ABSTRACT: Pneumocystis jirovecii pneumonia (PJP) remains a significant cause of morbidity and mortality in AIDS1. During AIDS and other immunosuppressive states, the absence of CD4 lymphocytic immunity results in serious PJP 2,3. In addition to CD4 cells, organism clearance requires a balanced macrophage response since excessive inflammation promotes lung injury and respiratory failure4. We have shown that polarization of AMs toward M1 versus M2 type phenotype, leads to detrimental lung inflammation during Pneumocystis pneumonia5. Corticosteroids given in addition to antibiotics significantly improves outcome during PJP 6. However, concerns exist that corticosteroids further suppress immunity and increase co-infections 7,8. New strategies to promote killing and clearance of Pneumocystis while balancing lung inflammation are required for patients with PJP, particularly for those who are refractory to antibiotic therapy alone. Our prior studies have shown that host innate immunity to Pneumocystis is mediated by C-Type Lectin Receptors (CLRs) on macrophages and involves downstream CARD9 activation 9. We have further shown that the CARD9 can be targeted by a novel specific small molecule inhibitor (BRD5529) that significantly reduces inflammatory signaling in macrophages stimulated with Pneumocystis 10. CARD9 serves as the central intracellular molecule through which Dectin-1, Dectin-2, Mincle, and other CLRs signal 11. Dectin-1 CLR is activated through its own intracytoplasmic ITAM domain12, while other innate CLRs (e.g. Dectin-2 and Mincle) require interactions with a common Fc-gamma receptor (FcγR) accessory chain to mediate responses to Pneumocystis 13,14. Strikingly, we observed that mice double deficient in both Dectin-1 and Fcer1g (which lack the FcγR gamma chain) demonstrated markedly reduced organism clearance compared to Card9-/- infected animals. These mice also possess deficiencies in immunoglobulin (Ig) Fc receptors directly mediating antibody uptake responses, further implicating altered humoral responses in Pneumocystis opsonization and killing. Thus, we hypothesize that innate immune responses through the CLR-CARD9 axis, humoral activity, and macrophage polarization act together to mediate effective responses resulting in optimal organism uptake, killing and generation of host inflammatory responses. This hypothesis will be addressed through three independent but interrelated Aims. In Aim #1. We will determine the relative roles of CLRs, CARD9, and FcRγ- mediated mechanisms in Pneumocystis organism clearance and inflammatory responses in vitro. We will study alveolar macrophages (AMs) and bone marrow-derived myeloid cells from wild-type, CARD9, and Dectin-1/FcRγ deficient animals. In addition, we will also now include mice deficient in all activating (FcγRI, FcγRIII, and FcγRIV, termed FcγRI/ FcγRIII/FcγRIV-/- mice) Fc receptors but retaining the functioning FcRγ chain signal transduction subunit. We will further study uptake and kill...