PROJECT SUMMARY. The prevalence of parasitic helminth infections is estimated at two billion individuals infected worldwide. Many helminths migrate through the lung as part of their developmental cycles and as a result promote substantial lung damage and tissue fibrosis. Our studies and those of others have shown that alveolar macrophages (AMs) with an alternatively activated or M2 phenotype are critical regulators of these responses. Alveolar macrophages are the most abundant immune cell in the parenchyma of the lung and play diverse metabolic and immunoregulatory roles that are needed to maintain lung function. Despite their importance, the molecular mechanisms that regulate AM activation and their M2 responses remain to be fully defined. Gaining a better understanding of the factors that regulate AM activation could reveal novel therapeutic targets to treat diverse forms of lung inflammation including fibrosis. Our new studies have shown that the enzyme carbonic anhydrase 4 (Car4) is a lineage-identifying feature of AMs. Carbonic anhydrases are a family of enzymes that are traditionally known for their ability to regulate pH and CO2 homeostasis. However, our published studies have recently identified that Car enzymes also play important roles in regulating immune cell activation and antihelminth immunity. Therefore, we sought to investigate whether Car4 regulates the phenotypes and functions of AMs. To test this, we generated a novel Car4-floxed mouse to selectively delete Car4 in AMs. Importantly, mice with Car4-deficient AMs (Car4-AM-/- mice) exhibited spontaneous induction of M2 and profibrotic pathways and presented with features of altered lipid metabolism and increased hypoxia-inducible factor (HIF) signaling. To further investigate the role of Car4 in regulating the M2 activation of AMs we infected Car4-AM-/- mice with Nippostrongylus brasiliensis (Nb), a parasitic infection that promotes robust M2 responses and lung pathology. Importantly, Car4-AM-/- mice infected with Nb exhibited dramatically elevated M2 responses, increased lung fibrosis and suffered from significantly reduced oxygen levels. Collectively, these data provoke the hypothesis that Car4 regulates the metabolism of AMs in a manner that restricts M2 activation and tissue fibrosis. This hypothesis will be tested in the three Aims. Aim 1 will focus on the importance of HIF-associated pathways in regulating the bioenergetic state of AMs and their M2/profibrotic phenotype. Aim 2 will investigate the role Car4 plays in regulating M2 activation and pulmonary fibrosis in the context of helminth-induce inflammation. Aim 3, will investigate whether Car4 also instructs the activation of newly seeded monocyte-derived alveolar macrophages that populate the lung post-Nb infection and acquire an M2 phenotype. Gaining better insight into these pathways may lead to the development of new therapeutic strategies to treat lung disease and tissue fibrosis.