Project description:Background: Pneumocystis jirovecii pneumonia (PJP) causes high morbidity and mortality in immunocompromised patients. Current therapeutic regimens for PJP are limited and thus alternative treatment options are needed. Results: In this study, we demonstrate that Pneumocystis murina (P. murina, a model of P. jirovecii infection) can survive and persist within bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and J774A macrophages. Modeling malnutrition, a risk factor for PJP, we found that nutrient deficient media enhanced the capacity for intracellular survival of the fungus. M-CSF boosted intracellular survival capability in starvation media in BMDMs. In vivo infection with P. murina promoted M2, not M1, macrophage polarization which was evident beginning. At 6 weeks post-infection, this polarization returned to baseline in wild type mice that clear the infection but remained elevated in IL-21R deficient mice, that fail to clear the infection. The activated M2 macrophages driven by IL4 and IL13 consisted of AMs and interstitial macrophages (IMs) and were derived from monocytes. AMs isolated from P. murina-infected mice supported continued survival of P. muria ex vivo, suggesting that AMs harbor P. murina in vivo. Immunofluorescence staining analysis showed co-localization of the P. murina antigen and the CD209-recognized mannan-containing major surface glycoprotein on the surface of P. murina within infected BMDMs and cultured AMs isolated from bronchoalveolar lavage fluid of infected mice. Immunohistochemistry analysis showed that P. murina staining colocalized with IMs in infected mouse lungs. The lung macrophage population and the lung burden are both significantly higher in Rag2-/- mice than that in WT mice at week 4. To determine if macrophages serve as a reservoir for the fungus we administered clodronate liposomes and observed a significant reduction of P. murina in vivo through depletion of lung macrophages. Conclusion: Our findings reveal that P. murina organisms are facultative intracellular fungal pathogens of macrophages. P. murina infection promoted monocyte-derived M2 macrophage activation, which comprises both AMs and IMs. Both Lung macrophage populations and fungal burdens in infected Rag2-/- mice are higher than that in infected C57BL/6 mice, suggestiong that lung macrophages are a reservoir and might be a proliferative niche for the fungus. Clodronate administration supports that macrophages are important host reservoir for the fungus, Therefore augmenting macrophage fungicidal activity is an important strategy for host directed therapies to enhance resolution of this infection.
Project description:Understanding immune mechanisms by which Pneumocystis is cleared in immunocompetent hosts can inform strategies for preventing and treating Pneumocystis pneumonia. We used a co-housing exposure model of mouse Pneumocystis (P. murina) infection to characterize the roles of pulmonary immune cell populations in immunocompetent (C57BL/6) mice at single-cell level. CD4+ TH1 cells showed the greatest increase and clonal expansion following P. murina infection, with less pronounced increases in TH17 and TfH cells. We also observed reduced TCR diversity post-P. murina infection, and identified expanded clonotypes with limited overlap between animals, suggesting antigenic variation. Infected mice showed differential expression of pro-inflammatory cytokines IL-21, Cxcl9, Ly6a, co-stimulatory molecule Ox-40 (CD134), and regulatory genes Lag3 and Ctla4, indicating a regulated immune response. Pathway analyses predicted IFN‐γ centered cytokine interactions which presumably enhance killing of P. murina organisms by macrophages. Thus, we demonstrate a predominant CD4+ TH1 cell response in immunocompetent hosts, and delineate expanded TCR clonotypes in Pneumocystis infected mice, providing insights for development of immunomodulatory therapies.