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Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice
Atypical lung neutrophils mediate TB pathogenesis in male Nox2-/- mice
Choi Eunsol Conceptualization Data curation Formal analysis Investigation Methodology Software Validation Visualization Writing – original draft Writing – review & editing 1
Choi Hong-Hee Conceptualization Investigation Methodology 1
Kwon Kee Woong Data curation Funding acquisition Investigation Methodology 1
Kim Hagyu Data curation Investigation Methodology 1
Ryu Ji-Hwan Methodology Resources 2
Hong Jung Joo Methodology Project administration Resources 3 4
https://orcid.org/0000-0003-0854-4582
Shin Sung Jae Conceptualization Funding acquisition Project administration Resources Supervision 1 *
1 Department of Microbiology, Institute for Immunology and Immunological Disease, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, South Korea
2 Department of Biomedical Sciences, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, Korea
3 National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, South Korea
4 KRIBB School of Bioscience, Korea University of Science & Technology (UST), Daejeon, South Korea
Sassetti Christopher M. Editor
University of Massachusetts Medical School, UNITED STATES OF AMERICA
The authors have declared that no competing interests exist.

* E-mail: sjshin@yuhs.ac
23 8 2024
8 2024
20 8 e101250014 11 2023
12 8 2024
© 2024 Choi et al
2024
Choi et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

NADPH oxidase 2 (NOX2) is an enzyme responsible for generating reactive oxygen species, primarily found in phagocytes. Chronic Granulomatous Disease (CGD), along with bacterial infections such as Mycobacterium tuberculosis (Mtb), is a representative NOX2-deficient X-linked disease characterized by uncontrolled inflammation. However, the precise roles of host-derived factors that induce infection-mediated hyperinflammation in NOX2-deficient condition remain incompletely understood. To address this, we compared Mtb-induced pathogenesis in Nox2-/- and wild type (WT) mice in a sex-dependent manner. Among age- and sex-matched mice subjected to Mtb infection, male Nox2-/- mice exhibited a notable increase in bacterial burden and lung inflammation. This was characterized by significantly elevated pro-inflammatory cytokines such as G-CSF, TNF-α, IL-1α, IL-1β, and IL-6, excessive neutrophil infiltration, and reduced pulmonary lymphocyte levels as tuberculosis (TB) progressed. Notably, lungs of male Nox2-/- mice were predominantly populated with CD11bintLy6GintCXCR2loCD62Llo immature neutrophils which featured mycobacterial permissiveness. By diminishing total lung neutrophils or reducing immature neutrophils, TB immunopathogenesis was notably abrogated in male Nox2-/- mice. Ultimately, we identified G-CSF as the pivotal trigger that exacerbates the generation of immature permissive neutrophils, leading to TB immunopathogenesis in male Nox2-/- mice. In contrast, neutralizing IL-1α and IL-1β, which are previously known factors responsible for TB pathogenesis in Nox2-/- mice, aggravated TB immunopathogenesis. Our study revealed that G-CSF-driven immature and permissive pulmonary neutrophils are the primary cause of TB immunopathogenesis and lung hyperinflammation in male Nox2-/- mice. This highlights the importance of quantitative and qualitative control of pulmonary neutrophils to alleviate TB progression in a phagocyte oxidase-deficient condition.

Author summary

While tuberculosis (TB) remains a global threat to public health, the immunologic factors contributing to TB susceptibility are not yet fully defined. To enhance our understanding of TB immunopathogenesis, we utilized TB-susceptible male Nox2-/- mice to dissect the immunologic factors accelerating TB pathogenesis. In this study, we observed that male Nox2-/- mice infected with Mycobacterium tuberculosis (Mtb) exhibited increased lung hyperinflammation and mycobacterial burden compared to female Nox2-/- mice and WT mice. Exacerbated TB immunopathogenesis in male Nox2-/- mice was strongly correlated with an extreme infiltration of pulmonary neutrophils and the loss of pulmonary lymphocytes. The pulmonary neutrophils in male Nox2-/- mice displayed immature phenotypes and mycobacterial permissiveness. The depletion of total neutrophils or the AM80-induced maturation of neutrophils ameliorated TB pathogenesis in Nox2-/- mice, reducing pulmonary neutrophil counts, lung hyperinflammation, and mycobacterial load. Furthermore, neutralization of G-CSF mitigated TB pathogenesis in male Nox2-/- mice by decreasing immature neutrophil counts. Thus, we identified that G-CSF-mediated generation of permissive immature neutrophils contributes to TB susceptibility in male Nox2-/- mice.

National Research Foundation of Korea RS-2023-00208115 https://orcid.org/0000-0003-0854-4582
Shin Sung Jae http://dx.doi.org/10.13039/501100003710 Korea Health Industry Development Institute HI22C0177 https://orcid.org/0000-0003-0854-4582
Shin Sung Jae http://dx.doi.org/10.13039/501100003715 Korea Research Institute of Bioscience and Biotechnology KGM4572431 https://orcid.org/0000-0003-0854-4582
Shin Sung Jae This work was supported by the National Research Foundation of Korea (NRF) grant, funded by the Ministry of Science and Information and Communication Technology (MSIT) under grant number RS-2023-00208115 to SJS and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program under grant number KGM4572431 to SJS. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS Publication Stagevor-update-to-uncorrected-proof
Publication Update2024-09-05
Data AvailabilityAll relevant data are presented in the paper and supporting information.
Data Availability

All relevant data are presented in the paper and supporting information.
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pmcIntroduction

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), which remains a significant public health concern worldwide. Approximately, one-quarter of the world’s population is estimated to have latent Mtb infection, with 5–10% of these individuals at risk of developing active TB at some point during their lifetime [1]. The factors that influence susceptibility to Mtb are still not completely understood, which hampers the control of this harmful pathogen [2–4]. There is extensive documentation indicating that males are more susceptible to TB than females, and this sexual bias is observed in various animal models including mice and humans [5,6]. The increased TB susceptibility in males is likely due to differences in immune cell functions as well as in their compositions [7,8]. Such host factor-mediated alterations in immune function may directly influence immune responses to Mtb infection. Considering the importance of immunological balance in maximizing anti-TB immunity, both excessive immune suppression or activation might be harmful to the host during TB progression [9–11]. These clues have led to the hypothesis that several host factors, such as biological sex and genetic distributions, which modulate inflammatory responses, may influence TB immunopathogenesis.

The phagocyte NADPH oxidase (NOX2, gp91phox) is located in the lumen of phagosomes. Its primary function is to produce reactive oxygen species (ROS), which play a crucial role in protecting the host against a wide range of pathogens [12,13]. NOX2 also plays a vital role in regulating autophagy and cytokine/chemokine signaling [14–16]. The NOX2 complex is a multimeric enzyme composed of a single catalytic transmembrane heterodimer (gp91phox / p22phox) and four cytosolic subunits (p40phox / p47phox / p67phox / Rac). Among the six major components of NOX2 complex, gp91phox acts as the catalytic core for ROS generation [17,18]. Individuals with impaired NOX2 function may develop chronic granulomatous disease (CGD), characterized by an increased susceptibility to fungal and bacterial infections and impaired inflammation control [19–21]. Since Nox2 is located at Xp21.1 of the X chromosome, CGD caused by Nox2 deficiency is inherited in an X-linked recessive manner, primarily affecting young males [22,23]. Furthermore, individuals with X-linked CGD have a heightened vulnerability to mycobacterial infections [24]. This poses significant challenges for TB therapy and prevention in X-linked CGD patients as BCG vaccination cannot be implemented due to the high risks of BCGosis, which is a disseminated mycobacterial infection that can occur following BCG vaccination [25–27].

Although phagocyte NADPH oxidase-originated ROS play a crucial role in clearing intracellular pathogens, studies have shown that phagocyte NADPH oxidase deficiencies did not significantly alter mycobacterial growth compared to wild type (WT) animals. According to Cooper et al., p47phox-deficient mice featured increase in lung mycobacterial load from 15 to 30 days post-infection. However, the increase in mycobacterial load was not sustained after 30 days post-infection [28]. Additionally, Olive et al. reported that male and female gp91phox-deficient mice did not show an increase in lung mycobacterial load until 60 days or 20 weeks post-infection [29,30]. These researches give clues that NOX2 deficiency may modulate immune responses against Mtb, independently of ROS-mediated bactericidal effects. Recent studies support this idea as Thomas et al. demonstrated that gp91phox-deficient mice displayed enhanced inflammation and lung neutrophil infiltration after Mtb challenge, which were mediated by IL-1 [12,31].

Dysregulated inflammatory responses and aberrant granuloma formation are hallmark features of X-linked CGD. X-linked CGD patients and NOX2-deficient mice exhibit uncontrolled formation of granulomas, characterized by an excessive influx of phagocytic myeloid cells to the site of infection [32,33]. NOX2-deficient phagocytes, such as macrophages and neutrophils, are major producers of pro-inflammatory cytokines [34,35], which can cause tissue damage [36,37]. Given that NOX2 deficiency-induced X-linked CGD can cause TB susceptibility along with lung hyperinflammation, we hypothesized that Mtb infection would cause uncontrolled inflammation and disruption of immune tolerance in NOX2-deficient mice. As the maintenance of immune tolerance is critical for effective anti-TB immunity, we further hypothesized that controlling the specific immunologic factor which causes Mtb infection-driven hyperinflammation would be crucial for ameliorating TB pathogenesis.

In this study, we established four experimental groups to investigate how two independent host factors (NOX2 deficiency and male sex) can modify the immunological responses against Mtb infection. As both male sex and NOX2 deficiency are risk factors for TB susceptibility, we hypothesized that male Nox2-/- mice would exhibit severer TB progression than female Nox2-/- mice and male WT mice. Additionally, we anticipated that specific immune factors would contribute to TB pathogenesis and hyperinflammation in male Nox2-/- mice, which show a clear correlation to disease progression. Finally, we aimed to discover novel interventions that can effectively regulate these disease-promoting factors in male Nox2-/- mice.

We found that male Nox2-/- mice exhibited higher levels of lung inflammation and mycobacterial burden compared to female Nox2-/- mice and WT mice following Mtb challenge. Male Nox2-/- mice also featured elevated production of pro-inflammatory cytokines in the lungs, along with excessive infiltration of immature neutrophils and reduction of lymphocytes at infection sites. We also dissected the properties of aberrant permissive immature neutrophils, which were distinct from mature neutrophils and constituted the majority of immune cell populations in male Nox2-/- mice. By reducing the number of total lung neutrophils or immature neutrophils, we could successfully mitigate TB pathogenesis in male Nox2-/- mice. Finally, we determined that G-CSF, rather than other cytokines, is the dominant controller of immature neutrophil generation through in vivo neutralization of each cytokine. Collectively, we identified G-CSF driven permissive neutrophils as the key immunologic factor that facilitates TB immunopathogenesis and lung hyperinflammation in male Nox2-/- mice. Furthermore, we suggested novel therapeutic interventions targeting immature neutrophils, which might be potential for controlling TB in NOX2-deficient condition.

Results

Male Nox2-/- mice feature increased mycobacterial burden and lung hyperinflammation following aerosol infection with the Mtb K strain

Female WT, male WT, female Nox2-/-, and male Nox2-/- mice were aerosol-infected with the Mtb K. At four weeks post-infection, the mice were autopsied to assess the severity of TB and immune function (Fig 1A). The mice were euthanized at the four weeks post-infection time point, when the increases in lung bacterial load (S1A Fig) and neutrophil infiltration (S1B Fig)—key pathological features of Mtb-infected Nox2-/- mice [12,31]—reached their peak. These measures began to increase from two weeks post infection, peaked at four weeks post-infection, and subsequently decreased until twelve weeks post-infection in Nox2-/- mice. Male Nox2-/- mice showed the highest lung bacterial load, spleen bacterial load, and lung inflammation. Female Nox2-/- mice featured the second-highest severity, while male and female WT mouse groups were ranked as 3rd and 4th, respectively (Fig 1B and 1C). Pro-inflammatory cytokines including IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF were significantly upregulated in the lungs of male Nox2-/- mice (Fig 1D). Conversely, anti-inflammatory cytokines including IL-4, IL-5, and TGF-β were not upregulated in the lungs of male Nox2-/- mice, although IL-10 was significantly upregulated (Fig 1E). This indicates that NOX2 deficiency plays a significant role in accelerating TB immunopathogenesis, as both male and female Nox2-/- mice feature exacerbated TB progression. Furthermore, we also found that male sex additionally promotes TB pathogenesis, as indicated by severer TB progression in male Nox2-/- mice.

10.1371/journal.ppat.1012500.g001 Fig 1 Male Nox2-/- mice featured severe lung hyperinflammation and increased bacterial load upon Mtb infection.

(A) Experimental design for in vivo analysis of TB susceptibility. Six to seven-week-old female WT, male WT, female Nox2-/-, and male Nox2-/- mice (n = 6 per group) were aerosol-infected with Mtb K strain. At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 288. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. The experiment was conducted twice. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05, **p <0.01.

Pulmonary infiltration of neutrophils and loss of B lymphocytes are correlated with TB pathogenesis of male Nox2-/- mice

To determine which immune cell population is associated with dysregulated pro-inflammatory cytokine production, live lung cell compositions of Mtb infected mice were examined according to the attached flow cytometry gating strategy (S1C Fig). Male Nox2-/- mice exhibited the highest numbers and percentages of CD11b+Ly6G+ lung neutrophils, while female WT mice featured the lowest among the four groups (Fig 2A). On the other hand, male Nox2-/- mice showed a significant reduction in pulmonary CD90.2+ T cell percentages (Fig 2B), CD19+B220+ B cell counts (Fig 2C), and CD19+B220+GL7+FAS+ Germinal Center B cell counts (Fig 2D).

The disparity in TB progression peaked at four weeks post-infection, as male Nox2-/- mice exhibited significantly increased lung bacterial loads and pulmonary neutrophil counts at this time, compared to male WT mice and female Nox2-/- mice. The increase in lung CFU (Colony-forming unit) s and pulmonary neutrophils in male Nox2-/- mice began between two and three weeks post-infection and reached its maximum at four weeks post-infection. Interestingly, the disparity in pulmonary neutrophil counts between male Nox2-/- mice and other groups was more pronounced than the differences in lung CFUs at two and three weeks post-infection, as the latter did not show significant statistical differences, suggesting that the influx of pulmonary neutrophils occurs earlier than the increase in lung bacterial load (Fig 2E). T cell responses against mycobacterial antigen stimulations were also examined, as IFN-γ producing CD4+ T cells take crucial roles in anti-TB immunity [38,39]. Although male Nox2-/- mice featured the most exacerbated TB progression among the four groups, T cell responses against Mtb antigens were strongly maintained (S2 Fig). The numbers of pulmonary CD11c+Siglec-F+ alveolar macrophages, CD11b+CD64+ macrophages, and CD11c+MHCII+ dendritic cells were also significantly reduced in male Nox2-/- mice (S3 Fig). The immune cell dynamics in the lungs of mice infected with Mtb were visually represented using pie-charts and t-SNE plots. We observed that neutrophils, B cells, and T cells comprise the majority of pulmonary immune cells (Fig 2E). Furthermore, we found strong correlations between these immune cell populations and TB severity. The percentages of neutrophils displayed a strong positive correlation with TB severity, with high correlation coefficients and low p-values. Conversely, the percentages of T cells did not exhibit a significant correlation with TB severity, while the percentages of B cells demonstrated a reverse correlation with TB severity. The loss of pulmonary B cells also showed a significant negative correlation with neutrophil infiltration (S4 Fig). Thus, we hypothesized that TB susceptibility of male Nox2-/- mice is highly associated with the increase of pulmonary neutrophils.

10.1371/journal.ppat.1012500.g002 Fig 2 Neutrophils are increased in the lungs of Mtb infected male Nox2-/- mice while B cells are diminished.

Pulmonary (A) CD11b+Ly6G+ neutrophil, (B) CD90.2+ T cell, (C) CD19+ B220+ B cell, and (D) CD19+ B220+ GL7+ FAS+ Germinal Center B cell populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45+ cells and total cell counts are presented in bar graphs. (E) Pulmonary CFUs and neutrophil counts of female WT, male WT, female Nox2-/-, and male Nox2-/- mice were enumerated in a time-course dependent manner. Six-week old mice (n = 5 per group) were aerosol infected with Mtb K strain. The number of lung neutrophils were calculated at 0, 2, 3, and 4 weeks post-infection. Red bars and symbols represent statistical analysis between male Nox2-/- mice and male WT mice. Black bars and symbols represent statistical analysis between male Nox2-/- mice and female Nox2-/- mice. Initial CFU = 300. The experiment was conducted once. The data are presented as the mean ± SD of five or six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. **p <0.01. (F) The compositions of the six major lung immune cell populations in Mtb-infected mice are presented in pie charts and t-SNE plots. The six major immune cells consist of neutrophils (red), macrophages (orange), alveolar macrophages (bright brown), dendritic cells (purple), B cells (blue), and T cells (green). The percentages of each immune cell among the sum of the six major immune cells are presented in pie charts.

Pulmonary neutrophils of Mtb infected male Nox2-/- mice are predominantly composed of aberrant CD11bintLy6GintCXCR2loCD62Llo permissive immature neutrophils

It was not only the number of neutrophils that were altered in male Nox2-/- mice. Pulmonary neutrophils of male Nox2-/- mice featured CD11bintLy6Gint phenotypes, while neutrophils of other mouse groups featured CD11bhiLy6Ghi phenotypes. Atypical lung neutrophils of male Nox2-/- mice showed relatively lower expression of CD11b, Ly6G, Ly6C, CXCR2, and CD62L compared to WT neutrophils, along with higher expression of CXCR4 (Fig 3A). We categorized CD11b+Ly6G+ pulmonary neutrophils into two distinct populations based on CXCR2 expression. According to previous studies, Ly6Ghi or CXCR2hi lung neutrophils are categorized as mature neutrophils, whereas Ly6Gint or CXCR2lo lung neutrophils are categorized as immature neutrophils [40–43]. We revealed that CD11b+Ly6G+ pulmonary neutrophils of Mtb infected mice are consisted of CXCR2hi and CXCR2lo populations. Moreover, we figured out that CXCR2hi mature pulmonary neutrophils highly expressed CD62L, while CXCR2lo immature neutrophils featured low expression of CD62L through our own gating strategy (Fig 3B). The majority of Nox2-/- pulmonary neutrophils were CXCR2loCD62Llo immature neutrophils, whereas WT pulmonary neutrophils comprised both immature and mature neutrophils. This suggests that the excessive accumulation of pulmonary neutrophils in male Nox2-/- mice is primarily mediated by immature neutrophil-specific lung infiltration (Fig 3C). We also analyzed neutrophil profiles from uninfected WT and Nox2-/- mice. The number of pulmonary total and immature neutrophils (S5A and S5B Fig), as well as bone marrow (BM) total and immature neutrophils (S5C and S5D Fig), were not increased in uninfected male Nox2-/- mice. Therefore, we concluded that the extreme pulmonary infiltration of CXCR2loCD62Llo immature neutrophils is initiated by the pathogenic stimulus triggered by Mtb infection. CXCR2loCD62Llo immature lung neutrophils displayed significantly lower expression levels of CD11b and Ly6G, which are major activation markers of neutrophils. They also featured lower FSC-A and SSC-A values compared to mature lung neutrophils, indicating that CXCR2loCD62Llo neutrophils are characterized by their smaller size and reduced granularity when compared to mature neutrophils (Fig 3D). To evaluate the permissiveness of pulmonary neutrophils, we isolated lung neutrophils from Mtb-infected mice and quantified intracellular bacterial counts. Nox2-/- pulmonary neutrophils, predominantly consisted of CXCR2loCD62Llo immature neutrophils, exhibited higher mycobacterial permissiveness compared to WT pulmonary neutrophils (Fig 3E). To examine if CXCR2loCD62Llo immature neutrophils are more permissive than CXCR2hiCD62Lhi mature neutrophils, we infected male WT and Nox2-/- mice with YFP-expressing Mtb K strain. At four weeks post-infection, the mice were euthanized, and neutrophil permissiveness was evaluated using flow cytometry analysis (S6A Fig). We noted that neutrophils constituted the majority of YFP+ cells in male Nox2-/- mice, and the number of Mtb-containing neutrophils were also increased. The mean fluorescence intensity (MFI) values of YFP in Nox2-/- lung neutrophils were substantially higher than those in WT mice, indicating increased mycobacterial permissiveness of Nox2-/- lung neutrophils (S6B Fig). Both immature and mature Mtb-containing neutrophil populations were enlarged in Nox2-/- mice. Notably, immature neutrophils displayed considerably higher YFP MFI values than mature neutrophils, suggesting their increased permissiveness to Mtb (S6C Fig). Conversely, the proportion of YFP+ alveolar macrophages was lower in male Nox2-/- mice, and there was no significant difference in the number of YFP+ alveolar macrophages between WT and Nox2-/- mice, although these cells exhibited relatively higher YFP MFI values compared to mature neutrophils (S6D Fig). The number of YFP+ macrophages was elevated in Nox2-/- mice, but their YFP MFI values were relatively lower than those of immature neutrophils and alveolar macrophages (S6E Fig). In conclusion, we hypothesized that immature neutrophils are the primary permissive phagocytes in male Nox2-/- mice, considering both their dominance in population and their significant bacterial permissiveness as indicated by MFI values. These results suggest that permissive immature lung neutrophils may play a key role in driving TB pathogenesis and lung hyperinflammation in male Nox2-/- mice.

10.1371/journal.ppat.1012500.g003 Fig 3 CD11bintLy6Gint CXCR2loCD62Llo immature neutrophils were dominant among the pulmonary neutrophils of Mtb infected male Nox2-/- mice.

(A) The expression levels of CD11b, Ly6G, Ly6C, CXCR4, CXCR2, and CD62L on CD11b+Ly6G+ neutrophils in the lungs of Mtb-infected mice at four weeks post-infection are presented. The mean fluorescent intensity (MFI) values of each molecule are presented in bar graphs. (B) The gating strategy used to distinguish immature and mature pulmonary neutrophils. CD11b+Ly6G+ neutrophils were classified into CXCR2hi mature neutrophils (red) and CXCR2lo immature neutrophils (bright blue). (C) CD62L expression levels of CXCR2hi neutrophils and CXCR2lo neutrophils are presented into contour plots, demonstrating that mature neutrophils feature CXCR2hiCD62Lhi phenotypes while immature neutrophils feature CXCR2loCD62Llophenotypes. The numbers and percentages of immature and mature neutrophils among total pulmonary neutrophils in each group are presented in bar graphs, along with flow cytometry plots. (D) The expression levels of CD11b and Ly6G on mature and immature pulmonary neutrophils are presented in flow cytometry plots and bar graphs, along with FSC-A and SSC-A values of mature and immature neutrophils. (E) The purity of mouse lung neutrophils after Ly6G specific magnetic sorting and their bacterial permissiveness are presented in flow cytometry plots and bar graphs. Equal numbers of WT and Nox2-/- lung neutrophils were lysed and plated onto 7H10 agar plates to enumerate bacterial permissiveness. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test and Mann-Whitney U test. *p < 0.05, **p <0.01.

Neutrophil depletion alleviates TB pathogenesis and lung hyperinflammation in male Nox2-/- mice

The exacerbated TB pathogenesis of male Nox2-/- mice was characterized by uncontrolled pro-inflammatory cytokine production and influx of permissive immature pulmonary neutrophils. Considering that neutrophils contribute to lung inflammation in Nox2-/- mice [20,44,45], and they are responsible for the increased mycobacterial burden in TB susceptible mouse models [46,47], we hypothesized that reducing Ly6G+ neutrophils would alleviate TB pathogenesis in male Nox2-/- mice. Starting from two weeks post-infection, a Ly6G-specific monoclonal antibody (mAb) was intraperitoneally injected to WT and Nox2-/- mice to deplete neutrophils. At four weeks post-infection, the mice were autopsied (Fig 4A). Neutrophil depletion alleviated TB pathogenesis in both WT and Nox2-/- mice, by effectively reducing bacterial load in the lungs and spleens (Fig 4B) and lung inflammation (Fig 4C). IFN-γ, IL-1 α, IL-1 β, IL-6, TNF-α, and G-CSF levels were significantly downregulated in the lungs of male Nox2-/- mouse after neutrophil depletion (Fig 4D), while IL-10, IL-4, IL-5, and TGF-β levels were not significantly altered (Fig 4E). Therefore, we clarified that Ly6G+ neutrophils are the actual cause of TB progression and lung hyperinflammation in male Nox2-/- mouse.

10.1371/journal.ppat.1012500.g004 Fig 4 Neutrophil depletion ameliorated TB pathogenesis in male Nox2-/- mice.

(A) Experimental design for in vivo depletion of neutrophils in Mtb infected mice. six-week old male WT and Nox2-/- mice (n = 4 per group) were aerosol infected with Mtb K strain. Starting from two weeks post-infection, 250 μg of anti-Ly6G mAb was intraperitoneally injected into each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 569. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. The experiment was conducted twice. The data are presented as the mean ± SD of four mice in each group. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05.

Neutrophil depletion reduces immature lung neutrophils and restores pulmonary lymphocytes in Mtb infected male Nox2-/- mice

Ly6G+ neutrophil depletion not only ameliorated TB pathogenesis, but also altered immune cell compositions in the lungs of male Nox2-/- mice. Neutrophil depletion removed most lung neutrophils in WT mice, and significantly reduced pulmonary neutrophil numbers in male Nox2-/- mice (Fig 5A). Surprisingly, it was highly effective at reducing CXCR2loCD62Llo pulmonary immature neutrophils in both WT and Nox2-/- mice (Fig 5B). Along with the reduction of immature pulmonary neutrophils, the number of B cells were increased in the lungs of Mtb infected male Nox2-/- mice (Fig 5C). However, neutrophil depletion did not significantly alter lung T cell population in male Nox2-/- mice (Fig 5D). This phenomenon was evident from the pie-charts and t-SNE plots illustrating lung immune cell dynamics, which clearly showed the reduction of pulmonary neutrophils and the restoration of B cells (Fig 5E). Consequently, we discovered that the reduction of pulmonary neutrophil counts can restore the balance between lung lymphocytes and neutrophils in male Nox2-/- mice.

10.1371/journal.ppat.1012500.g005 Fig 5 Neutrophil depletion removed immature neutrophils and restored lymphocytes in the lungs of Mtb infected Nox2-/- mice.

Pulmonary (A) CD11b+Ly6G+ neutrophil, (B) CD11b+Ly6G+CXCR2loCD62Llo immature neutrophil, CD11b+Ly6G+CXCR2hiCD62Lhi mature neutrophil, (C) CD19+MHC-II+ B cell, and (D) CD90.2+ T cell populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45+ cells or total neutrophils, and total cell counts are presented in bar graphs, along with flow cytometry plots. (E) The compositions of the six major lung immune cell populations in Mtb-infected mice are presented in pie charts and t-SNE plots. The six major immune cells consist of neutrophils (red), macrophages (orange), alveolar macrophages (bright brown), dendritic cells (purple), B cells (blue), and T cells (green). The percentages of each immune cell among the sum of the six major immune cells are presented in pie charts. The data are presented as the mean ± SD of four mice in each group. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05.

AM80 administration mitigates TB pathogenesis in male Nox2-/- mice and reduces pulmonary immature neutrophils

Among various strategies for the specific control of immature neutrophils, we considered RA treatment as a promising option. Previous studies have examined that retinoic acid (RA) or retinoic acid receptor (RAR) agonist administration can effectively promote the generation of fully differentiated mature neutrophils, which feature segmented nuclei and enhanced bactericidal capacity [48,49]. Moreover, Yamada et al. suggested the potential of RAs such as all-trans retinoic acid (ATRA) as a therapeutic agent against mycobacterial infection [50,51]. To investigate whether immature neutrophil-specific intervention can ameliorate TB pathogenesis in male Nox2-/- mouse, we utilized tamibarotene (AM80), a synthetic retinoid and a RAR agonist demonstrating 10 times greater potency than ATRA. AM80 induces the expression of key molecules, such as C/EBP, CD11b, and CXCR2 through the activation of RARα and RARβ [52], triggering a cascade of cellular pathways that generate fully functional mature neutrophils [53,54]. Starting from one-week post-infection, we orally administered 40 μg of AM80 per mouse, with a total of nine injections given over three weeks. At four weeks post-infection, the mice were autopsied (Fig 6A). AM80 administration significantly reduced mycobacterial load in the lungs and spleens (Fig 6B) and alleviated lung inflammation (Fig 6C) in male Nox2-/- mice. However, despite a reduction in pulmonary bacterial load, its effects were not prominent in reducing lung inflammation of male WT mice. AM80 administration downregulated pulmonary expression of IFN-γ, IL-1 β, IL-17A, and G-CSF in male Nox2-/- mice. It did not substantially alter the expression of pro-inflammatory cytokines in male WT mice, although the expression of TNF-α was significantly reduced (Fig 6D). AM80 intervention did not alter the expression of IL-10, IL-4, IL-5, and TGF-β in both WT and Nox2-/- mice (Fig 6E). Total lung neutrophil counts (Fig 6F) and CXCR2loCD62Llo immature lung neutrophil counts (Fig 6G) were significantly decreased, and the portion of mature neutrophils among total lung neutrophils were increased in AM80-treated WT and Nox2-/- mice. While AM80 treatment effectively modified the accumulation of immature lung neutrophils, it did not exhibit significant effects in promoting Mtb-specific T cell responses in WT and Nox2-/- mice (S7 Fig). This suggests that the therapeutic potential of AM80 intervention in male Nox2-/- mice is largely contingent on the conversion of neutrophil maturity. To summarize, we specified immature lung neutrophils as the key subpopulation responsible for accelerating TB pathogenesis in male Nox2-/- mice.

10.1371/journal.ppat.1012500.g006 Fig 6 AM80 administration mitigated TB pathogenesis in male Nox2-/- mice by reducing immature neutrophils.

(A) Experimental design for in vivo administration of AM80 in Mtb infected mice. Six-week old male WT and Nox2-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 20 μg of AM80 was orally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 320. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b+Ly6G+ neutrophil, (G) CD11b+Ly6G+CXCR2loCD62Llo immature neutrophil, and CD11b+Ly6G+CXCR2hiCD62Lhi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45+ cells or total neutrophils, and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted twice. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05. **p < 0.01.

G-CSF predominantly regulates the generation of permissive immature pulmonary neutrophils in Mtb infected male Nox2-/- mice

To conclude the study, we aimed to identify specific immunologic factors responsible for the generation of CXCR2loCD62Llo permissive immature pulmonary neutrophils. Considering that pro-inflammatory cytokines upregulated in the lungs of Mtb-infected male Nox2-/- mice, such as IL-1, IL-6, and G-CSF accelerate the production of immature neutrophils through emergency granulopoiesis [55–57], we hypothesized that uncontrolled production of pro-inflammatory cytokines may trigger an explosive generation of immature neutrophils. To identify the causative cytokines, we administered mAbs to neutralize each cytokine in Nox2-/- mice. We confirmed that IFN-γ and IL-17A, key cytokines in anti-TB immunity for both innate and adaptive immune responses, are not associated with the generation of immature neutrophils. In vivo neutralization of IFN-γ or IL-17A increased mycobacterial burden, lung inflammation, pro-inflammatory cytokine levels, and pulmonary influx of immature neutrophils in female Nox2-/- mice (S8 Fig). Furthermore, in vivo neutralization of IL-6, IL-1α, and IL-1β also aggravated immature neutrophil-mediated TB pathogenesis and lung hyperinflammation in Nox2-/- mice, although IL-1 is reported to be crucial for mediating neutrophilic inflammation in NOX2-deficient conditions [12,36,37,44] (S9 Fig). Blockade of IL-1 receptor (IL-1R), which inhibits the function of both IL-1α, and IL-1β, exacerbated TB progression in Nox2-/- mice as well. We implemented two different in vivo blockade models: a three-week IL-1R blockade on female Nox2-/- mice and a two-week IL-1R blockade on male Nox2-/- mice, following the protocols from Olive et al. [12]. Both blockade models resulted in exacerbated TB progression, as evidenced by increased pulmonary bacterial load, lung inflammation, and neutrophil counts in both female and male Nox2-/- mice (S10 Fig). In contrast, in vivo neutralization of G-CSF, the main controller of granulopoiesis, remarkably ameliorated immature neutrophil-mediated TB pathogenesis in male Nox2-/- mice. Starting from one-week post-infection, G-CSF specific mAb was intraperitoneally administered, with a total of nine injections over a three-week period. At four weeks post-infection, the mice were autopsied (Fig 7A). While G-CSF neutralization did not alleviate TB progression in WT mice, it significantly reduced mycobacterial load in the lungs and spleens (Fig 7B) and mitigated lung inflammation (Fig 7C) of male Nox2-/- mice. G-CSF neutralization also downregulated IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels specifically in the lungs of male Nox2-/- mice (Fig 7D), while IL-10. IL-4, IL-5, and TGF-β (Fig 7E) levels did not change. G-CSF neutralization decreased total lung neutrophil counts (Fig 7F) and CXCR2loCD62Llo immature lung neutrophil counts (Fig 7G) in male WT and Nox2-/- mice. The reduction of immature lung neutrophils was particularly pronounced and dramatically reflected in G-CSF neutralized male Nox2-/- mice. This underscores that the excessive G-CSF-mediated accumulation of pathogenic immature neutrophils is a specific event observed in TB susceptible male Nox2-/- mice Ultimately, we concluded that upregulated G-CSF is the immunologic trigger for the generation of permissive immature pulmonary neutrophils, which facilitates TB immunopathogenesis and lung hyperinflammation in male Nox2-/- mice (Fig 8).

10.1371/journal.ppat.1012500.g007 Fig 7 G-CSF neutralization ameliorated TB pathogenesis of male Nox2-/- mice by suppressing generation of immature pulmonary neutrophils.

(A) Experimental design for in vivo neutralization of G-CSF in Mtb infected mice. Six-week old male WT and Nox2-/- mice (n = 6 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 30 μg of anti-G-CSF mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 225. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex. The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b+Ly6G+ neutrophil, (G) CD11b+Ly6G+CXCR2loCD62Llo immature neutrophil, and CD11b+Ly6G+CXCR2hiCD62Lhi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45+ cells or total neutrophils, and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted three times. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05. **p < 0.01.

10.1371/journal.ppat.1012500.g008 Fig 8 The results of the study are summarized in a visual abstract.

CD11bintLy6GintCXCR2loCD62Llo permissive immature pulmonary neutrophils are responsible for lung hyperinflammation and increased mycobacterial load in male Nox2-/- mice. Among the upregulated pro-inflammatory cytokines, G-CSF, rather than IL-1, plays a dominant role in generating immature neutrophils. The control of immature neutrophils through neutrophil depletion, AM80 intervention, and G-CSF neutralization has shown promising results in ameliorating TB immunopathogenesis in male Nox2-/- mice.

Discussion

Our study aimed to identify disease-promoting factors that are responsible for triggering Mtb-induced immunopathogenesis in the absence of phagocyte oxidase. We found that pulmonary infiltration of aberrant immature neutrophils is strongly associated with increased mycobacterial burden, lung hyperinflammation, and the loss of pulmonary lymphocytes in male Nox2-/- mice. We were able to remarkably alleviate TB progression in male Nox2-/- mice by reducing immature neutrophil counts. Overall, we identified G-CSF, rather than other host factors, as the initiator of immature pulmonary neutrophil-mediated TB immunopathogenesis in male Nox2-/- mice.

Progressed from previous studies on the roles of phagocyte oxidases in TB pathogenesis, our study proposed novel advocative findings. First, we have demonstrated that both sex difference and NOX2 deficiency can affect TB susceptibility in mice. In previous studies, gp91phox-deficient mice did not feature a significant increase in mycobacterial load when compared to WT mice [12,29–31]. Similarly, Duox1 or p47phox-deficient mice did not show an increase in lung mycobacterial load around four weeks post-infection [28,58]. Moreover, Olive et al. indicated that there was no variation in TB susceptibility between male and female gp91phox-deficient mice [12,31]. However, we displayed that male Nox2-/- mice exhibited a significantly higher lung mycobacterial load compared to WT mice and female Nox2-/- mice. This could be attributed to the use of the Mtb K strain, recognized for its heightened virulence in comparison to the H37Rv and H37Ra strains [59,60]. Additionally, as we focused in vivo analysis at the four weeks post-infection period, when TB severity in Nox2-/- mice is optimized, we were able to evaluate the exacerbated TB susceptibility of male Nox2-/- mice. This observation of time-point-dependent alterations in TB progression in Nox2-/- mice is particularly significant, as the pulmonary influx of neutrophils occurs before the explosive increase in mycobacterial CFUs. Such findings highlight the crucial role of constant pulmonary neutrophil influx, serving as a source of uncontrolled inflammation and a superior niche for Mtb, consequently driving TB pathogenesis. Our results newly indicate that biological sex is one of the crucial factors that accelerate TB pathogenesis in mice lacking phagocyte NADPH oxidase, which is inherited in an X-linked manner. Additionally, to the best of our knowledge, our study is the first to investigate the populations and functions of pulmonary lymphocytes in Nox2-/- mice during Mtb infection. We revealed that IFN-γ-producing CD4+ T cells were abundant, and antigen-specific Th1 responses were highly maintained in the lungs of male Nox2-/- mice. Hence, we concluded that the TB susceptibility of male Nox2-/- mice is not solely due to the inadequacy of Th1 responses, which contributes to TB susceptibility in various mouse models [61–63]. Lastly, we emphasized G-CSF as the primary inducer of immature neutrophil-mediated TB pathogenesis in male Nox2-/- mice, instead of other cytokines which remain essential for proper defense against Mtb infection. Chao et al. reported that the blockade of IL-1β or IL-1R can alleviate mycobacterial infection in phagocyte oxidase-deficient mice [12,37]. However, our data indicated that both IL-1α and IL-1β are required for proper defense against Mtb. IL-1α and IL-1β act as the upstream controllers of excessive G-CSF production in Nox2-/- mouse model [36,44], but they also play protective roles of in anti-TB immunity, such as activating myeloid cells in general, beyond just neutrophils [64–66]. We suggest that such protective roles of IL-1 signaling are especially crucial in the early phases of mycobacterial infection. By demonstrating that initiating IL-1R blockade at either one week or two weeks post-infection time point exacerbates TB progression in Nox2-/- mice, we highlighted the dominance of G-CSF over IL-1 in neutrophil-mediated TB pathogenesis.

Although Nox2-/- mice are prone to autoimmune diseases such as rheumatoid arthritis [67,68], Mtb-induced lung hyperinflammation in Nox2-/- mice was not primarily associated with impaired Th1 responses or autoreactive T cells. We observed that pulmonary neutrophils are the actual cause of lung hyperminflammation, and the loss of pulmonary B cells is more closely associated with neutrophilic inflammation rather than alterations in T cell responses. Our research may offer deeper insights into TB susceptible animal models that are not associated with altered T cell function. We explicitly characterized immature lung neutrophils and identified them as the primary drivers of TB immunopathology in male Nox2-/- mice. While various studies have emphasized the importance of neutrophils in promoting lung inflammation in Nox2-/- mice undergoing bacterial, parasitic, or fungal challenge [36,45,69–71], their properties were not dissected in detail. We uncovered that Nox2-/- immature pulmonary neutrophils feature CD11bintLy6Gint and CXCR2loCD62Llo phenotypes, altered size and granularity, and increased mycobacterial permissiveness. Still, we need transcriptomic or metabolomic analysis of CD11bintLy6GintCXCR2loCD62Llo immature neutrophils to elucidate the precise mechanisms underlying neutrophilic inflammation and mycobacterial permissiveness. We proposed novel interventions with therapeutic potentials to enable proper control of immature neutrophils. The effectiveness of neutrophil depletion in alleviating TB pathogenesis and Mtb-induced lung hyperinflammation was formerly demonstrated in TB susceptible Nos2-/- and TLR2-/- mice [72,73], as well as in WT mice suffering from chronic TB [74]. We firstly provided the therapeutic potential of neutrophil depletion in alleviating TB pathogenesis specifically in Nox2-/- mice. We also demonstrated the effectiveness of AM80 in alleviating TB pathogenesis. While Yamada et al. illustrated that ATRA intervention mitigated TB progression by increasing pulmonary T cell counts or reducing pulmonary myeloid-derived suppressor cells (MDSCs) [50,51,75–77], AM80 has not been tested on animal models as a therapeutic agent against TB. We highlighted the efficacy of AM80 in controlling TB pathogenesis in a susceptible mouse model, focusing on its role in reducing inflammatory neutrophils rather than altering T cell or macrophage responses. We have newly revealed that AM80 intervention does not alter Mtb-specific T cell responses in Nox2-/- mice. Although AM80 may affect the phagocytic capacities of Nox2-/- macrophages, we have excluded the effects on macrophages because AM80 did not alter the phagocytosis of Nox2-/- BMDMs in our setup studies. Since AM80 shows enhanced potency compared to ATRA and is free from the side effects associated with RAR-γ activation [78,79], AM80 intervention may offer superior outcomes for establishing host-directed TB therapies in susceptible models compared to ATRA. Furthermore, our study newly discovered that G-CSF neutralization can alleviate TB pathogenesis in Nox2-/- mice. These attempts may provide insights into controlling TB progression in susceptible animal models with excessive pulmonary granulocyte influx.

Still, our study requires discussions on the following limitations. AM80 intervention was effective at mitigating TB pathogenesis in WT mice, while G-CSF neutralization did not clearly alter TB progression in this mouse model. This is considered to be originated from two different reasons. Firstly, unlike male Nox2-/- mouse model, WT mice do not show excessive and constant G-CSF production in response to Mtb infection. Therefore, Mtb infected WT mice may have an insufficient amount of circulating G-CSF which should be depleted through G-CSF neutralization. The second reason is that G-CSF and AM80 promotes myeloid cell generation via independent signaling pathways. AM80 induces neutrophil differentiation and maturation through the activation of RAR, while G-CSF is not involved in RAR signaling or C/EBPβ activation, rapidly generating immature neutrophils [52]. Therefore, we conclude that AM80 featured therapeutic capacity in both mouse strains, while G-CSF neutralization specifically showed potency in male Nox2-/- mouse model. While anti-Ly6G mAb injection successfully removed pulmonary neutrophils in WT mice, it failed to fully eliminate the cells in Nox2-/- mice. This outcome is thought to stem from the nature of the intraperitoneal injection of anti-Ly6G mAb (1A8). Boivin et al. noted that intraperitoneal injection of anti-GR1 or Ly6G monoclonal antibodies may not fully eliminate neutrophils, as newly synthesized young neutrophils can replenish the blood or organs even after depletion [80]. Furthermore, anti-Ly6G mAb mediates slow neutrophil depletion through Fc-dependent opsonization and phagocytosis of neutrophils by macrophages. Considering that NOX2 deficiency leads to both G-CSF mediated constant neutrophil production and macrophage malfunction, we conclude that these two factors contributed to the incomplete depletion of pulmonary neutrophils in male Nox2-/- mice. A similar explanation can be given for the persistence pro-inflammatory cytokines in the lungs, even after mAb-mediated cytokine neutralization. This inefficiency is likely due to the nature of intraperitoneal injections, which effectively target circulating cells or cytokines but may not completely reach lung-residing targets. Additionally, since Nox2-/- neutrophils are inflammatory, they are likely the source of the cytokines that were still detected after intraperitoneal depletion. To optimally deplete such targets, a combination of intraperitoneal and intratracheal injections is likely necessary to effectively target and deplete cytokines residing in the lung. The disparity in the effects of IL-1R blockade on Nox2-/- mice also warrants discussion. Despite following protocols from Olive et al. [12], initiating IL-1R blockade from two weeks post-infection exacerbated TB in male Nox2-/- mice. We conclude that differences in the infection model system contributed to this discrepancy. Specifically, our study differed from previous ones in terms of the mycobacterial strain virulence, initial infectious doses, and the ages of the mice used. IL-1R blockade may suppress neutrophil-mediated TB pathogenesis in a less severe infection model, but it was ineffective in our system, which involved a severe infection with a high dose of the highly virulent Mtb K strain.

Despite the abundance of T cell responses, male Nox2-/- mice displayed severe TB progression. Accumulating evidences suggest that disrupted T cell responses during Mtb infection may diminish protection against the infection. For instance, the deficiency of the T cell inhibitory receptor PD-1 leads to a significant enhancement of T cell responses and an increase in the pulmonary mycobacterial burden [81,82]. Given that neutrophil depletion and G-CSF neutralization decreased the pulmonary levels of IFN-γ and IL-17A in male Nox2-/- mice, maintaining the balance between immune activation and suppression might be more crucial for the proper control of TB than the amount of cytokines produced. To gain a deeper understanding of TB pathogenesis, it is crucial to identify immunologic factors that disrupt the proper activation of Th1 responses. The absence of pulmonary B cells in the presence of BAFF, TNF-α, and IL-6, which are B cell activating cytokines, remains a question to be solved. As pulmonary B cells play crucial roles in suppressing neutrophil-mediated lung inflammation through direct interaction [83,84], their absence may contribute to TB susceptibility in Nox2-/- mice. Thus, elucidating the association between the increase in neutrophils and the decrease in B cells may contribute to a better understanding of anti-TB immunity. We investigated the impacts of AM80 intervention at four weeks post-infection. However, considering the broad roles of retinoic acids in modulating macrophage and T cell responses [50,51], understanding the significance of non-neutrophil factors affected by AM80, such as T cell responses, recruited macrophage functions, and alveolar macrophage functions at early stages of Mtb infection, can significantly enhance our understanding of the pathogenesis of TB in susceptible animal models.

Collectively, our study revealed that phagocyte NADPH oxidase deficiency contributes to exacerbated TB immunopathogenesis in a sex-dependent manner. This is attributed to the excessive pulmonary infiltration of immature neutrophils in the presence of sufficient Th1 responses. Therapeutic interventions targeting the generation of G-CSF driven immature pulmonary neutrophils successfully ameliorated TB immunopathogenesis in male Nox2-/- mice. Our study provides a comprehensive understanding of TB pathogenesis and highlights potential therapeutic targets for proper TB control in the absence of phagocyte-specific NADPH oxidase.

Materials and methods

Ethics statements and study approval

All animal experiments followed the regulations set by the Korean Food and Drug Administration. The Ethics Committee and Institutional Animal Care and Use Committee (2019–0174; C57BL/6N, Nox2-/-, 2022–0140; C57BL/6N, Nox2-/-) at Yonsei University Health System (Seoul, Korea) granted approval for each experimental protocol.

Mice

Six- to seven-week-old female and male C57BL/6N mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). Six- to seven-week-old female and male Nox2-/- mice were provided by Dr. Yun Soo Bae (Ewha Womans University, Seoul, South Korea). The mice were housed in a specific pathogen-free (SPF) environment with barriered conditions at the Yonsei University Medical Research Center SPF facility.

Bacterial culture and infection protocols

Purchase of the Mtb K strain, mycobacterial culture, and aerosol infection processes were executed according to the referenced studies [85,86]. The mycobacteria were cultured in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC; Difco Laboratories, MD) and 2 μg/ml of mycobactin J (Allied Monitor, Fayette, MO) for 4 weeks at 37°C. Following cultivation, all bacteria were washed three times with 10 mM phosphate-buffered saline (PBS; pH 7.4). Bacterial cell pellets were collected after centrifugation, and small aliquots were stored at -80°C until use. The Mtb K strain used for in vivo challenges was PDIM positive. YFP vectors for the generation of YFP+ Mtb K strain were kindly gifted by Dr. Christopher Sassetti. In order to deliver bacteria to the lungs of each mouse, all mice were exposed to at least 200 viable mycobacteria in an inhalation chamber for 60 minutes.

In vivo treatments for neutrophil control

From two weeks post-infection, 250 μg/mouse of anti-Ly6G mAb (clone: 1A8, Bio X Cell, West Lebanon, NH, USA), 200 μg/mouse of anti-IL-1R mAb (clone: JAMA-147, Bio X Cell, West Lebanon, NH, USA), 200 μg/mouse of anti-IFN-γ mAb (clone: H22, Bio X Cell, West Lebanon, NH, USA), and 250 μg/mouse of anti-IL-17A mAb (clone: 17F3, Bio X Cell, West Lebanon, NH, USA) mAb were intraperitoneally administered. The antibodies were diluted in PBS and administered three times a week for a total duration of two weeks. From one-week post-infection, 30 μg/mouse of anti-G-CSF mAb (clone: #67604, R&D Systems, Minneapolis, MN, USA), 200 μg/mouse of anti-IL-1R mAb (clone: JAMA-147, Bio X Cell, West Lebanon, NH, USA), 400 μg/mouse of anti-IL-6 mAb (clone: MP5-20F3, Bio X Cell, West Lebanon, NH, USA), 200 μg/mouse of anti-IL-1α mAb (clone: ALF-161, Bio X Cell, West Lebanon, NH, USA), and 200 μg/mouse of anti-IL-1β mAb (clone: B122, Bio X Cell, West Lebanon, NH, USA) were intraperitoneally administered. The antibodies were diluted in PBS and administered three times a week for a total duration of three weeks. Rat IgG2a, rat IgG1, Armenian hamster IgG, and mouse IgG1 isotype control antibodies were purchased from Bio X Cell (West Lebanon, NH, USA). Isotype control antibodies were administered to control mice in correspondence with the original antibodies. From one-week post-infection, 20 μg/mouse of AM80 (Sigma-Aldrich, St. Louis, MO, USA) was orally administered. AM80 was diluted in PBS and administered three times a week for a total duration of three weeks.

Antibodies and flow cytometry

Flow cytometric analysis of immune cells was conducted using the subsequent antibodies. LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, Green Dead Cell Stain Kit, and Aqua Dead Cell Stain Kit were purchased from Molecular Probes (Carlsbad, CA, USA). Brilliant violet (BV) 605-conjugated mAb against Thy1.2 and CD19; Allophycocyanin (APC)-conjugated mAb against CD45R (B220); BV 421-conjugated mAb against CD45; APC-R700-conjugated mAb against Siglec-F; Fluorescein isothiocyanate (FITC)-conjugated mAb against CD95 (FAS); BV 786-conjugated mAb against CXCR2; and V450- conjugated mAb against Ly6G were purchased from BD Bioscience (San Jose, CA, USA). Phycoerythrin (PE)-conjugated mAb against CXCR2, CD64, IFN-γ, and GL7; Peridinin chlorophyll (PerCP)-Cy5.5-conjugated mAb against CD11b; APC-Cy7-conjugated mAb against MHC-II; Alexa fluor 700-conjugated mAb against CD62L; and PE-Dazzle conjugated mAb against CD11c were purchased from Biolegend (San Diego, CA, USA). Surface and intracellular staining processes were executed according to the referenced study [87].

Preparation of single cell suspensions and immune cell analysis

Four weeks post-infection, the mice were euthanized via inhalation of carbon dioxide and then underwent an autopsy. The mouse lungs were perfused before undergoing flow cytometry analysis. Using the methods described in previous studies [61,88], single-cell suspensions were obtained from the entire lung tissue. These cells were then stained with the mAbs mentioned in the previous section and subjected to flow cytometry analysis.

Measurement of cytokines

For the analysis of lung cytokines, lung lysates from the autopsied mice were collected, homogenized, dissolved into PBS, and then stored in a -80°C freezer. For the analysis of T cell cytokines, lung single cell suspensions were stimulated with two different Mtb antigens, PPD and ESAT-6 according to the referenced studies [87,88]. To detect cytokines, sandwich enzyme-linked immunosorbent assay (ELISA) and LEGENDplex kits were utilized. Briefly, mouse G-CSF and IL-1α ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Mouse IFN-γ, IL-1β, IL-17A, and IL-5 ELISA kits were purchased from Invitrogen (San Diego, CA, USA). Mouse IL-10 ELISA kit, LEGENDplex mouse B cell panel (for the detection of TNF-α, IL-4, IL-6, BAFF, and TGF-β), and LEGENDplex mouse inflammation panel (for the detection of IL-1α, IL-1β) were purchased from Biolegend (San Diego, CA, USA).

Quantification of lung inflammation and mycobacterial CFU

The lungs and spleens of Mtb infected mice were harvested four weeks post-infection. The right superior lobes of the lungs were kept in 10% formalin overnight for preservation and later embedded in paraffin. To perform histopathologic analysis, the lungs were sectioned at 4–5 μm and stained with hematoxylin and eosin (H&E). To quantify pulmonary inflammation in each mouse, we employed Adobe Photoshop (Adobe, San Jose, California) and ImageJ (National Institutes of Health, USA) programs, following a previously described reference [89]. The complete lung lesion image was isolated using Adobe Photoshop and saved as a separate file. Another image, identical in size to the original lung image but filled with black, was also saved as a separate file. These images were then opened in ImageJ. By using the ’Split Channels’ function, the image from the green channel was processed to calculate the size of the area showing green positivity, which was originally purple-blue due to H&E staining. The black-colored image was analyzed through the green channel in the same manner to calculate the size of the entire lung lesion. The ’inflamed area (%)’ value, which indicates the proportion of inflamed tissue relative to the entire lung lesion, was computed by dividing the green positivity values of the first image by the green positivity values of the black-colored image. Subsequently, the actual size of the inflamed lesion was calculated by determining the pixel values in Adobe Photoshop. The pixel value corresponding to a single millimeter was calculated based on a 2mm scale bar included in each images. The pixel value for the entire lung lesion was also enumerated, and the value was divided by the square of the pixel value representing one millimeter to quantify the size (mm2) of each lung lesion. By multiplying the inflamed area (%) values by the total lung sizes (mm2) for each sample, we were able to precisely enumerate the exact size of each inflamed area (mm2) (S11 Fig). To enumerate mycobacterial growth, lung and spleen tissues were plated onto Middlebrook 7H10 agar (Becton Dickinson, Franklin Lakes, NJ, USA) after homogenization, following preparation methods from previous studies [61,90]. After a four-week incubation period at 37°C, mycobacterial colonies were counted.

Neutrophil purification and assessment of mycobacterial permissiveness

MACS magnetic cell sorting kit including anti-Ly6G magnetic beads and LS MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to enrich Ly6G+ cells from the lungs of Mtb-infected mice. The manufacturer’s protocols were followed. After separating Ly6G+ lung cells, the cells were treated with Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and plated onto Middlebrook 7H10 agar (Becton Dickinson, Franklin Lakes, NJ, USA). After a three-week incubation period at 37°C, mycobacterial colonies were counted.

Statistical analyses

The results were presented as the mean ± standard deviation (SD). To analyze the significance of differences between two selected groups, One-way ANOVA and Mann-Whitney U test were conducted using GraphPad Prism version 8 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com). The statistical significance was determined using the following definitions: n.s.: not significant, *p < 0.05, and **p < 0.01. Correlation analysis and unpaired t-test were conducted using GraphPad Prism, and the correlation coefficients and p-values were reported for each graph. Flowjo V10 (Flowjo, Ashland, OR, USA) was employed to perform t-distributed stochastic neighbor embedding (t-SNE) analysis.

Supporting information

S1 Fig The gating strategy for immune cell populations and the kinetics time-course data of pulmonary CFUs and neutrophils are provided.

(A) Pulmonary CFUs of female WT and Nox2-/- mice were enumerated in a time-course dependent manner. six-week old female WT and Nox2-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Mycobacterial CFUs in the lungs of Mtb infected mice were calculated at 0, 2, 3, 4, 8, and 12 weeks post-infection. Initial CFU = 373. The experiment was conducted once. (B) Pulmonary neutrophil counts were calculated at 0, 2, 3, 4, 8, and 12 weeks post-infection. The experiment was conducted once. The significance of differences was determined, using the One-way ANOVA and Mann-Whitney U test. n.s., not significant. *p < 0.05. **p < 0.01. (C) The gating strategy for immune cell populations is presented as flow cytometry plots. Firstly, Live cells stained with the LIVE/DEAD Aqua Dead Cell Stain Kit were gated among single cells. CD45+ immune cells were then gated in order to investigate lung immune cell compositions. Among CD45+ immune cells, CD19+CD90.2+ SSC-Alo lymphocytes and CD19-CD90.2-SSC-Ahi myeloid cells were discriminated. Lymphocytes consisted of CD90.2+ T cells, CD19+B220+ B cells, and CD19+B220+GL7+FAS+ germinal center B cells. Myeloid cells consisted of CD11b+Ly6G+ neutrophils, CD11c+Siglec-F+ alveolar macrophages, CD11b+CD64+ macrophages, and CD11c+MHC-II+ dendritic cells.

(TIF)

S2 Fig Pulmonary T cells of male Nox2-/- mice maintained strong responses against mycobacterial antigens.

Live lung cell suspensions were cultured with or without the mycobacterial antigens ESAT-6 and PPD for 8 hours. (A) Lung IFN-γ levels and (B) Lung IL-17A levels after mycobacterial antigen stimulus are presented in bar graphs. (C) IFN-γ positive CD90.2+ CD4+ CD44+ CD62L+ effecter T cell populations are presented in bar graphs and flow cytometry plots. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. **p <0.01.

(TIF)

S3 Fig Alveolar macrophages, macrophages, and dendritic cells were reduced in the lungs of Mtb infected male Nox2-/- mice.

Pulmonary (A) CD11c+Siglec-F+ alveolar macrophages, (B) CD11b+CD64+ macrophages, and (C) CD11c+MHCII+ dendritic cell populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45+ cells and total cell counts are presented in bar graphs. The data are presented as the mean ± SD of six mice in each group. The significance of differences was determined, using the One-way ANOVA test. **p <0.01.

(TIF)

S4 Fig TB severity featured strong correlation to increase of lung neutrophils and decrease of lung B cells.

The percentages of (A) neutrophils, (B) T cells, and (C) B cells among lung CD45+ cells were individually correlated with inflamed lung percentages, inflamed lung area, and lung bacterial loads of each mouse and presented in correlation graphs. Additionally, (D) the ratio of pulmonary B cells to neutrophils (B:N ratio) was also individually correlated with inflamed lung percentages, inflamed lung area, and lung bacterial loads of each mouse and presented in correlation graphs. The significance of differences was determined by unpaired t-test and correlation analysis, featuring r and p values of each correlation.

(TIF)

S5 Fig BM and lung neutrophil profiles of uninfected WT and Nox2-/- mice are illustrated.

Neutrophil counts and percentages from the lungs and bone marrows of uninfected WT and Nox2-/- mice are provided (n = 4 per group). (A) The percentages of neutrophils among lung CD45+ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. (B) Total cell counts and the percentages of CXCR2loCD62Llo immature neutrophils and CXCR2hiCD62Lhi mature neutrophils among total lung neutrophils are presented in bar graphs, along with flow cytometry plots. (C) The percentages of neutrophils among CD45+ bone marrow cells and total cell counts are presented in bar graphs, along with flow cytometry plots. (D) Total cell counts and the percentages of CXCR2loCD62Llo immature neutrophils and CXCR2hiCD62Lhi mature neutrophils among total bone marrow neutrophils are presented in bar graphs, along with flow cytometry plots. The data are presented as the mean ± SD of four mice in each group. The significance of differences was determined using the One-way ANOVA test and Mann-Whitney-U test. n.s., not significant. *p < 0.05. **p < 0.01. significant.

(TIF)

S6 Fig Immature neutrophils were the most permissive cells to mycobacterial infection among phagocytes infected with YFP+ Mtb.

(A) Experimental design for the in vivo enumeration of permissiveness in phagocytes. Male WT and Nox2-/- mice (n = 5 per group) were aerosol infected with YFP-expressing Mtb K strain. At four weeks post-infection, all mice were autopsied, and permissiveness of neutrophils were analysed via flow cytometry (indicated by red arrow). Initial CFU = 742. (B) The percentage of neutrophils among YFP+ cells are presented in bar graphs, along with flow cytometry plots. The number of YFP+ neutrophils and MFI values of YFP in concatenated neutrophils (103 cells) are presented in bar graphs. (C) Total cell counts, percentages among YFP+ neutrophils, and MFI values of YFP (per 103 concatenated cells) in CXCR2loCD62Llo immature neutrophils and CXCR2hiCD62Lhi mature neutrophils are presented in bar graphs, along with flow cytometry plots. Total cell counts, percentages among YFP+ cells, and MFI values of YFP (per 103 concatenated cells) in (D) CD11c+Siglec-F+ alveolar macrophages and (E) CD11b+CD64+ recruited macrophages are presented in bar graphs, along with flow cytometry plots. The experiment was conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined using the One-way ANOVA test and Mann-Whitney-U test. n.s., not significant. *p < 0.05. **p < 0.01.

(TIF)

S7 Fig AM80 treatment did not significantly alter T cells responses against mycobacterial antigens.

Live lung cell suspensions were cultured with or without mycobacterial antigens ESAT-6 and PPD for 8 hours. (A) Lung IFN-γ levels and (B) Lung IL-17A levels after mycobacterial antigen stimulus are presented in bar graphs. (C) IFN-γ positive CD90.2+ CD4+ CD44+ CD62L+ effecter T cell populations are presented in bar graphs and flow cytometry plots. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA test and Mann-Whitney test. n.s., not significant. *p < 0.05. **p < 0.01.

(TIF)

S8 Fig Neutralization of IFN-γ and IL-17A exacerbated TB pathogenesis of female Nox2-/- mice.

(A) Experimental design for in vivo neutralization of IFN-γ and IL-17A in Mtb infected mice. six-week old female Nox2-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from two weeks post-infection, 200 μg of anti-IFN-γ mAb or 250 μg of anti-IL-17A mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 225. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b+Ly6G+ neutrophil, (G) CD11b+Ly6G+CXCR2loCD62Llo immature neutrophil, and CD11b+Ly6G+CXCR2hiCD62Lhi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45+ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05. **p < 0.01.

(TIF)

S9 Fig Neutralization of IL-6, IL-1α, and IL-1β exacerbated TB pathogenesis of male Nox2-/- mice.

(A) Experimental design for in vivo neutralization of IL-6, IL-1α, and IL-1β in Mtb infected mice. six-week old male Nox2-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 400 μg of anti-IL-6 mAb or 200 μg of anti-IL-1α mAb or 200 μg of anti-IL-1β mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and immunological analysis, bacterial counting, and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 330. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) IFN-γ, IL-1α, IL-1β, IL-17A, IL-6, TNF-α, G-CSF, and BAFF levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex (E) IL-10, IL-4, IL-5, and TGF-β levels in Mtb-infected mouse lung lysates were measured by ELISA and LEGENDplex The cytokine levels are presented in bar graphs. Pulmonary (F) CD11b+Ly6G+ neutrophil, (G) CD11b+Ly6G+CXCR2loCD62Llo immature neutrophil, and CD11b+Ly6G+CXCR2hiCD62Lhi mature neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of each immune cell among lung CD45+ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiment was conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the One-way ANOVA test. n.s., not significant. *p < 0.05. **p < 0.01.

(TIF)

S10 Fig Blockade of IL-1R exacerbated TB pathogenesis of Nox2-/- mice.

(A) Experimental design for in vivo blockade of IL-1R in Mtb infected female Nox2-/- mice. six-week old female Nox2-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from one week post-infection, 200 μg of anti-IL-1R mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and bacterial counting and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 300. (B) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (C) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (D) Pulmonary CD11b+Ly6G+ neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of neutrophils among lung CD45+ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. (E) Experimental design for in vivo blockade of IL-1R in Mtb infected male Nox2-/- mice. six-week old male Nox2-/- mice (n = 5 per group) were aerosol infected with Mtb K strain. Starting from two weeks post-infection, 200 μg of anti-IL-1R mAb was intraperitoneally administered to each mouse three times a week (indicated by blue bars). At four weeks post-infection, all mice were autopsied, and bacterial counting and histopathological analysis were conducted (indicated by red arrow). Initial CFU = 342. (F) Mycobacterial CFUs in the lungs and spleens of each group at four weeks post-infection were analyzed by calculating the number of colonies and presented in bar graphs. (G) H&E staining was performed on the superior lobes of the right lung at four weeks post-infection to visualize the gross lung pathology. The inflamed area of the H&E-stained samples was quantified in terms of percentage and square millimeters and presented in bar graphs. (H) Pulmonary CD11b+Ly6G+ neutrophil populations of Mtb-infected mice at four weeks post-infection. The percentages of neutrophils among lung CD45+ cells and total cell counts are presented in bar graphs, along with flow cytometry plots. The experiments were conducted once. The data are presented as the mean ± SD of five mice in each group. The significance of differences was determined, using the Mann-Whitney test. n.s., not significant. *p < 0.05. **p < 0.01.

(TIF)

S11 Fig Quantification method of lung inflammation is briefly described.

(A) As outlined in the Materials and Methods section, the inflamed area of the H&E-stained lung samples were quantified in terms of percentage and square millimetres. The values were calculated using Adobe Photoshop and ImageJ programs. New image files were created to determine the percentage of the inflamed (purple-stained) lesions, and the actual size of both total lung lesions and inflamed lesions was quantified in mm2.

(TIF)

We appreciate the Medical Illustration & Design(MID) team, a member of Medical Research Support Services of Yonsei University College of Medicine, for their excellent support with medical illustration.

10.1371/journal.ppat.1012500.r001
Decision Letter 0
Sassetti Christopher M. Academic Editor
Otto Michael Section Editor
© 2024 Sassetti, Otto
2024
Sassetti, Otto
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
26 Jan 2024

Dear Dr. Shin,

We apologize for the delay in evaluating your manuscript, "Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice".  Given the initial comments we received, we felt that it was particularly important to secure three thorough reviews, and this delayed our decision.   In light of these reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

You will note that the reviewers appreciated the importance of the topic and noted a number of interesting observations.  They also shared several concerns that would require additional experimental work to alleviate.  We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Michael Otto

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Kasturi Haldar

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Editor-in-Chief

PLOS Pathogens

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In this manuscript “Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice,” Choi et al. explore the effects of NOX2 deficiency on Mycobacterium tuberculosis infection. They find that NOX2 deficiency leads to increased lung inflammation and bacterial burdens, which is associated with increased inflammation and immature neutrophilia, all with a greater difference in male mice. They next go on to evaluate the potential mechanistic underpinning for these observations, showing that this susceptibility in the absence of NOX2 is in part dependent on neutrophils, and associated with immature neutrophils and G-CSF, further this can be ameliorated with a drug which stimulates the retinoic acid receptor. Overall, this study highlights a key role for NOX2 in the regulation of pathology during Mycobacterium tuberculosis infection and uses a diverse array of immunologic tools to characterize its effects. Enthusiasm for this manuscript is significantly tempered by three key points: 1) Even in the presence of anti-Ly6G treatment, NOX2 deficiency results in a nearly 1 log increase in CFU (fig 4B, green vs blue bars), suggesting a significant contribution of non-neutrophil factors. The authors do not address this, instead their interpretation is that neutrophils account for all of differences seen in NOX2 deficiency; 2) Related to this, NOX2 deficiency has a significant effect on other phagocytes, including macrophages, which are examined only superficially; 3) At the relatively late timepoint evaluated, there are already significant differences in bacterial burden and it is difficult to discern cause versus effect. This caveat could be mitigated by examining earlier timepoints. Lastly, it is not clear whether any of the experiments were repeated, which is critical to evaluate these findings, especially in light of the difference of these findings with published tuberculosis literature on gp91-/- mice (ref 29 in this publication), which as the authors state could be due to strain differences.

Reviewer #2: In this manuscript by Choi et al the investigators examine differences in role of phagocyte oxidase mediated protection in male and female mice. They find that male Nox2-/- mice have higher bacterial burden following infection with a virulent Strain K, while female mice do not. They find increased immature neutrophil recruitment and lung damage at 4 weeks post-infection that is associated with higher Mtb levels and a hyperinflammatory response. Depleting neutrophils in male mice reversed the increased CFU and hyperinflammation while IL1 blockade did not. In contrast, treatment with AM80 and anti-G-CSF lowered bacterial levels in male Nox2-/- mice and reversed the inflammatory state. The authors conclude that increased G-CSF in male mice specifically results in the recruitment of immature neutrophils that drive inflammation and increased bacterial burden. The paper is generally clear and the experiments are mostly well controlled with a few caveats. Several of the key observations including the role of increased neutrophil recruitment and the role of immature neutrophils in driving disease have previously been described. Furthermore, several of the data presented appear to conflict with other previous reports using various knockouts of distinct subunits of the NADPH phagocyte oxidase, yet the reasons for these discrepancies are not fully understood/examined. Without further experiments or controls this leaves some of the conclusions not fully supported by the data as is.

Reviewer #3: Choi and colleagues in this study have characterized the lung neutrophils phenotype in female and male Mtb infected wild type (WT) and Nox2-KO mice; and showed that increased immature CXCR2lo CD62Llo Ly6G+ lung neutrophils were associated with enhanced TB pathology in male Nox2-kO mice. They performed depletion experiments using antibodies against various cytokines and found that GCSF-driven differentiation of immature neutrophils was responsible for exacerbated TB pathology in male Nox2-KO mice. These kinds of studies are important to dissect the pathologic and protective role of neutrophils in TB and possible in other infectious diseases. What is the phenotype of depleting immature neutrophils in WT mice? Does depleting GCSF in WT mice has an effect on TB pathology? These biological questions and significant technical issues lowered the interest of this reviewer.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Given the large differences in bacterial burdens, it is difficult to know which changes in immune cell number/proportion and increased inflammatory cytokines seen in the male NOX2 deficient mice are reflective of increased bacterial burdens, or responsible for driving the pathology. To gain better insight into the mechanistic determinants for NOX2-driven pathology, the authors could evaluate responses at an earlier timepoint, when there is no (or less) discrepancy in bacterial burdens. Also, why is this data different from the published gp91-/-, as above do the authors think this is solely related to bacterial strain, or potentially could infectious dose also influence these responses?

The methods outlined for determining extent of inflammation are not sufficient to understand what this is measuring. The listed reference also does not have enough detail (overall there is not enough detail in the methods). In the text, authors refer to “necrotic inflammation” whereas the figure axes are labeled “inflamed area,” which are quite different. Also, from thin tissue sections of a region of interest, I’m not sure how useful the absolute area of inflammation is, the percent inflammation controls for the size of the section, which is the most relevant to analyze. Overall, the images aren’t at a sufficient resolution to evaluate in the uploaded tif files. Perhaps the authors could include images from all evaluated animals in a supplemental figure?

The authors propose that immature lung neutrophils are permissive, but never show this specifically on immature neutrophils in the setting of NOX2 deficiency. In an above (or related) experiment this could be shown by sorting and plating immature and mature neutrophils. Otherwise, the authors should make a statement more generally about neutrophils, which they have shown have higher bacterial burdens.

AM80 treatment has a relatively modest effect on the absolute number of neutrophils present. Given the mechanism proposed, is there a difference in the percentage of immature neutrophils? Also, the proportion of neutrophils does not seem to change, which is similar to other cell populations. Are the absolute numbers of all cell populations decreased? If so, it doesn’t seem fair to conclude that immature neutrophils are the key cell population, if many other cell populations changed as well. Given the effects of ATRA treatment in reference 58, were responses of T cells and macrophages evaluated further?

Reviewer #2: Major Comments:

1. Several previous papers cited by the authors show that infection of Phox deficient mice does not result in major changes to CFU over time in contrast to what is shown. The authors suggest this is due to difference strains however, but this is not directly tested. The authors only examine a single time point in their studies (4 weeks post infection) and do not examine the kinetics of bacterial growth over time which were shown to have some variability during infection or the impact on survival long term. The authors use a wild type C57BL6N background which also differs from previous studies. It is unclear from the methods whether the Nox2-/- mouse is also on the C57BL6N or the C57BL6J background. This should be made clearer as a potential confounder.

2. For several of the treatment studies including AM80, G-CSF, IL1a, IL1b, IL6 there are no wild type controls to understand changes in control mice. This is particularly important for AM80 which the authors conclude is driving better neutrophil maturation and control, however, this could also be directly acting on infected macrophages to lower bacterial burden as has been shown previously in human macrophages. Without knowing if AM80 is specifically driving neutrophil mediated control these conclusions are not supported by the current data. The authors also conclude the changes in Male Nox2-/- mice are not due to IL1a or IL1b but a co-treatment is not done even though these cytokines can act redundantly. Furthermore, it is known that IL1 signaling is important for early control of Mtb infection yet depletion beings very early following aerosol infection which again, may be driving changes in macrophage bacterial levels that then amplify differences in neutrophil responses. This is not addressed through experiments.

3. The majority of the statistics seem to use the Mann-Whitney U test which from my understanding is a comparison between two groups and in several experiments there are more than two comparisons being made. A more appropriate test should be used for these experiments to account for multiple comparisons. Furthermore, in the figure legends it is unclear how many replicate experiments were done for each data panel making it difficult to judge the reproducibility of the results.

4. The methods in general are not sufficient for reproducible studies. While previous studies can be cited the manuscript should contain enough information for readers to fully understand the results. For example, how was the Strain K cultured? Is it PDIM positive? For the aerosol infections, was the day 0 lung CFU value calculated for each experiment (if so this should be explicitly written in the figure legends)? Were there differences in these values between male and female mice that may drive downstream phenotypes? These data are important for the readers to understand all the parameters of the experiments.

Reviewer #3: 1. Thomas et al. 2023 have earlier demonstrated that gp91phox-deficient mice displayed enhanced inflammation and lung neutrophil infiltration after Mtb challenge, which were mediated by IL-1. In this context a Fig 1 and Fig 2 of this manuscript is just repeating what was published before. They can be merged into oone figure. Having said that the current manuscript though brings the angle of sex, which is interesting.

2. Immature neutrophils and pathology: Regarding the observation that Nox2-KO mice exhibited higher levels of immature lung neutrophils, inflammation and mycobacterial burden compared to female Nox2-KO mice and WT mice after Mtb challenge. Is this due to the inherent nature of higher immature neutrophils in the lung of mice at the time of infection, or those increased immature neutrophils arrive only upon infection (granulopoisis in bone marrow). Thus, It is important to know the neutrophil phenotype (possibly in lung and bone marrow) in uninfected female / male WT and Nox2-KO mice.

3. In Fig S4 authors showed that “The loss of pulmonary B cells showed a significant negative correlation with neutrophil infiltration”. The negative correlation of neutrophils and B cells was again depicted in neutrophil depletion experiments in Fig. 5. Authors have not provided much explanation on this; it will be good to discuss this aspect in detail and its impact on TB pathogenesis.

4. For immature neutrophils I would suggest including Evrard et al., 2018 Immunity article as reference; This was one of the first seminal paper to characterize different neutrophil subtypes.

5. Authors mentioned that “CXCR2loCD62Llo immature lung neutrophils were phenotypically heterogeneous from CXCR2hiCD62Lhi mature lung neutrophils”. This is not clear. Which figure panel shows this? Was any sub-gating of these immature and mature neutrophils (using other markers) was performed? How the heterogenity was measured / assessed?

6. Fig 5E/6H – Not sure what kind of information tsne plots are adding in this panel. Same goes for other figures where tsne plots have been shown. For example in Fig 6H - I can see many green colour clusters (all are T cells), but what is what; which is CD4, CD8, MAIT, etc etc. If the authors do not have that granular information, avoid including tsne plots. This will be very confusing for the readers.

7. Fig 6 – Does AM80 treatment also ameliorate TB pathology / bacterial load in wild type mice? Why this experiment was performed? Good to make it more clear.

8. Fig S5 – I could not get whether this experiment (depletion of IFNg and IL17A) was performed with male or female mice. In the text its mentioned male Nox2-KO mice however in the Fig S5 legend its mentioned female Nox2-KO mice.

9. Does depleting GCSF in wild type mice also has an effect on lung immature neutrophils, bacillary load and pathology? This is very important to investigate as this will generalize the pathological role of immature neutrophils in TB; In my view this study will then have a more clinical relevance.

10. Figure legend – please include how many times each experiment was repeated.

11. Statistical analysis – I am surprised to see that authors have used Mann-Whitney test, though they have more than 2 groups in maximum experimental settings. This is not correct. Whenever there are more than 2 groups ANOVA should be used.

12. Depletion experiments - I could not find the info (neither in method section nor in figure legend) on the control Ab used in all depletion studies. Were the control mice receive any antibody?

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The sex differences in NOX2 deficiency are very interesting. However, it may appear that there could be a difference in female mice as well, potentially with lung CFU and neutrophil number. It is not clear from the plots or text if a statistical test has been applied to these differences, which should be addressed on the figures and in the text as appropriate. Related to this: the plots for CFU and inflamed area should include points for individual mice, as there are in every other figure panel.

Is the same clone of antibody used to deplete neutrophils as it is to detect neutrophils in figures 4 and 5? If so, it is important to see a method of neutrophil quantification that does not rely on Ly6G. This may be able to be accomplished with existing flow markers. As a minor note in this figure, it is a circular argument to say that neutrophil depletion restores the balance between neutrophils and lymphocytes. What does it do to the absolute numbers of lymphocytes?

Would the authors address why there is so much IFNg, IL1, IL6, and IL17 detected in the setting of the respective cytokine depletion experiments? Was there another method implemented to show that neutralization was effective?

G-CSF is a nice way addition to specifically target neutrophils. It can have multiple effects in addition to granulopoiesis, including on mature neutrophil function and migration into tissues. Evaluating these mechanisms may be outside of the scope of this study, however the authors should address that it might not be as simple as they propose. Also to the above point, did the authors perfuse lungs to ensure that most cells evaluated were within the lung parenchyma? It does not appear that intravascular antibody labeling was completed.

Displaying cell proportions as pie charts makes is impossible to evaluate statistical relationships. Please include statistical measurements for key comparisons,

When the labels of the graphs are at the bottom require a lot of scrolling to evaluate all panels. Please include labels under each graph.

The key for supplemental figure S6 is missing the anti-IL6 only group.

Reviewer #2: (No Response)

Reviewer #3: 1. Antibodies – Please provide the clone information.

2. This article needs an English editing.

3. Enumerating CFU in isolated neutrophils – Did the cells were counted after MACS sorting?

4. References – Some of the references are not needed can be removed, for example 47 and 49. There are other examples in the text.

5. The way four groups are depicted in Fig 3B is very confusing. Please relabel them.

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Reviewer #2: No

Reviewer #3: No

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10.1371/journal.ppat.1012500.r002
Author response to Decision Letter 0
Submission Version1
31 Mar 2024

Attachment Submitted filename: Choi ES et al._response to reviewers.docx

10.1371/journal.ppat.1012500.r003
Decision Letter 1
Sassetti Christopher M. Academic Editor
Otto Michael Section Editor
© 2024 Sassetti, Otto
2024
Sassetti, Otto
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
13 May 2024

Dear Dr. Shin,

Thank you very much for submitting your manuscript "Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The reviewers appreciated your revisions and additional experimental data, but note a number of points that remain to be addressed.  While we always endeavor to avoid multiple rounds of review/revision, these remaining issues are significant and must be addressed before publication.  Specifically, reviewers 1 and 2 note several issues to address by revising the text and/or providing additional flow cytometry data.  We recognize that these revisions may be significant, but trust that this constructive will produce a more impactful manuscript.  

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael Otto

Section Editor

PLOS Pathogens

Michael Otto

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Choi et al. have done a nice job generating and providing additional data, and making progress in addressing the reviewers’ comments in the following areas. First, the analysis of the permissiveness of immature neutrophils using a fluorescent Mtb strain is valuable and supports the authors’ conclusions. Second, showing the effects of G-CSF and AM80 in WT mice, and other necessary comparisons such as analysis of uninfected mice is a very welcome addition. Lastly, the figure clarity and readability has improved.

However, this reviewer still has substantial concerns about many of the conclusions reached by the authors in this manuscript. The article does highlight a major role for neutrophil-driven pathology during Mtb infection in the setting of NOX2 deficiency. However, it is unlikely that this is the only process affected by NOX2 deficiency, and non-neutrophil alternative explanations are only superficially addressed / dismissed. Additionally, the AM80 treatment results in WT mice, together with other data presented in the paper, call into question the interpretation that this treatment acts primarily through neutrophils. Lastly, the fact that findings were observed at high doses with a highly virulent strain, when others have found different results with different strains, could either suggest interesting biology and/or limit the broad applicability of these findings. Given this, the article as written needs some reframing and significant revision of the conclusions drawn from these studies.

Reviewer #2: In this revised manuscript by Choi et al, the authors examine an increase in CFU in male Nox2 mice at 4 weeks post infection and if blocking G-CSF can prevent the neutropenia seen in these animals. The revised manuscript aims to address reviewer concerns, and some of the data are informative, including the YFP-Mtb and the AM80 BMDM experiments and the topically is of great general interest and finding that G-CSF blockade may help block deleterious neutrophil influx is important. However, some of the data provided undercut key conclusions the authors are making in the discussion (and figure legends). These include conclusions around the antimicrobial role of Nox2 in mice and the durability of this CFU difference versus the inflammatory difference. These issues ultimately limit the scope of the paper and limit several of the key conclusions.

Reviewer #3: In the revised version Choi and colleagues have performed additional experiments and re-analysed their data (with ANOVA) to support their conclusion and respond to not only my comments (example: adding new data on bone marrow and lung neutrophils profiling in uninfected KO and WT mice, AMD treatment of infected WT mice), but other reviewer comments as well. Together this new data has improved the manuscript. I am particularly excited to see the data of an in vivo experiment (Fig S5) where authors have infected WT and KO mice by YFP+ Mtb and analysed the uptake of the florescent bacteria by mature and immature neutrophils. They showed (by looking at YFP MFI) the increased permissiveness of immature neutrophils compared to mature one.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: This reviewer appreciates the inclusion of the experiment using fluorescently labeled bacteria to demonstrate the increased permissiveness in immature neutrophils. Given the caveats introduced by the differences in bacterial burden at this timepoint, additional analysis is required to support the claim that immature neutrophils are more permissive in the setting of NOX2 deficiency (or if there is increased MFI simply because the bacterial burden in the lung is increased). Looking at additional myeloid populations, such as alveolar macrophages and recruited macrophages, and comparing the fold changes in percent infected cells and YFP MFI in these populations as compared to immature neutrophils in WT and KO mice would give insight into these differences. Additionally, this may give some insight into the roles of macrophages during Mtb infection in NOX2 deficiency.

The time course is valuable information to have. However, this time course is in female mice and as the authors state, a major advance of the paper is sex differences in NOX2 deficiency, and the major conclusions of the paper are in male mice. This reviewer realizes that the likely reason this data was shown is that the experiment was already done, but I wonder if the results would be the same in male mice. This is not necessarily required to do, but a mention of this time course being done in female mice and the rationale should be called out in the text, not in a supplemental figure legend where it is hard to find. On a related note, from this time course (albeit in female mice) it appears that week 3 is a very interesting timepoint, since differences in neutrophils are already apparent at a time there is similar CFU between KO and WT mice. This would appear to support the authors’ main conclusion of the paper.

How do the authors reconcile the new results shown of AM80 treatment in WT mice, which had an effect, and anti-G-CSF in WT mice, which did not have an effect? The model proposed is that both primarily mediate their effects through decreasing the numbers of immature neutrophils. If this were true, wouldn’t similar effects from both treatments be expected in WT mice? Was macrophage activation in vivo evaluated during treatment, or just in vitro data? Lastly, the conclusion that T cell responses weren’t affected has the caveat that there are significant differences in bacterial burden at the timepoint evaluated. Couldn’t AM80 have induced an earlier/stronger Th1 response, which is diminished at week 4 due to decreased CFU? Given these caveats, and the strength of the G-CSF results, this reviewer wonders whether this story would be stronger without the inclusion of this experiment.

Reviewer #2: 1. Central to the conclusions of this paper are the CFU differences in male mice shown generally at 4 weeks. At the request of the reviewers a timecourse is now shown that shows what has previously been observed, that the small CFU differences are only seen significantly at 4 weeks post-infection. While the authors say 3 weeks is an increase there are no statistics included and the differences look minor. Importantly, while the authors say the difference in neutrophils goes back to normal, these actually seem to persist with no differences in CFU. In line with other previous studies using Nox2 mice. With these data in the revision, the only supported conclusion is that male Nox2 mice have a transiently higher CFU at 4 weeks, but the paper is written to make these differences in CFU a larger conclusion which is not robustly supported by the data shown.

2. The inhibition of IL1R included in the rebuttal is also confusing. IL1 has a key role in early antimicrobial responses, so blocking IL1R as early as a week following infection is going to have severe effects, as seen by the authors. It is interesting that the male mice succumbed to disease and couldn’t be used in this experiment, but using females complicates comparisons across experiments. Thus, with the way this experiment was done I’m not convinced the data can really discern what the role of IL1a or IL1b is in this model.

Reviewer #3: None

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The authors should entertain additional possibilities (such as other cell types playing a role) as to why the neutrophil depletion did not fully mitigate pathology and decrease bacterial burdens to WT mice. Also, why was the IA8 depletion not efficacious in this setting, when others have depleted pulmonary neutrophils during Mtb infection with much greater effect (DOIs: 10.1038/nmicrobiol.2017.72; 10.1172/JCI130546)?

The methods describing quantification of lung inflammation are still lacking in specifics. How was inflammation determined? Was it with a specific plugin in ImageJ? By eye? If the latter, was the scorer blinded? This information is crucial to ensure reproducibility.

In instances where there is a significant difference between groups, they should not be concluded to be the same. Specific figures to note are: Fig 1E (IL-10 is significantly upregulated in male KO vs WT); Fig S5A-B (the number of total, immature, and mature neutrophils are slightly increased in naïve NOX2 deficient male mice as compared to WT, please change corresponding test); Fig 6D (TNF significantly decreased with AM80 in WT, perhaps choose another word such as “substantially” here?).

Reviewer #2: (No Response)

Reviewer #3: N/A

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Amit Singhal

Figure Files:

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10.1371/journal.ppat.1012500.r004
Author response to Decision Letter 1
Submission Version2
8 Jul 2024

Attachment Submitted filename: Choi ES et al._response to reviewers_Second Revision.docx

10.1371/journal.ppat.1012500.r005
Decision Letter 2
Sassetti Christopher M. Academic Editor
Otto Michael Section Editor
© 2024 Sassetti, Otto
2024
Sassetti, Otto
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
12 Aug 2024

Dear Dr. Shin,

We are pleased to inform you that your manuscript 'Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Christopher M. Sassetti

Academic Editor

PLOS Pathogens

Michael Otto

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This study is conceptually exciting, and of interest to the field. I appreciate the detailed responses and careful considerations of the many comments, questions, and criticisms posed by reviewers. I have three relatively minor lingering issues for the authors to address:

1. Language about the role of additional cell types has been included about the AM80 study, yet the title for Figure 6 still states "AM80 administration mitigated TB pathogenesis in male Nox2-/- mice by reducing immature neutrophils." This mechanism of action of AM80 is not supported by the data shown here, and the language should be modified to reflect that this relationship is an association.

2. The image analysis methods are a little hard to follow. Could the authors show example images of processing steps in a supplemental figure?

3. On lines 204 and 206, the authors state there is a discrepancy in CFU values, when Figure 1E shows no significant difference. This language should be modified to reflect that there is no significant difference.

Reviewer #2: The authors have done a good job at addressing my concerns and highlighting possible caveats to their results in the discussion. This is an interesting story that will be appreciated by the TB community.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

10.1371/journal.ppat.1012500.r006
Acceptance letter
Sassetti Christopher M. Academic Editor
Otto Michael Section Editor
© 2024 Sassetti, Otto
2024
Sassetti, Otto
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
19 Aug 2024

Dear Dr. Shin,

We are delighted to inform you that your manuscript, "Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064
==== Refs
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