Pneumocystis murina promotes inflammasome formation and NETosis during Pneumocystis pneumonia

Abstract

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30%-60% mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed neutrophil extracellular trap (NET) presence in the lungs of the P. murina-infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina. Isolated NETs compromised P. murina viability in vitro, highlighting the potential role of neutrophils in controlling fungal growth and promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP.IMPORTANCEPneumocystis jirovecii pneumonia (PjP) affects individuals with weakened immunity, such as HIV/AIDS, cancer, and organ transplant patients. Severe PjP triggers lung inflammation, impairing function and potentially causing acute respiratory distress syndrome. Non-HIV individuals face a 30%-60% mortality rate, underscoring the need for deeper insight into PjP's inflammatory responses. Past research focused on macrophages in managing Pneumocystis infection and its inflammation, while the role of neutrophils was generally overlooked. In contrast, our findings in P. murina-infected mouse lungs showed neutrophil involvement during inflammation and increased expression of NLRP3 inflammasome and NETosis pathways. Detection of neutrophil extracellular traps further indicated their involvement in the inflammatory process. Although beneficial in combating infection, unregulated neutrophil activation poses a potential threat to lung tissues. Understanding the behavior of neutrophils in Pneumocystis infections is crucial for controlling detrimental reactions and formulating treatments to reduce lung damage, ultimately improving the survival rates of individuals with PjP.

Keywords: NETosis; Pneumocystis; Pneumocystis murina; Pneumocystis pneumonia; immunity; infectious disease; inflammation; neutrophils; opportunistic fungi.

Fig 1

Signaling pathways associated with inflammatory processes are up-regulated as Pneumocystis murina infection progressed in mice. Immunosuppressed mice were exposed to uninfected or previously P. murina-infected mice. (A) After 5 or 7 weeks of exposure, the fungal burden in the lungs was enumerated. Dashed line, limit of microscopic enumeration. ***, P < 0.001. ****, P < 0.0001. (B) Heatmap of sample-to-sample distances. 5wC and 7wC uninfected samples cluster near the bottom, and 5wI clusters in the middle, while 7wI clusters at the top of the heatmap. (C) Heatmap comparison analysis of gene set enrichment analysis on KEGG pathways relating to signaling. Data are represented as logFC.

Fig 2

NOD-like receptor signaling and NETosis-related transcripts are up-regulated as P. murina infection progressed in mice. Data are represented as log2 fold change between the displayed groups. Data are represented as logFC. Bold, FDR-adjusted P < 0.05.

Fig 3

NETosis-related proteins are present in the lungs of Pneumocystis murina-infected mice. (A) ELISA reveals increased expression of NET components, NE and (B) MPO, in the lung tissue from P. murina-infected (Pm+) mice over uninfected (UI) mice. *t-test, P < 0.05. (C) Immunohistochemistry of the lungs of P. murina-infected mice. The NETosis markers, NE and MPO, are expressed throughout the thickened alveolar spaces of Pm+ mice. Scale, 100 µm.

Fig 4

Pnuemocystis murina stimulates NET production in bone marrow-derived neutrophils. Bone marrow-derived neutrophils were stimulated with vehicle, phorbol myristate acetate (PMA; 25 nM; positive control), and P. murina at different multiplicities of infection (1, 2, or 5). Controls were established using the same concentrations of P. murina organisms without neutrophils.(A) Culture supernatant was assessed for extracellular DNA released from neutrophils. (B) Culture supernatant assessed for MPO-DNA complexes by ELISA. One-way ANOVA. *, P < 0.05 compared to vehicle.

Fig 5

Pneumocystis murina directly stimulates NET production in bone marrow-derived neutrophils. Immunofluorescence of the bone marrow-derived neutrophils stimulated with vehicle, phorbol myristate acetate (PMA; 25 nM), or P. murina. The NET production was seen in PMA- and P. murina-treated neutrophils as shown by neutrophil elastase (green), citrullinated histone H3 (yellow), MPO (magenta) expression. DAPI, blue. Scale, 50 µm.

Fig 6

NETs are detrimental to Pneumocystis murina viability. P. murina (n = 3; 5 × 10⁷ nuclei) were inoculated into 96-well plates (Costar 3548, Corning, New York). Isolated NETs from PMA (25 nM)-stimulated neutrophils or salmon sperm DNA samples, used as control for non-complexed DNA, were added to the wells. Plates were incubated at 5% CO₂, 37°C. After 24 hours, P. murina was quantified by large subunit (LSU) rRNA copy number by qPCR. One-way ANOVA. *, P < 0.05 compared to vehicle.