Cobalt is a transition metal utilized frequently in industry for the production of alloys, such as tungsten carbide. Human exposures to cobalt result mostly from workers inhaling cobalt-containing dusts. Many of these workers develop hard metal lung disease (HMLD or "cobalt lung"). Cobalt has been shown to be the necessary component in tungsten carbide dust to cause HMLD, though the mechanism is largely unknown. Interestingly, cobalt is a well-known hypoxia mimic and activates hypoxia-inducible factor (HIF)-mediated signaling. Our previous studies using a doxycycline inducible, lung epithelial-specific knockout of HIF1alpha showed a switch from neutrophilic to eosinophilic recruitment during cobalt-induced inflammation, suggesting a role for epithelial HIF1alpha in modulating the immune response of the lung. Little is known, however, about the role of HIF2alpha in cobalt-induced lung injury or the possible overlap of HIF1alpha and HIF2alpha in this context. With this in mind, several experiments have been performed to elucidate the mechanism of HIF1alpha-deficient induced eosinophilia and the role of HIF1alpha and HIF2alpha in cobalt-induced lung inflammation. To characterize the mechanism of change in cobalt-induced inflammatory response in the HIF1alpha-deficient mice, several experiments were performed, including flow cytometry of resident lung immune cells, different temporal HIF1alpha deletions, use of specific pharmacologic inhibitors of the suspected effector pathway of NF-kappaB and the protective pathway of adenosine receptor A2B (Adora2b). These studies revealed that cobalt-treated HIF1alpha-deficient mice produced more GATA3+ T-helper cells, confirming that the eosinophilia involved a T-helper type 2 (TH2) response. Also, inducing the HIF1alpha deletion in the early postnatal time period (P4-14), and not the adult (P32 onward) was required in establishing a predisposition for eosinophilic inflammation. Adora2b exerts protective effects on inflamed tissues, and inhibition of this receptor prior to cobalt dosing decreased the eosinophilia seen in HIF1alpha-deficient mice to control levels, suggesting a role for adenosine signaling in promoting eosinophilic inflammation. Use of the proteasome inhibitor TCH013, thought to selectively inhibit NF-kappaB, immediately prior to cobalt dosing, was ineffective in altering the expected eosinophilia. Overall these data imply that HIF1alpha in alveolar epithelial type II and Club (Clara) cells plays a vital role in establishing the lung's immune environment early in postnatal lung development. HIF2alpha-deficient mice treated with cobalt display eosinophilia which peaks at the 14 day time point, later than eosinophilia observed at 2-5 days in HIF1alpha-deficient mice. When mice lose both HIF1alpha and HIF2alpha by recombination, results are similar to the HIF1alpha-deficient mice at 5 days, suggesting that the effects of HIF1alpha loss are more important in driving earlier eosinophilia. Gene expression, histopathological analyses and cytokine profiling of lungs showed increases in classic TH2 markers correlated with eosinophilic inflammation. All together these data show that both HIF1alpha and HIF2alpha are likely involved in post-natal development of proper immune responses to cobalt-induced lung inflammation.
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