Characterization of gene expression by single-cell transcriptomics during Pneumocystis infection in immunocompetent mice

Authors

• AC Niemiec
• GO Joe
• AD Kokkinis
• KH Fischbeck
• C Grunseich
• JA Shrader

Abstract

Pneumocystis is an opportunistic fungal pathogen that causes asymptomatic infection in immunocompetent hosts but life-threatening pneumonia (PCP) in immunosuppressed hosts. Understanding immune mechanisms by which Pneumocystis is cleared will give valuable insights into strategies for reducing risk of PCP.
We used a co-housing exposure model of mouse Pneumocystis infection to determine gene expression levels of host cells from the lungs of immunocompetent (C57BL/6) mice. We applied single-cell RNA sequencing combined with flow cytometry, qPCR, ELISA, and pathway analyses, to better characterize host immune responses during infection.
We observed a peak in Pneumocystis organism load at day 35 post-infection. All infected mice cleared Pneumocystis and developed anti-Pneumocystis antibodies. CD4+ T cells increased at days 35 and 42 post-infection, primarily involving Th1-effector cells during clearance. An IFN‐γ centered cytokine signaling pathway was identified at peak of infection. The top differentially expressed genes in infected mice included pro-inflammatory cytokines IL-21 and CXCL9, co-stimulatory molecule Ox-40 (CD134), and regulatory genes CTLA4, LY6A, and IRF4.
These data support a CD4 Th1 cell-driven response in immunocompetent hosts and suggest that a regulated IFN‐γ‐rich milieu is necessary to enhance phagocytosis of Pneumocystis organisms by macrophages, leading to efficient clearance of Pneumocystis without the development of severe symptoms. More research on the roles of B cells, epithelial cells, and endothelial cells will help to further elucidate the diverse host-pathogen interactions required to prevent PCP in immunosuppressed hosts.

Scientific Focus Area: RNA Biology

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