stimulatory cytokines that elicit IFNg production and cytotoxicity.242 While PMA/ionomycin can activate numerous immune cell types by activation of protein kinase C by PMA and robust calcium influx by ionomycin (a calcium ionophore), which leads to the activation of several intracellular signaling pathways.243 Differences in genotype and tBHQ treatment effects between PMA/ionomycin and IL-12/IL-18 activated NK cells are likely due to the different modes of activation. Additionally, other cell types activated by PMA/ionomycin may influence NK cell responses, whereas IL-12/IL-18 activation is more specific to NK cell activation and may have less outside influence from other cell types. Notably, there was a striking difference in maturation between wildtype and Nrf2-null NK cells. Wildtype NK cells displayed a more mature phenotype (Figure 9A-D), which is important because as NK cells mature, they acquire effector function. This may also help explain the genotype differences in NK cell effector functions, such as granzyme B and perforin (Figure 12). Maturation of NK cells are defined by their expression of CD27 and CD11b surface markers. CD27+CD11b- and CD27+CD11b+ NK cells have a higher capacity for secreting cytokines, such as IFNg.244 In contrast to perforin and granzyme B, IFNg and FasL expression in NK cells did not show significant genotype effects, which suggests that factors separate from or in addition to NK cell maturation may modulate expression of these effector molecules. Interestingly, and in contrast to NK cells, IFNg expression is increased in activated T cells derived 61 from Nrf2-null mice as compared to wild-type.142 Taken together, the data suggest that while the role of Nrf2 in modulating expression of perforin and granzyme B expression may be tied to its effects on NK cell maturation, there is no genotype effect in the expression of IFNg and FasL, suggesting that other (unidentified) factors may play a role. Although tBHQ is a potent Nrf2 activator, Nrf2-independent effects of tBHQ may be explained by alternative mechanisms. For example, tBHQ may impact reactive oxygen species, which are known to play an important role in the regulation of the redox-sensitive transcription factor, NFkB.230 In particular, antioxidants structurally similar to tBHQ have been shown to inhibit NFkB.245 Therefore, Nrf2-independent effects of tBHQ may be directly due to its antioxidant capacity. Additionally, tBHQ has been shown to cause a delay and a decrease in calcium influx in Jurkat T cells, which is relevant because intracellular calcium levels are critical in NK cell activation.146 tBHQ has also been shown to activate aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that has many immunomodulatory effects.246 Overall, there are a number of potential alternative mechanisms that could play a role in the Nrf2-independent effects of tBHQ. Together, tBHQ- mediated effects may be attributed to mechanisms independent of Nrf2. Although Nrf2 is well known for its cytoprotective effects, the role for Nrf2 in NK cell activity remains uncertain. In models of inflammatory disease, studies have demonstrated an anti-inflammatory role for Nrf2. The current study is the first to elucidate the Nrf2-dependent and -independent effects of tBHQ on activated NK cells. Notably, this study suggest that Nrf2 represents a novel mechanism for the regulation of NK cell activation. Further studies will be needed to identify the mechanisms underlying the Nrf2-independent effects of tBHQ on NK cells. 62 CHAPTER 4 The Nrf2 activator, tBHQ, inhibits early NK cell responses to influenza 63 Abstract Influenza virus is estimated to cause millions of illnesses and thousands of hospitalizations and deaths yearly in the United States, making influenza a major public health concern. It is well known that an early natural killer (NK) cell response against influenza is vital for effective viral clearance. Tert-butylhydroquinone (tBHQ) is a widely used food preservative with known immunomodulatory activity. Our lab has shown tBHQ to negatively impacts NK cell activation, effector function, and maturation ex vivo. However, little is known regarding the effects of tBHQ consumption on NK cells in vivo, specifically in response to influenza infection. In the current study, we examined whether the consumption of tBHQ impairs NK cell response two and three days after primary influenza virus infection. Female C57Bl/6J mice were fed a diet containing either 0.0014% tBHQ or control diet 10 days prior to infection. Mice were intranasally infected with influenza A/PR/8/34 (H1N1), and at day two and three post-infection, lungs were collected and processed for analysis by flow cytometry, qPCR, and ELISA. Mice exhibited similar weight loss throughout infection regardless of their respective diet. There were no statistical differences in the infiltration or maturation of NK cells in the lungs between the groups at day two of infection. At day three, tBHQ modestly decreased the percentage of NK cells in the lung, and NK cells adopted a more immature phenotype. Notably, tBHQ significantly reduced expression of FasL and production of IFNg in NK cells compared to those on a control diet at day two. NK cell cytotoxicity decreased with tBHQ at day three, as demonstrated by reduced CD107a expression. Likewise, induction of antiviral effector genes was decreased in the lungs of mice on a tBHQ diet at days two and three of infection. Taken together, the data suggests the food additive, tBHQ, negatively impacts early NK cell responses 64 to influenza infection by inhibiting FasL, CD107a, and IFNg expression. Furthermore, tBHQ impaired the induction of genes associated with NK cell effector function in influenza-infected mice. 65 Introduction Influenza virus is a contagious respiratory illness that causes a persistent threat to society as it infects approximately 8% of the US population each year.4 Although efforts in influenza vaccination coverage and effectiveness have increased, the burden of disease remains high, affecting millions in the United States alone.247 Increased susceptibility to influenza infection and risk of developing severe influenza-related diseases are determined by a wide range of viral, host, and environmental factors. The CDC defines individuals with higher risk for severe influenza A virus-related health outcomes to include people of advanced age (>65 years old), young children (<5 years old), pregnant people, and people with chronic diseases. However, there are discrepancies between susceptibility of healthy individuals infected with influenza, which have been attributed to several factors, including climate, occupation, and geography, among others.248 Therefore, the importance for identifying components that may contribute to the range in susceptibility and severity in infection is necessary, especially when vaccination efficacy is poor or unavailable. Natural killer (NK) cells are large, granular innate lymphocytes that play a critical role in host antiviral responses by rapidly producing cytokines, such as IFNg, and directly killing virally infected cells through the release of granzymes and perforin. Early on in infection, NK cells can limit viral replication while allowing time for the adaptive immune response to mount.249,250 However, defects in NK cell responses have been linked to increased pathology and worsened outcomes. Our group has shown increased mortality and decreased NK cell cytotoxicity in mice on a calorie-restricted mice infected with influenza A.73 Furthermore, in aged mice, calorie restriction further diminished the response to influenza resulting in decreased survival, 66 increased virus titers, and reduced NK cell activity compared to aged mice fed ad libitum and calorie restricted, young mice.72 In diet-induced obese mice, influenza-infected mice had increased mortality while NK cells exhibited reduced cytotoxicity and antiviral cytokine production.74 In contrast, supplementation of active hexose correlated compound increased NK cell activity and NK cell number in the lungs following infection.75 Previous studies have shown altered NK cell activity in response to influenza infection after implementation of other nutritional interventions to the diet including, cocoa and fortified yogurt.251,252 In the absence of influenza, studies have demonstrated NK cell activity and effector function to be influenced by vitamin supplementation and alcohol consumption.76,77 Together, these studies suggest that NK cells are highly influenced by environmental factors, and impairment during influenza infection may lead to decreased viral clearance and survival. Tert-butylhydroquinone (tBHQ) is a widely used food additive that is found in a variety of products to prevent the oxidation and rancidification of fats, such as cooking oils, cereals, and crackers, among others.185 Our lab has also shown T cell modulation with tBHQ treatment in numerous models, including primary mouse CD4+ T cells, Jurkat T cells, and primary human CD4+ T cells.142,143,145,147 Additionally, studies from our lab and others have demonstrated immunomodulatory effects of tBHQ in B cells, dendritic cells, and macrophages.139,235–237 Of particular interest, our lab has demonstrated tBHQ greatly impacts primary NK cell maturation, activation, and effector function in vitro. Specifically, we found tBHQ reduced expression of early activation markers, CD69 and CD25, and induction of IFNg and the cytotoxic molecules, granzyme B and perforin, by activated NK cells.137 Therefore, the following study aims to 67 identify whether dietary tBHQ influences the early immune cell response to primary influenza A infection by NK cells. Materials and Methods Materials Materials were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise stated. Animals, Diets, and Virus Influenza A/PR/8/34 (H1N1) was generously gifted by Dr. Kymberly Gowdy and East Carolina University in Greenville, North Carolina. The aliquot of virus was propagated and quantified following methods previously published.253 Using specific pathogen-free, embryonated chicken eggs (Charles River Laboratories, Wilmington, MA), the virus was inserted into the allantoic fluid and incubated for 48 hours at 37°C. Afterwards, infected eggs were incubated for another 24 hours at 4°C. Supernatants from allantoic fluid were collected, distributed into single-use aliquots, and stored at -80°C. Tissue culture infectious dose 50 (TCID50) and hemagglutinin assays were used to quantify propagated virus stock. The TCID50 assay was performed by adding a serial dilution of virus sample to confluent monolayers of MDCK cells (ATCC, Manassas, VA) in a 96-well plate. Cells were inspected daily for cytopathic effect (CPE) or cell death, and the dilution, at which point 50% of wells show CPE, was used to calculate TCID50 via the Reed-Muench method.254 For hemagglutinin assay, virus stock was serially diluted across a 96-well plate containing 0.5% chicken red blood cells, followed by a 30- minute incubation at room temperature. After, wells were analyzed for agglutination, and the highest dilution of virus that caused complete hemagglutination was used to determine virus 68 titer. The virus stock used in this experiment was determined to be 2.5 x 105 TCID50/mL and 7260 HAU/mL. All animal studies were performed in the accordance with the Guide for Care and Use of Animals as adopted by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Michigan State University. Female C57BL/6 mice (12 weeks old) were purchased from Jackson Laboratories (Bay Harbor, ME) and housed in cages containing 3- 5 animals per cage. Upon arrival, mice were given AIN-93G purified rodent diet containing 0% or 0.0014% tBHQ and water ad libitum. Food was monitored daily, and diets were replaced every three days to avoid spoilage. After 2 weeks of acclimation on their respective diets, initial body weights were recorded, followed by anesthetization via intraperitoneal injection with 2,2,2-tribromoethanol, avertin (Alfa Aesar, Ward Hill, MA). Subsequently, mice were intranasally instilled with either 30 µL of sterile saline (CTRL) or influenza A/PR/8/34 (H1N1) (VIR) at a titer of 2.5 x 103 TCID50/mL (72.6 HAU/mL), which resulted in a total amount of 75 TCID50 or 2.18 HAU per mouse. After recovering from anesthesia, mice were returned to their respective cages. Daily health checks were performed and changes in food and body weights were recorded. Two separate experiments were completed to assess NK cells responses at early timepoints of infection. A schematic of the timeline for the following experiments is shown in Figure 15. 69 Figure 15: Schematic of Experimental Design. Mice arrived at Michigan State University from Jackson Laboratories and were placed on their respective diets for an acclimation period of 14 days. Mice were infected with influenza A/PR/8/34 (H1N1), and after 2- or 3-days post infection, tissues were collected for analysis. Tissue Collection and Cell Isolation At day two or three post-infection, mice were anesthetized with avertin and euthanized via exsanguination by severing the vena cava. Lungs were excised and placed in DMEM containing 1 mg/mL collagenase D (Roche, Indianapolis, IN) and dissociated twice using the gentleMacs dissociator (Miltenyi Biotec, Auburn, CA) with a 30 min incubation at 37°C between dissociations. Subsequently, 1 mL of dissociated lung was removed, centrifuged, and placed in TRIzol reagent for later RNA isolation. Remaining sample was washed with FACS buffer (1% FBS in dPBS) and filtered through a 40 µm strainer. Red blood cells from lung were lysed using ammonium chloride-potassium (ACK) lysis buffer (Lonza, Walkersville, MD). Single-cell suspensions were subsequently used for flow cytometry analysis. Immunophenotyping Single-cell suspensions from the lungs were added to a 96-well V-bottom plate and washed with dPBS prior to assessing cell viability by Zombie Aqua Fixable Viability Kit (Biolegend, San Diego, CA) following manufacturer’s protocol. Afterward, cells were washed with FACS buffer and incubated for 10 minutes with anti-CD32/16 antibody (BD Pharmingen, San Diego, CA), to block non-specific binding. Cells were then labeled with fluorochrome 70 conjugated antibodies for 30 minutes in the dark at 4°C. The surface antibodies used are listed in Table 2. Cells were washed, fixed using BD Cytofix Fixation Buffer (BD Biosciences, Franklin Lakes, NJ) per manufacturer’s protocol, and stored at 4°C in the dark until later FACs analysis by Attune Nxt (Thermo Scientific, Waltham, MA). Ex Vivo Stimulation and Intracellular Labeling Following lung cell isolation, single-cell suspensions were cultured in complete media, containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 25 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid solution (HEPES), 10% fetal bovine serum (FBS), non-essential amino acid (1X final concentration from 100X), 100 U/mL penicillin, 100 U/mL Streptomycin, and 100 U/mL 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA). Cells were stimulated using 10 ng/mL of recombinant mouse IL-12 (P70) (Biolegend, San Diego, CA) for 5 hours in the presence of monensin (Biolegend, San Diego, CA). After, samples were transferred to a 96-well V-bottom plate and labeled with Zombie Aqua Fixable Viability Kit. Cells were then incubated with anti-CD32/CD16 and labeled with fluorochrome-conjugated antibodies for surface markers, NK1.1 and CD3. Following surface labeling, cells were permeabilized using FoxP3/Transcription Buffer Staining Kit (Invitrogen, Waltham, MA) per manufacturer’s protocol and labeled with intracellular antibodies listed in Table 2. Cells were fixed with 1% formaldehyde fixative and stored in the dark at 4°C until FACS analysis on the Attune NxT. 71 Table 2: List of Antibodies Antibody Fluorochrome Clone Supplier Live/Dead Zombie Aqua Dye Biolegend CD3 Alexa Fluor 488 17A2 Biolegend CD3 Alexa Fluor 700 eBio500A2 eBioscience NK1.1 Brilliant Violet 711 PK136 Biolegend CD27 PerCP-eFluor 710 LG.7F9 eBioscience CD11b Alexa Fluor 647 M1/70 Biolegend CD25 Pacific Blue PC61 Invitrogen CD69 Alexa Fluor 700 H1.2F3 Biolegend CD107a PE-Cy7 1D4B BD Biosciences CD178 (FasL) PE MFL3 Biolegend NKG2D APC CX5 Biolegend Granzyme B FITC NGZB Invitrogen T-bet PE eBio4B10 Biolegend IFN-𝛾 APC XMG1.2 Biolegend Perforin PE S16009B Biolegend RNA Isolation and Quantitative PCR RNA was isolated from homogenized lung tissues using TRIzol reagent (Life Technologies, Grand Island, NY). RNA was quantified using the Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and cDNA was synthesized by M- MLV Reverse Transcriptase (Promega, Madison, WI) following the manufacturer’s protocol. 72 Real-time was performed with SYBR green PCR master mix using the QuantStudio 7 Flex Real- Time PCR System (Applied Biosystems, Foster City, CA). Expression values were normalized to expression of ribosomal protein L13A (RPL13A) and quantified by the ΔΔCt method. Gene- specific primers are listed in Table 3. Table 3: Primer Sequences Gene Forward Primer Sequence Reverse Primer Sequence Influenza M1 CAAAGCGTCTACGCTGCAGTCC AAGACCAATCCTGTCACCTCTGA IFN𝛾 TGAGCTCATTGAATGCTTGG ACAGCAAGGCGAAAAGGAT Granzyme B AGTGGGGCTTGACTTCATGT AGTGGGGCTTGACTTCATGT Perforin TGGAGGTTTTTGTACCAGGC TAGCCAATTTTGCAGCTGAG T-bet CAAGGGGGCTTCCAACAATG GGCTCTCCATCATTCACCTCC IL-18 TCCTTGAAGTTGACGCAAGA TCCAGCATCAGGACAAAGAA IL-1β GGTCAAAGGTTTGGAAGCAG TGTGAAATGCCACCTTTTGA IFNα TAGGAGGGTTGCATTCCAAG CCAGCAGATCAAGAAGGCTC RPL13a GTTGATGCCTTCACAGCGTA AGATGGCGGAGGTGCAG Statistical Analysis Treatment groups are presented as mean ± SEM for each individual experiment. In virus-infected groups, differences in treatment (CTRL vs tBHQ) were assessed using two-tailed T-tests with GraphPad PRISM software version 8.4.2 (GraphPad Software, La Jolla, CA). Data sets with unequal variances were analyzed using Welch’s t-tests. Saline-instilled mice were not included in the statistical analysis, as their purpose for the current study was to serve as a 73 reference and to confirm that absence of immune response in uninfected mice. Animals which had no detectable viral RNA were excluded from statistical analysis. Results tBHQ Modestly Decreased Viral RNA but did not Affect Weight Loss. Indication of infection in mice is often characterized by a decrease in appetite followed by weight loss. Upon arrival, mice were given diets containing either no tBHQ or 0.0014% tBHQ, and daily consumption was monitored for the duration of the study. Since mice were not singly housed, the average amount of diet consumed per mouse was estimated based upon consumption from the whole cage. Dietary tBHQ did not impact weight loss in mice infected with influenza with an average weight loss of 4.56% and 5.24% at day two (Figure 16A) and 9.53% and 9.07% at day three (Figure 16B) for CTRL and tBHQ groups, respectively. Interestingly, tBHQ had a significant decrease in viral RNA at day two of infection (Figure 17A) and trended downward at day 3 (Figure 17B), however this was not significant. Although viral RNA was decreased with dietary tBHQ, similar food consumption patterns and weight loss were observed between virus-infected tBHQ and CTRL groups throughout the infection. 74 Figure 16: Dietary tBHQ did not affect weight change during early infection. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to influenza A/PR/8/34 infection. Weights were recorded daily up to (A) 2- or (B) 3-days post infection, and percent weight change was calculated Equation 1: Calculation of Daily tBHQ Intake per Mouse !""# %&'%( )%* +&,% (,) 3.356 1, '789 5 1":;% 5333 , 1, '789 # "0 12+% )%* +&,% 𝑥 5 , 0""# 𝑥 <=%*&,% >%2,?' "0 12+% 2( +&,% (,) 𝑥 5 @, = @, A"#B>%2,?' Table 4: tBHQ Consumption during 2-day Influenza Infection (mg/kg) Primary Exposure Day 0 Day 1 Day 2 with H1N1 tBHQ Saline 1.47727273 1.35333333 1.52224053 tBHQ Virus 1.08229426 1.17223814 0.83815789 Table 5: tBHQ Consumption during 3-day Influenza Infection (mg/kg) Primary Exposure Day 0 Day 1 Day 2 Day 3 with H1N1 tBHQ Saline 1.95081967 1.66993464 1.53548387 1.92283951 tBHQ Virus 1.54901326 1.50762245 0.85088011 0.05976521 75 Figure 17: Dietary tBHQ decreased viral titer in lungs of infected mice. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to intranasal instillation with influenza A/PR/8/34 infection. After (A) 2- to (B) 3-days post infection, lungs were collected and processed for viral RNA quantification by real-time PCR. Viral quantification was performed using viral matrix protein, M1, and was normalized using housekeeper gene, RPL13a. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 Dietary tBHQ had Minimal Influence on NK cell Population in the Lungs of Infected Mice. At days 2 and 3 post-infection, lungs were collected and processed for flow cytometry analysis. Single-cell suspensions were labeled with fluorochrome-conjugated antibodies against CD3 and NK1.1. NK cells were identified as viable CD3-NK1.1+ cells. In agreement with previous reports, the NK cell population ranged from 10-20% of lung lymphocytes (Figure 18).255 Although the percent of NK cells in the lung at days two and three of infection displayed a modest decrease with tBHQ, the difference did not reach significance (Figure 18B). 76 Figure 18: tBHQ had no effect on lung NK cell population at days 2 and 3 post-infection. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to intranasal instillation with either saline or influenza A/PR/8/34 infection. After (A) 2- to (B) 3- days post infection, lungs were collected and processed for flow cytometry analysis. Single-cell suspensions were labeled with antibodies against CD3 and NK1.1 and quantified by flow cytometry. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 tBHQ Decreased the Percentage of Terminally Mature NK cells at Day 3 of Influenza Infection. Maturation of NK cells in the periphery are divided into 4 stages defined by the expression of CD27 and CD11b, starting with immature NK cells, CD27-CD11b-. As NK cells mature, they express CD27 and gain effector function capabilities. CD11b expression is later acquired signifying double positive, CD27+CD11b+, NK cells, which are highly cytotoxic and produce large amounts of cytokines. Terminally mature NK cells are identified by the loss of CD27 expression and are comparable in effector responses to CD27+CD11b+ NK cells.21 In the current study, the majority of NK cells in the lung were terminally mature, as indicated by CD27-CD11b+ phenotype (Figure 19D and 19H). There were no differences in maturation at day 2 post-infection. However, at day 3 of infection, tBHQ modestly, but significantly, decreased terminally mature NK cells (Figure 19H) while increasing CD27+CD11b- NK cells (Figure 19F). Potential explanations for altered maturation at day 3 but not day 2 of infection 77 include the increased proliferative capacity of CD27+CD11b- NK cells or potential differences in phenotype of migrating NK cells.21 Figure 19: tBHQ caused a minor decrease in terminally mature NK cells at day 3 of infection. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to intranasal instillation with either saline or influenza A/PR/8/34 infection. After (A-D) 2- to (E-H) 3-days post infection, lungs were collected and processed for flow cytometry analysis. Single-cell suspensions were labeled with antibodies against CD3, NK1.1, CD27, and CD11b and quantified by flow cytometry. NK cells were identified as NK1.1+CD3- prior to analysis of maturation using CD27 and CD11b. Maturation occurs in the following order: (A, E) CD27- CD11b-, (B, F) CD27+CD11b-, (C, G) CD27+CD11b+, (D, H) CD27-CD11b+. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 Dietary tBHQ Impaired NK cell Effector Functions and Diminished Expression of Genes Associated with NK Cell Effector Function. The ability for NK cells to elicit a sufficient response is crucial in limiting viral replication early on in infection. To assess NK cell effector functions, we analyzed the surface expression of CD107a and FasL on NK cells, as well as the intracellular expression of IFNg, granzyme B, and perforin by flow cytometry. At day 2 of infection, FasL expression on NK cells was decreased with tBHQ exposure (Figure 20B). However, this decrease was insignificant by day 3, by which 78 time FasL expression was decreased as compared to day 2 (Figure 20D). NK cell cytotoxicity was assessed by CD107a expression, a marker for active degranulation, and production of granzyme and perforin in NK cells. CD107a was greater on day 3 as compared to day 2 following influenza infection. Dietary tBHQ significantly reduced CD107a at day three of infection (Figure 20C). Interestingly, intracellular expression of granzyme B were not affected by tBHQ at either timepoints (Figure 21B and 21E), and perforin was slightly decreased at day 2 of influenza infection (Figure 21C). Notably, NK cell production of cytokine, IFNg, was significantly diminished at day 2 of infection with dietary tBHQ but had recovered by day 3 (Figure 21A and 21D). Coinciding with our flow cytometry results, several genes associated with NK cell effector functions were significantly reduced at day 2 and 3 of influenza infection (Figure 22 and 23). Specifically, at day 2 of infection tBHQ markedly diminished IFNg mRNA expression, which was consistent with the decrease in IFNg protein expression by NK cells at this time point. In contrast, tBHQ also decreased granzyme B mRNA expression, whereas granzyme B protein expression by NK cells was not affected by tBHQ at this time point. It is important to note, however, that whole lung tissue was used to quantify gene expression. Therefore, differences in gene expression cannot be attributed solely to NK cells. Collectively, the data suggest tBHQ decreases the expression of a number of important effector molecules by NK cells at early time points following influenza infection. 79 Figure 20: NK cell expression of FasL decreased at day 2 of infection while CD107a expression decreased at day 3 of infection with dietary tBHQ. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to intranasal instillation with either saline or influenza A/PR/8/34 infection. After (A-B) 2- to (C-D) 3-days post infection, lungs were collected and processed for flow cytometry analysis. Single-cell suspensions were labeled with antibodies against CD3, NK1.1, CD107a, and FasL and quantified by flow cytometry. NK cells were identified as NK1.1+CD3- prior to analysis percent NK cells expressing (A, C) CD107a and (B, D) FasL. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 80 Figure 21: tBHQ decreased NK cell expression of IFN𝛾 and perforin at day 2 post-infection. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to intranasal instillation with either saline or influenza A/PR/8/34 infection. After (A-C) 2- to (D-F) 3-days post-infection, lungs were collected and processed for flow cytometry analysis. Single-cell suspensions were cultured with 10 ng/mL IL-12 in the presence of monensin for 5 hours. After, cells were labeled with antibodies against CD3, NK1.1, IFN𝛾, granzyme B, and perforin and quantified by flow cytometry. NK cells were identified as NK1.1+CD3- prior to analysis of percent NK cells expressing (A, D) IFN𝛾, (B, E) granzyme B, and (C, F) perforin. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 Figure 22: Low concentration of tBHQ in the diet decreased mRNA expression of granzyme B and IFN𝛾 at day 2 post-infection. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to intranasal instillation with either saline or influenza A/PR/8/34 infection. After 2-days post-infection, lungs were collected and processed for RNA 81 Figure 22 (cont’d): quantification. Real-time PCR was done to quantify (A) granzyme B and (B) IFN𝛾 mRNA. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 Figure 23: tBHQ in the diet decreased mRNA expression of genes associated with NK cell effector function at day 3 post-infection. Mice were placed on a diet either containing 0.0014% tBHQ or no tBHQ (CTRL) for two weeks prior to intranasal instillation with either saline or influenza A/PR/8/34 infection. After 3-days post-infection, lungs were collected and processed for RNA quantification. Real-time PCR was done to quantify (A) IFN𝛾, (B) perforin, (C) IL-18, (D) IL-1β, (E) T-bet, and (F) IFNα mRNA. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 Discussion Collectively, these results suggest consumption of tBHQ greatly influences the primary NK cell response to influenza at days two and three of infection. The percentage of NK cells trended toward a modest reduction in mice fed a diet containing tBHQ (Figure 18). In addition, tBHQ caused NK cells to adopt a more immature phenotype, which is known to be less functional at day three of infection (Figure 19E-H). At day two of infection, expression of FasL, 82 which is involved in initiating apoptosis, was significantly reduced in NK cells of mice on a tBHQ diet (Figure 20B). Additionally, tBHQ markedly reduced IFN𝛾 production in NK cells at day two of infection (Figure 21A). IFN𝛾 and granzyme B gene expression was also reduced at day two of infection with tBHQ (Figure 22). At day three, NK cell cytotoxicity decreased with tBHQ, as demonstrated by reduced CD107a expression (Figure 20C). Notably, at day three, tBHQ suppressed the expression of several genes important in antiviral immunity, including IFN𝛼, IFN𝛾, T-bet, IL-1b, IL-18, and perforin (Figure 23). In murine NK cells, maturation in the periphery occurs in four stages depending on expression of CD27 and CD11b. Maturation occurs in the following order: CD27-CD11b-, CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+. It is well known that NK cells with a phenotype of CD27+CD11b+ and CD27-CD11b+ are more functional, as they can rapidly release cytotoxic molecules and cytokines. In contrast, more immature NK cells, including CD27- CD11b- and CD27+CD11b-, have greater proliferative capacity compared to mature NK cells.21,256,257 In the current study, dietary tBHQ modestly increased CD27+CD11b- NK cells and decreased CD27-CD11b+ NK cells at day 3 of infection (Figure 19F and 19H), while no difference was found at day 2 (Figure 19A-B), which could be due to differential levels of proliferation and/or recruitment. Previously, a study found lung NK cell proliferation to peak by day 4 of influenza infection with 2% of NK cells in the lung proliferating, which is comparably low to other cell types at similar timepoints. However, the accumulation in lung NK cells was due to NK cell recruitment during influenza infection with CD27-CD11b+ subset having the greatest increase in the lung compared to other subsets.53 Therefore, it is possible that tBHQ impairs NK cell recruitment to the lung at day 3 of influenza infection as demonstrated by a decrease in the 83 percent of terminally mature NK cells. Further studies elucidating the effects of tBHQ on NK cells at later timepoints are essential to understand the impact on NK cell recruitment. Notably, the current study examined the effects of low dose tBHQ on NK cell responses to a relatively high concentration of virus at two early time points during infection. Others have suggested NK cells to exacerbate pathology of influenza virus when infected with lethal amount of virus. They found mice depleted of NK cells in vivo and infected with a high viral dose of influenza A/PR/8/34 virus to have increased survival rates.69 Furthermore, adoptively transferred NK cells from mice infected with a high dose of virus had higher morbidity and mortality. Whereas NK1.1+ cell-depleted mice were protected from lethal influenza virus infection, as demonstrated by minimal morbidity.70 While the results in the current study suggest that in particular cytokine response was strongly inhibited by tBHQ under these conditions, future studies are needed to investigate the effects of higher concentrations of tBHQ, which might serve as a model for high consumers of tBHQ. Additionally, the effects of tBHQ in NK cells following infection at a lower dose of influenza virus is necessary to model seasonal influenza virus infections rather than pandemic-like responses. Of importance to human relevance, the amount of tBHQ consumed per animal in the current study equated to approximately 1-2 mg/kg/day (Table 4 and 5), which exceeds the recommended acceptable daily intake of 0.7 mg/kg/day for tBHQ. However, tBHQ intake was well below the set NOAEL of 72 mg/kg/day, which was used in establishing the current ADI. Additionally, model diets estimating tBHQ consumption have shown high consumers of tBHQ to substantially exceed the ADI with 7.7 mg/kg/day of tBHQ.186 By day 3 of infection, tBHQ consumption was well below the ADI, suggesting the effects of dietary tBHQ persist with levels 84 below the ADI. Future work is necessary to understand whether tBHQ has long-term effects on NK cell responses, especially when tBHQ is removed from the diet prior to influenza infection. Overall, this study demonstrates that low concentrations of the commonly used food additive tBHQ inhibits IFNg, and to a lesser extent, FasL and CD107a, by NK cells. These data suggest that consumption of tBHQ may impact early innate immunity to influenza. 85 CHAPTER 5 Summary, Significance, and Future Directions 86 Summary of Findings Previous studies from our lab have examined the role of tBHQ and Nrf2 activation on T cell responses. More specifically, these studies demonstrate that tBHQ inhibits CD4+ T cell activity via Nrf2-dependent and -independent mechanisms. However, investigation of the impact of the Nrf2 activator, tBHQ, remains limited in NK cells. The central hypothesis for the current studies is that the food preservative, tBHQ, impairs NK cell function through a mechanism involving the activation of the transcription factor, Nrf2. Initial studies assessed the impact of tBHQ on primary murine NK cells in vitro. Preliminary data showed inhibition of activation and effector function in NK cells following treatment of primary murine splenocytes with tBHQ. Importantly, PMA/ionomycin activation of NK cells in the presence of tBHQ significantly decreased production of IFNg, granzyme B, and perforin in a concentration dependent manner. Additionally, induction of activation markers, CD69 and CD25, was significantly reduced in response to tBHQ. Maturation was also altered with tBHQ, which was demonstrated by an increase in terminally mature NK cells with 5 µM tBHQ. As we hypothesized tBHQ would impair NK cell activation with tBHQ treatment via Nrf2- dependent mechanisms, we used a Nrf2-null mouse model to assess the role of Nrf2 in the tBHQ-mediated effects on NK cell activation. We utilized two NK cell activators in this study, including PMA/ionomycin and IL-12/IL-18. We used PMA/ionomycin to be consistent with our previous published study and IL-12/IL-18 because, in contrast to PMA/ionomycin, it is a relatively selective NK cell activator. Notably, the presence of Nrf2 promoted NK cell maturation towards the terminally mature phenotype, CD27-CD11b+, whereas the absence of Nrf2 resulted in a more immature NK cell phenotype. Furthermore, NK cell expression of the 87 activating markers, CD25 and CD69, was decreased via a partially Nrf2-dependent mechanism when the NK cells were activated with PMA/ionomycin, but not IL-12/IL-18. FasL expression in NK cells activated with IL-12/IL-18 was inhibited in a Nrf2-dependent manner. Similarly, IFNg expression was also inhibited by tBHQ in NK cells through a partially Nrf2-dependent mechanism at 1 µM, but not 5 µM, tBHQ. Whereas IFNg protein accumulation in the supernatant was diminished by tBHQ in a partially Nrf2-dependent manner in the 5 µM tBHQ group. This study demonstrates a differential role of Nrf2 activation by tBHQ on NK cell responses with two different NK cell activators. Based on these findings, we wanted to assess whether these tBHQ-mediated effects on NK cells in vitro could translate into an in vivo model. We hypothesized that tBHQ would impair NK cell responses against influenza A infection. In our influenza model, mice were fed diets containing either no tBHQ or tBHQ, at a concentration relevant to human exposure, prior to influenza infection. NK cell responses were analyzed at days 2 and 3 after infection. At day two of influenza infection, dietary tBHQ decreased expression of IFNg and FasL in NK cells. Additionally, induction of genes important in NK cell effector function, such as IFNg and granzyme B, were diminished in mice fed tBHQ at day two. At day three of infection, tBHQ suppressed the expression of CD107a expression in NK cells. Furthermore, tBHQ decreased expression of a number of genes important in antiviral immunity, including IFNa, IFNg, T-bet, and perforin. Taken together, these data suggest that exposure to a low dose of tBHQ in the diet reduces NK cell response to influenza A virus. 88 Significance of Findings In the United States, influenza vaccinations have been estimated to prevent millions of illnesses and medical visits and thousands of hospitalizations and deaths each season.8 Despite the advancements in vaccination effectiveness and coverage, the burden of disease remains high, affecting millions in the United States alone.9 Individual factors, including demographic, genetic, and environmental, are known to influence immune responses, which provides an explanation for the wide variations in susceptibility and severity of infection.10 Impaired immune responses often result in increased morbidity and mortality from influenza, and therefore, the identification of factors and mechanisms involved in the impairment of host immunity warrants further investigation. tBHQ is a commonly used preservative found in foods to prevent rancidification and oxidation of fats.185 tBHQ was first approved in the 1970’s with an ADI of 0.7 mg/kg/day, which was based on toxicity findings in dogs. However, model diets estimating tBHQ consumption have found high consumers of fats, oils, and frozen fish products to exceed recommended ADI for tBHQ with levels up to 7.7 mg/kg/day.186 Additionally, studies in healthy volunteers have shown serum levels of tBHQ reaching the high micromolar range following ingestion of 150 mg tBHQ.191 To our knowledge, the following studies are the first to demonstrate the negative impact of dietary tBHQ on NK cell responses to influenza A virus infection. These studies agree with other experiments suggesting that NK cells are highly influenced by a variety of environmental factors, and impairment of NK cells during influenza infection may lead to decreased viral clearance and survival. For instance, in calorie-restricted mice, influenza infection resulted in higher mortality rates and decreased NK cell cytotoxicity. In addition, aged 89 mice put on a calorie-restricted diet had more severe mortality and morbidity rates compared to young, calorie-restricted mice and aged mice fed ad libitum.71–73,258,259 Diet-induced obese mice also display hampered NK cell responses against influenza infection, which resulted in decreased NK cell cytotoxicity and anti-viral cytokines.74 Accordingly, it is clear that environmental factors influence NK cell antiviral response and it is important to identify such factors that may impair NK cell function and compromise host defense to influenza. The current studies suggest tBHQ impairs NK cell responses at early timepoints in infection. In addition to the effects of tBHQ on NK cells in vivo, we also gained insight on the role of Nrf2 in NK cells in vitro. Our lab has previously shown T cell modulation through the activation of the transcription factor, Nrf2, by tBHQ in numerous models, including primary mouse CD4+ T cells, Jurkat T cells, and primary human CD4+ T cells.142,143,145–147 Studies from our lab and others have demonstrated the immunomodulatory effects of tBHQ through Nrf2 activation in other immune cell types.134,139,236,237 However, the current findings are the first to elucidate the role of Nrf2 in NK cell activation in primary murine splenocytes. Future Directions The current studies identify the effects of tBHQ on NK cell activation and a potential role for Nrf2. Additionally, these findings suggest dietary tBHQ dampens early NK cell responses to influenza infection. However, further studies are necessary to understand the role of Nrf2 activation in NK cells and how these effects occur. The following in vitro studies help identify the molecular effects of tBHQ. However, the studies do not directly show Nrf2 activation in NK cells. Previous studies in T cells show increased Nrf2 DNA binding and upregulation of Nrf2 target genes.142 The current studies could be bolstered by quantifying Nrf2 target gene 90 induction following tBHQ treatment in isolated NK cells derived from wild-type and Nrf2-null mice. Additionally, NK cells are considered a rare immune population. Therefore, the in vitro experiments assessed NK cell population within the lymphocyte populations of the spleen, which is rich with immune cells. Future studies on the effects of tBHQ on isolated NK cells without outside stimuli from surrounding immune cells are needed. It is reported that three average sized spleens from C57Bl/6 mice are estimated to return an average of approximately 2.5 x 106 purified NK cells.260 Thus, although large studies identifying the effects of tBHQ at various concentrations would require a large number of mice, a limited number of experiments could be performed with one or two concentrations of tBHQ to determine effects in isolated NK cells. For the influenza experiments, tBHQ was fed to mice for the duration of the studies. It is unclear whether tBHQ has lasting effects on NK cell responses to influenza A virus infection. For example, previous study on calorie-restricted mice showed short-term refeeding to restore body weight and attenuate the decline in NK cell function following primary influenza infection.261 Although diet consumption decreases during infection, it is unclear from these studies if tBHQ exposure is sufficient to decrease response to infection. Future studies should address whether tBHQ has long term effects or if stopping tBHQ consumption rescues these effects. It is well established that NK cells are recruited to the lung following influenza infection.262 However, the current studies examine NK cell responses at day two and three of infection where induction of cytokines and effector molecules is high, but NK cell lung 91 infiltration is minimal. Therefore, experiments examining the effects of tBHQ at later timepoints in infection are necessary to determine if tBHQ impacts NK cell recruitment to the lung. Lastly, experiments assessing human relevance of the tBHQ-mediated effects in NK cells is needed. As previously discussed, tBHQ exposure data is limited in humans. Therefore, experts have estimated tBHQ consumption using model diets. This limitation makes identifying an epidemiological link between influenza infections and tBHQ consumption unlikely. Furthermore, tBHQ is not found on food labels. Where tBHQ is listed on labels, the amount of tBHQ is often not available. Humanized immune system mice could be an option in assessing the effects of tBHQ on human NK cell responses against influenza. Additionally, using an in vitro model to assess NK cell responses from PBMCS following tBHQ treatment can be performed. However, depending on the individual diet and baseline consumption of tBHQ, tBHQ-treatment may have an additive effect. Together, the studies presented provide an outline to assess Nrf2 activation in NK cells with tBHQ-treatment and to further expand the effects of tBHQ on NK cell immune responses in the context of influenza A virus infection. 92 WORKS CITED 1. Samji T. Influenza A: Understanding the Viral Life Cycle. Yale J Biol Med. 2009;82(4):153. Accessed January 1, 2023. /pmc/articles/PMC2794490/ 2. Shim JM, Kim J, Tenson T, Min JY, Kainov DE. Influenza Virus Infection, Interferon Response, Viral Counter-Response, and Apoptosis. 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Toxicol Appl Pharmacol. 2016;304:79-89. doi:10.1016/J.TAAP.2016.05.017 295. Keil DE, Buck B, Goossens D, et al. Health effects from exposure to atmospheric mineral dust near Las Vegas, NV, USA. Toxicol Rep. 2016;3:785-795. doi:10.1016/J.TOXREP.2016.09.009 296. Liao CMM, Chio CPP, Cheng YHH, Hsieh NHH, Chen WYY, Chen SCC. Quantitative links between arsenic exposure and influenza A (H1N1) infection-associated lung function exacerbations risk. Risk Analysis. 2011;31(8):1281-1294. doi:10.1111/j.1539- 6924.2010.01575.x 297. Alex AA, Ganesan S, Palani HK, et al. Arsenic trioxide enhances the NK cell cytotoxicity against acute promyelocytic leukemia while simultaneously inhibiting its bio-genesis. Front Immunol. 2018;9(JUN). doi:10.3389/fimmu.2018.01357 119 APPENDIX Arsenic trioxide impairs primary human NK cell responses against influenza A virus 120 Abstract Arsenic is a naturally occurring contaminant that is commonly found in water supplies. Chronic exposure to arsenic has been associated with multisystem disease, including respiratory diseases such as viral infections. Arsenic is known to regulate immune cell activity and has been shown to alter NK cell frequency and function. However, our knowledge on effects of arsenic on NK cell response to influenza remains limited. The following study aims to identify the effects of arsenic trioxide on NK cell activation and function against influenza A virus in primary human immune cells. Primary human peripheral blood mononuclear cells were treated with environmentally relevant concentrations of arsenic trioxide prior to an influenza A virus challenge. Flow cytometric analysis of NK cells showed arsenic trioxide significantly decreased NK cell viability. Additionally, NK cell activation markers, CD69 and NKp46, were reduced with increasing concentrations of arsenic. Consistently, the production of NK cell effector mediators, including IFNg, granzyme B and perforin, were significantly impaired with treatment of arsenic trioxide. Overall, these data suggest that environmentally relevant concentrations of arsenic trioxide impaired NK cell responses to influenza A virus, which could negatively impact NK cell-mediated host defense. 121 Introduction Arsenic is a naturally occurring chemical that is commonly found in drinking water, and long-term exposure has been link to numerous diseases including cancer.263 Although the maximum contaminant levels for arsenic in public-water supplies has been lowered from 50 µg/L to 10 µg/L, domestic wells in the U.S. have been found to contain arsenic at levels greater than 10 µg/L.264 It is estimated that 14% of the total population in the U.S. relies on domestic wells for water use.265 Chronic exposure to arsenic can lead to multisystem disease, including skin changes, diarrhea, cardiovascular disease, neuropathy, prostate cancer, bladder cancer, lung cancer, kidney cancer, skin cancer, and diabetes.266 In addition, arsenic has been associated with a wide spectrum of respiratory diseases.267 A meta-analysis found arsenic to impair pulmonary function 268, which agrees with several epidemiological studies that have shown a negative relation between arsenic exposure and respiratory infection.269–271 In addition to the epidemiological studies, arsenic has been reported to impact several immune cells in response to influenza infection. A study found sodium arsenite to enhance influenza infection in epithelial cells and decrease effectiveness of anti-influenza drug, oseltamivir.272 In mice chronically exposed to arsenic, influenza infection resulted in higher influenza virus titers and decreased the number of dendritic cells early in influenza infection.273 Additionally, mice exposed to sodium arsenite in utero and postnatal resulted in enhanced inflammatory responses which were attributed to the dysregulation of innate immune functions, including natural killer (NK) cells.274,275 More recently, our group showed arsenic trioxide to impair human T cell responses against influenza A virus ex vivo.276 122 NK cells are innate lymphocytes that play a vital role in limiting viral spread through the rapid release of cytokines and cytotoxic granules.249 Currently, several studies have assessed the effects of arsenic in human and mouse NK cells. Numerous cancer disease models have shown arsenic trioxide to enhance NK cell cytolytic activity in both human cell lines and mouse models.277,278 Conversely, pretreatment of NK-92 cells and primary murine NK cells with sodium arsenite significantly reduced NK cell cytotoxicity in response to target cells.279 In mice, prenatal exposure to sodium arsenite was shown to increase NK cell activity and enhance inflammation in response to IAV infection.275 Additionally, women chronically exposed to arsenic via ground water had significantly less circulating CD56+ NK cells compared to women with acute exposure.280 Despite these findings, the effects of arsenic on NK cells in response to influenza in humans remain unclear. Therefore, the objective of this study is to determine how arsenic, in the form of arsenic trioxide impacts human NK cell responses against IAV infection ex vivo. Materials and Methods Materials All reagents, including arsenic (III) oxide (arsenic trioxide, ATO, catalog number A1010) were purchased from Sigma-Alrdich (Saint Louis, MO) except where otherwise stated. Complete PBMC medium used in this study consisted of RPMI 1640 (Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Biowest LLC, Kansas City, MO), 25 mM HEPES, 1 mM sodium pyruvate, 1x nonessential amino acids, 100 U/mL penicillin (Life Technologies, Carlsbad, CA), and 100 U/mL streptomycin (Life Technologies, Carlsbad, CA). Peripheral Blood Mononuclear Cell (PBMC) Isolation and Treatment 123 Source leukocytes were purchased from the Gulf Coast Regional Blood Center (Houston, TX). Isolation of PBMCs was done by Lymphoprep (Stem Cell Technologies, Vancouver, BC) density gradient centrifugation following the manufacturer’s protocol. Cells were resuspended in complete medium to 1x106 cells/mL and plated in triplicates at 0.2 mL/well in a 96-well U- bottom plate. Cells were either left untreated (background, BKG) or treated with vehicle (VEH, 0.004% PBS), 0.5 µM, 1 µM, or 2 µM ATO. Concentrations of arsenic were based on high environmental levels of arsenic in drinking water (10 ppb to 1000 ppb) where pathogenic effects have been observed with concentrations greater than 5 µM [375-750 ppb AS(III)].281,282 Additionally, blood arsenic in humans exposed via drinking water containing levels >50 ppb resulted in blood levels of 2-23 ppb.283 After 30 min, VEH- and ATO-exposed cells were infected with 0.5 hemagglutination units (HAU) of influenza A/PR/8/34 (H1N1) while the BKG cells had no virus added. The amount of virus added to cells was based on a previous study.284 Cells were maintained at 37°C, 5% CO2 for 96 h. Monensin (1x) (Biolegend, San Diego, CA) was added to all cells for the remaining 6 h of stimulation for intracellular cytokine accumulation. Flow Cytometry The following anti-human mAbs used in this study are listed in Table 6. After 96 h of stimulation, supernatants were collected for later cytokine analysis and cells were processed for FACs analysis. To assess viability, cells were initially washed in PBS and stained with Zombie Aqua Fixable Viability Dye (Biolegend, San Diego, CA). After, cells were washed in FACS buffer (1% FBS in PBS) and labeled for surface markers for 30 min at 4°C. For intracellular cytokine detection, cells previously stained for surface markers were permeabilized using FoxP3/Transcription Buffer Staining Kit (Invitrogen, Waltham, MA) following the manufacturer’s 124 protocol, then labeled with antibodies against interferon gamma (IFNg), granzyme B (GZMB), and perforin for 30 min at room temperature. Cells were then washed and immediately analyzed on an Attune NxT Flow Cytometer (ThermoFisher, Waltham, MA). Compensation for this study was done with Ultra-Comp eBeads following manufacturer’s protocol (Invitrogen, Waltham, MA). Table 6: List of Antibodies Antibody Fluorochrome Clone Supplier Live/Dead Zombie Aqua Dye Biolegend CD3 PerCP/Cyanine5.5 OKT3 Biolegend CD56 (NCAM) FITC HCD56 Biolegend CD335 (NKp46) Brilliant Violet 711 9E2 Biolegend CD69 Brilliant Violet 605 FN50 Biolegend CD25 APC BC96 Invitrogen CD107a (LAMP-1) APC/Cyanine7 H4A3 Biolegend CD16 PE/Cy5 3G8 Biolegend Granzyme B Pacific Blue QA16A02 Biolegend IFN-𝛾 PE 4S.B3 Biolegend Perforin PE/Cyanine7 B-D48 Biolegend IFNg ELISA Supernatants were collected from samples 96 hours after treatment. Secreted IFNg was measured using the human IFNg ELISA MAX Standard Kit (Biolegend, San Diego, CA) following 125 manufacturer’s protocol. Tecan Infinite M1000 Pro Microplate Reader (Tecan, San Jose, CA) was used to quantify absorbance at 450 nm. Statistical Analysis Individual data points were converted to a percentage of the vehicle mean for each independent experiment. The mean ± standard error (SE) was determined for each treatment group and analyzed by one-way ANOVA followed by a Dunnett’s post hoc test to compare treatments to the VEH group. Analyses were done using GraphPad PRISM software version 8.4.2 (GraphPad Software, La Jolla, CA). Results Arsenic Trioxide Reduces Viability of PBMCs. The percentage of viable PBMCs was determined since arsenic has been shown to induce apoptosis in PBMCs.285 We assessed the viability of cells by flow cytometry using Zombie Aqua fixable viability dye. The percentage of viable cells was significantly attenuated at concentrations greater than 1 µM ATO (Figure 24A), which occurred in a concentration- dependent manner. 126 Figure 24: Arsenic trioxide reduced overall PBMC and NK cell viability. Human PBMCs were isolated and treated with arsenic trioxide (0.5- 2 µM As2O3), dPBS (VEH), or left untreated (BKG). Samples were either unactivated (BKG) or activated with influenza A virus at 5 HAU/sample (VEH and 0.5- 2 µM As2O3) for 96 h. Cells were stained with viability dye and fluorescently conjugated antibodies against CD3 and CD56 and were analyzed by flow cytometry. (A) PBMC viability was assessed using Live/Dead stain. (B) Viability of NK cells was determined by identifying CD56+CD3- cells followed by Live/Dead stain. Percent vehicle was calculated by dividing individual cell count by the average cell count of the vehicle group. Three experiments were performed in triplicates. * Denotes P ≤ 0.5, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, **** denotes P ≤ 0.0001 compared to VEH. Arsenic Decreased NK Cell Population. In addition to overall cell viability, we examined the effects of ATO on CD56+CD3- NK cells, as previous studies have shown arsenic to induce DNA damage in murine NK cells.286,287 NK cells were identified as CD56+CD3- prior to gating of NK cell subsets, CD56brightCD16- and CD56dimCD16+. ATO caused a concentration-dependent decrease in viable NK cells (Figure 24B). When further divided into NK cell subsets, most NK cells displayed CD56dimCD16+ phenotype (data not shown). This was expected since CD56dimCD16+ NK cells are the main population of NK cells found in peripheral blood.288 In CD56dimCD16+ NK cells, a reduction was observed with 1 µM and 2 µM ATO, whereas CD56brightCD16- NK cells were decreased with all 127 ATO-treatments (Figure 25). These data suggest that the NK cell subset, CD56brightCD16-, are more sensitive to ATO at lower concentrations. Figure 25: NK cell subsets decreased with arsenic trioxide. Human PBMCs were isolated and treated with arsenic trioxide (0.5- 2 µM As2O3), dPBS (VEH), or left untreated (BKG). Samples were either unactivated (BKG) or activated with influenza A virus at 5 HAU/sample (VEH and 0.5- 2 µM As2O3) for 96 h. Cells were stained with viability dye and fluorescently conjugated antibodies against CD3, CD56, and CD16 were analyzed by flow cytometry. NK cells were identified as viable CD56+CD3- prior to analysis of subsets, (A) CD56brightCD16- and (B) CD56dimCD16+. Percent vehicle was calculated by dividing individual cell count by the average cell count of the vehicle group. Three experiments were performed in triplicates. * Denotes P ≤ 0.5, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, **** denotes P ≤ 0.0001 compared to VEH. Arsenic Trioxide Suppressed Expression of the Activating Receptor, NKp46, and Early Activation Marker, CD69, in NK Cells. NKp46 is an important NK cell activating receptor which is involved in the recognition of hemagglutinin which is expressed on virally infected cells. Although NKp46 is constitutively expressed on NK cells, the level of expression has been shown to correlate with NK cell cytolytic activity.289 In the present study, we found IAV exposure to modestly increase NKp46 expression in NK cells (Figure 26). However, ATO-treatment diminished NKp46 expression to levels 128 comparable to the BKG group, which suggests impaired NK cell activation in response to influenza (Figure 26B). Likewise, ATO-treatment significantly reduced expression of the activation marker, CD69, in NK cells compared to the vehicle group (Figure 27A). Overall, the data suggest that ATO inhibits NK cell activation. Figure 26: Arsenic trioxide suppressed expression of activating receptor NKp46. Human PBMCs were isolated and treated with arsenic trioxide (0.5- 2 µM As2O3), dPBS (VEH), or left untreated (BKG). Samples were either unactivated (BKG) or activated with influenza A virus5 HAU/sample (VEH and 0.5- 2 µM As2O3) for 96 h. Cells were stained with viability dye and fluorescently conjugated antibodies against CD3, CD56, and NKp46 and were analyzed by flow cytometry. NK cells were identified as viable CD56+CD3- cells prior to analysis of NKp46. Percent vehicle was calculated by dividing individual (A) cell count or (B) median fluorescence intensity (MFI) by the average of the vehicle group. Three experiments were performed in triplicates. * Denotes P ≤ 0.5, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, **** denotes P ≤ 0.0001 compared to VEH. 129 Figure 27: Arsenic trioxide reduced activation and active degranulation in NK cells. Human PBMCs were isolated and treated with arsenic trioxide (0.5- 2 µM As2O3), dPBS (VEH), or left untreated (BKG). Samples were either unactivated (BKG) or activated with influenza A virus at 5 HAU/sample (VEH and 0.5- 2 µM As2O3) for 96 h. Cells were stained with viability dye and fluorescently conjugated antibodies against CD3, CD56, CD69, and CD107a and were analyzed by flow cytometry. NK cells were identified as viable CD56+CD3- cells prior to analysis of surface markers (A) CD69 and (B) CD107a. Percent vehicle was calculated by dividing individual cell count by the average cell count of the vehicle group. Three experiments were performed in triplicates. * Denotes P ≤ 0.5, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, **** denotes P ≤ 0.0001 compared to VEH. NK Cell Cytotoxicity is Impaired with Arsenic Trioxide. NK cell activation is a necessary precursor to NK cell effector function. Thus, we investigated the effect of ATO on NK cell effector function. NK cells play an essential role in limiting viral replication by rapidly killing virally infected cells through the release of perforin and granzyme.249 A previous study found arsenic to enhanced NK cell activation and effector potential in response to influenza A infection in mice. More specifically, cell surface expression of CD107a, a marker of active degranulation, was greater in mice exposed to arsenic.275 In comparison, our study demonstrated a significant decrease in NK cell expression of CD107a at 1 130 µM and 2 µM ATO compared to the vehicle group (Figure 27B). Similarly, intracellular granzyme and perforin, were also reduced in NK cells at 1 µM and 2 µM ATO (Figure 28). Figure 28: NK cell cytotoxicity was decreased with arsenic trioxide. Human PBMCs were isolated and treated with arsenic trioxide (0.5- 2 µM As2O3), dPBS (VEH), or left untreated (BKG). Samples were either unactivated (BKG) or activated with influenza A virus at 5 HAU/sample (VEH and 0.5- 2 µM As2O3) for 96 h. Monensin was added 6 h prior to flow cytometry analysis (90 h post treatment). Cells were stained with viability dye and fluorescently conjugated antibodies against CD3, CD56, granzyme B, and perforin and were analyzed by flow cytometry. NK cells were identified as viable CD56+CD3- cells prior to analysis of intracellular markers: (A) granzyme B and (B) perforin. Percent vehicle was calculated by dividing individual median fluorescence intensity (MFI) by the average MFI of the vehicle group. Three experiments were performed in triplicates. * Denotes P ≤ 0.5, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, **** denotes P ≤ 0.0001 compared to VEH. Arsenic Trioxide Diminishes IFNg Expression in NK Cells. IFNg is an important antiviral cytokine secreted by NK cells during influenza infection to limit viral replication.249 Therefore, we also wanted to assess the effects of ATO on NK cell production of IFNg when exposed to IAV. With influenza, IFNg expression in NK cells sharply increased in the vehicle group, which confirms NK cell activation. However, cells treated with 131 ATO exhibited a concentration-dependent decrease in IFNg to levels similar to the BKG group (Figure 29A). Consistent with our FACS analysis, IFNg secretion detected in the supernatant was also markedly reduced in ATO-treated cells (Figure 29B). Figure 29: Arsenic diminishes NK cell expression of IFNg. Human PBMCs were isolated and treated with arsenic trioxide (0.5- 2 µM As2O3), dPBS (VEH), or left untreated (BKG). Samples were either unactivated (BKG) or activated with influenza A virus at 5 HAU/sample (VEH and 0.5- 2 µM As2O3) for 96 h. Monensin was added 6 h prior to flow cytometry analysis (90 h post treatment). Cells were stained with viability dye and fluorescently conjugated antibodies against CD3, CD56, and IFN𝛾 and were analyzed by flow cytometry. NK cells were identified as viable CD56+CD3- cells prior to analysis of intracellular marker, (A) IFN𝛾. Percent vehicle was calculated by dividing individual cell count by the average cell count of the vehicle group. Three experiments were performed in triplicates. Supernatants collected prior to FACS analysis were used to quantify (B) secreted IFN𝛾 protein by ELISA. Data are representative of an individual experiment done in triplicate. * Denotes P ≤ 0.5, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, **** denotes P ≤ 0.0001 compared to VEH. Discussion Overall, this study demonstrated ATO decreased overall PBMC and NK cell viability. Additionally, ATO significantly reduced NK cell activation, as demonstrated by decreased 132 CD107a and CD69 expression and reduced NKp46 MFI in NK cells exposed to influenza virus. Consistent with NK cell activation, ATO significantly reduced important antiviral effector functions, including the production of IFNg and induction of the cytotoxic molecules, granzyme B and perforin, in NK cells. In addition to the decrease in intracellular expression of IFNg in NK cells, we also showed an overall decrease in secreted IFNg protein from supernatants treated with ATO. However, the reduction in IFNg may also be attributed to other immune cell populations, such as T cells since it was previously reported that ATO attenuated IFNg production in T cells from IAV-exposed PBMCs.276 NK cells are divided into subsets based on the density of surface markers CD56 and CD16. CD56dimCD16+ subset compromises about 90% of circulating NK cells, whereas CD56brightCD16- subset make up the remaining 10%, which aligned with what we observed (data not shown).288 CD56dimCD16+ NK cells are known to express higher levels of natural cytotoxicity receptors (NCRs), have increased granularity, and are more cytotoxic compared to CD56brightCD16- NK cells.289 Human NCRs include the activating receptor, NKp46, which is constitutively expressed on resting and activated NK cells.290 However, studies have shown a positive correlation between the levels of NK cell surface expression of NKp46 and NK cell cytolytic activity.291–293 In agreement with our NK viability data (Figure 24B), we found NK cells identified as NKp46+ to significantly decrease with ATO-treatment (Figure 26A). However, when examining the MFI of NKp46, ATO-treatment decreased induction of NKp46 expression in NK cells compared to the vehicle group (Figure 26B), which may indicate diminished NK cell activity in response to IAV infection. 133 In addition, we found arsenic to decrease NK cell activation, CD69 (Figure 27A) and CD107a (Figure 27B), and effector function, as demonstrated by reduced NK cell expression of IFNg, granzyme B, and perforin (Figures 28 and 29). In contrast, a previous study found arsenic to enhance expression of genes relating to NK cell effector function and increase cytolytic activity in mice infected with influenza A virus.275 Specifically, single-cell RNA sequencing showed enhanced inflammatory response which was mediated by dysregulation of innate immune cells.275 However, arsenic exposure via dust particles impaired NK cell lysis of target cells in mice.294,295 Disparities in outcomes across studies are likely due to differences in the form of arsenic exposure, species, and experimental model used. This study is novel because it is the first to investigate the effects of arsenic, at concentrations achievable in humans, on the response of primary human NK cells to influenza. Although there are conflicting studies showing increased NK cell activity with arsenic exposure in mice and NK cell lines, it should be noted that our study is specific to primary human NK cells and aligns with epidemiological studies evaluating arsenic exposure and respiratory disease.268,270,275,277,296,297 Specifically, in utero arsenic exposure was associated with increased risk of infection during the first year of life.270 Additionally, children with higher urinary arsenic concentrations were more likely to have pneumonia.271 Lastly, chronic arsenic exposure was found to exacerbate risk of lung infection.296 Together, this study furthers our knowledge of the impacts of arsenic trioxide exposure on primary human NK cells in response to influenza A virus, which could negatively impact NK cell-mediated host defense. This study is also unique because we developed an in vitro model that can be used to screen the effects of chemicals on the antiviral response of primary human NK cell in vitro. 134