Methods: We used RNA sequencing of whole lung tissue from C57Bl/6 mice over the course of PC infection in both immunocompetent and CD4-depleted animals to evaluate specific cellular signatures. Analysis of bronchoalveolar lavage fluid by flow cytometry was also conducted to examine recruited cellular populations. Hydrodynamic injection of a plasmid expressing IL-5 (pIL5) was used to induce hypereosinophilia in C57Bl/6, BALB/c and double GATA1 knockouts, which are deficient in eosinophilopoiesis. To evaluate Pneumocystis burden, qRT-PCR of the large subunit rRNA was used.
Results: Strikingly, PC induced a potent CD4-dependent type II immune response with significant increases in IL-4, IL-5, and IL-13. This type II immune response resulted in a strong eosinophil signature by RNA sequencing at day 14, which was corroborated by the presence of eosinophils in bronchoalveolar lavage fluid (BAL). Immunocompromised mice (e.g. Rag1-/- or CD4-depleted) receiving pIL5 had increased eosinophils in the lung and decreased PC burden. Finally, double GATA knockout mice deficient in eosinophilopoiesis, had no change in burden with pIL5 treatment following CD4-depletion, further implicating eosinophils as novel mediators of protective immunity against PC.
Conclusion: These results suggest that one role of CD4+ T-cells in clearance of PC is to recruit eosinophils to the lung, which then have fungicidal activity. Eosinophils may therefore be a viable therapeutic option, although concomitant pathology needs to be explored. Future studies will examine the mechanisms of pathology induced by PC infection and will dissect the mechanisms of pathologic and protective immunity to PC.
B. Campfield, None
J. Kolls, None