Review 2: "Pneumocystis Murina Promotes Inflammasome Formation and NETosis during Pneumocystis Pneumonia"

Terry W. Wright

doi:doi:10.1162/2e3983f5.21aa1648

Pneumocystis murina Promotes Inflammasome Formation and NETosis during Pneumocystis Pneumonia

by Steven G. Sayson, Alan Ashbaugh, Aleksey Porollo, and Melanie T. Cushion

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Published on Feb 18, 2024
www.biorxiv.org

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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. While isolated NETs did not compromise P. murina viability, our data highlight the potential role of neutrophils in 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.IMPORTANCE Pneumocystis 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.

RR:C19 Evidence Scale rating by reviewer:

Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: Pneumocystis pneumonia (PJP) remains a clinically important respiratory fungal infection of immunocompromised patients. In addition to its role as an AIDS-related opportunistic infection, the incidence of non-HIV PJP has shown a dramatic increase in the past decade. In the current manuscript the authors explore the role of neutrophil NETosis in an animal model of PJP. Lung neutrophil recruitment is a hallmark of PJP in both patients and animal models. While several studies have found that the number of neutrophils correlates with the degree of respiratory impairment, other work suggests that they do not contribute meaningfully to either lung injury or host defense. However, the multiple potential roles for neutrophils during the different stages of PJP have not been thoroughly examined. The current work suggests that neutrophils undergo NETosis during PJP in vivo, and after direct stimulation with Pneumocystis in vitro. Although neutrophil NETs were not directly toxic to Pneumocystis, the authors propose that NET formation may contribute to lung inflammation and injury during PJP. This study is the first to show that Pneumocystis induces NETosis and may open new areas of investigation into PJP.

The studies are limited in that they neither directly show NET formation and NETosis in vivo during PJP, nor prove a role for neutrophils and NETs in lung injury or antifungal host defense. The immunofluorescence staining for NE and CitH3 is consistent with NET formation but does not directly prove that this process is occurring in vivo. Furthermore, the percentage of neutrophils undergoing NETosis in vitro is not quantified. Is this a rare occurrence, or is Pneumocystis a potent inducer of NETosis? This data would aid in evaluating the biological relevance of NETosis during PJP. Overall, this is an interesting initial observation with potential to further advance our understanding of PJP and how to treat this disease more effectively.

Review 2: "Pneumocystis Murina Promotes Inflammasome Formation and NETosis during Pneumocystis Pneumonia"

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