ARTICLE https://doi.org/10.1038/s41467-020-19412-6 OPEN Type I IFN exacerbates disease in tuberculosis- susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis Lúcia Moreira-Teixeira 1 ✉, Philippa J. Stimpson1, Evangelos Stavropoulos1, Sabelo Hadebe 1, Probir Chakravarty2, Marianna Ioannou 3, Iker Valle Aramburu3, Eleanor Herbert 4,5, Simon L. Priestnall 4,5, Alejandro Suarez-Bonnet 4,5, Jeremy Sousa6,7,8, Kaori L. Fonseca6,7,8,9, Qian Wang3, 1234567890():,; Sergo Vashakidze10, Paula Rodríguez-Martínez11, Cristina Vilaplana 12, Margarida Saraiva 6,7,14, Venizelos Papayannopoulos3,14 & Anne O’Garra 1,13,14 Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infec- tion upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are pre- sent in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine- based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection. 1 Laboratory of Immunoregulation and Infection, The Francis Crick Institute, London NW1 1AT, UK. 2 Bioinformatics and Biostatistics Team, The Francis Crick Institute, London NW1 1AT, UK. 3 Laboratory of Antimicrobial Defence, The Francis Crick Institute, London NW1 1AT, UK. 4 Department of Pathobiology and Population Sciences, Royal Veterinary College, Hatfield AL9 7TA, UK. 5 Experimental Histopathology Team, The Francis Crick Institute, London NW1 1AT, UK. 6 i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. 7 IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal. 8 ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal. 9 Programa de Pós-Graduação Ciência para o Desenvolvimento (PGCD), Instituto Gulbenkian de Ciência (IGC), Oeiras, Portugal. 10 National Center for Tuberculosis and Lung Diseases (NCTLD), 50, Maruashvili Str, 0101 Tbilisi, Georgia. 11 Pathology Department, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona (UAB), Crtra. Del Canyet, s/n, 08916 Badalona, Catalonia, Spain. 12 Experimental Tuberculosis Unit (UTE), Fundació Institut Germans Trias i Pujol (IGTP), Universitat Autònoma de Barcelona (UAB), CIBER Enfermedades Respiratorias. Edifici Laboratoris de Recerca. Can Ruti Campus, Crtra. de Can Ruti, Camí de les Escoles, s/n, 08916 Badalona, Catalonia, Spain. 13 National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London W2 1PG, UK. 14 These authors jointly supervised this work: Margarida Saraiva, Venizelos Papayannopoulos, Anne O’Garra. ✉email: luciamoreirateixeira@gmail.com NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 T uberculosis (TB) remains a major health problem world- Mechanistically, our findings reveal that increased type I IFN sig- wide with 1.2 million reported deaths in 20181. Despite its nalling induces pulmonary NETosis and promotes mycobacterial clinical significance, there are still significant gaps in our growth in the absence of GM-CSF. We show that type I IFN- understanding of the mechanisms underlying protection or induced NETosis is also associated with disease severity in TB- pathogenesis following Mycobacterium tuberculosis infection that susceptible C3HeB/FeJ mice, supporting a more generalised role of could be exploited to improve TB treatment and thus reduce type I IFN-induced NETosis in TB pathogenesis over and above disease severity and mortality2–4. GM-CSF blockade. Importantly, we demonstrate for the first time Transcriptional studies of human TB have been instrumental that NETs are present in necrotic lung lesions from patients with in unveiling new mediators of the immune response to M. nonresolving pulmonary TB, supporting the clinical revelance of tuberculosis infection5. Blood transcriptional signature of active NETs in TB pathogenesis. TB revealed a dominance of type I IFN-inducible genes6 (and reviewed in7), which correlated with the extent of radiographic Results lung disease and was diminished upon successful treatment6,8,9. GM-CSF blockade increases host susceptibility to TB. To These studies highlighted an important role for type I IFN in address the mechanisms of pathogenesis resulting from M. driving TB pathogenesis. Consistently, studies in mouse models tuberculosis infection in the absence of GM-CSF signalling, of TB demonstrated that induction of high and sustained levels of without the confounding factors associated with the lack of GM- type I IFN by different mechanisms result in increased host CSF during host development33,34, we used monoclonal anti- susceptibility to M. tuberculosis infection7,10–15. Transcriptomic bodies (mAbs) directed against GM-CSF (αGM-CSF) to block analyses in human and mouse studies have also revealed an over- GM-CSF function during the course of infection. Mice were abundance of type I IFN and granulocyte-associated genes cor- treated with αGM-CSF or isotype control (labelled as Ctrl Ab) relating with lung disease severity in both active TB patients and mAbs the day prior to aerosol infection with the M. tuberculosis TB-susceptible mice16. In humans, neutrophils are the pre- clinical isolate, HN878, and then twice weekly for 3 weeks after dominant cell type infected with M. tuberculosis in the airways infection (Fig. 1a). GM-CSF blockade during M. tuberculosis and are detected within inflammatory lung lesions of patients infection resulted in enhanced host susceptibility, similar to the with active disease17,18. In mice, neutrophils are abundant in the findings reported for GM-CSF-deficient mice27,31, with sig- lung lesions of M. tuberculosis-infected susceptible mice and have nificant weight loss (Fig. 1b), increased lung mycobacterial loads also been reported to contribute to TB progression promoting (Fig. 1c) and exacerbated lung pathology (Fig. 1d and Supple- both tissue damage and bacterial replication18–20. However, the mentary Data 1), when compared to infected Ctrl Ab treated mechanisms of neutrophil-mediated pathology in TB-susceptible mice. Scoring of histological sections showed an increase in lung mice and patients with active TB remain unclear. lesion burden and severity in infected mice treated with αGM- Macrophages and CD4+ T cells, and several cytokines such as CSF mAbs (Fig. 1e and Supplementary Data 1). This was char- IL-12, IFN-γ, TNF and IL-1 have been shown to play a major acterized by enhanced necrosis and intra-alveolar necrotic debris role in protection against M. tuberculosis infection2–4,13,21–24. (Fig. 1f and Supplementary Data 1), with greater numbers of acid- More recently, GM-CSF has also been increasingly recognised as fast bacilli in the lungs of M. tuberculosis-infected mice treated a key mediator for TB resistance25–31, and is in the pipeline for with αGM-CSF mAbs as compared to infected Ctrl Ab treated host-directed therapy as an adjuvant treatment of TB32. The mice (Fig. 1g). presence of GM-CSF specific autoantibodies that block GM-CSF Since increased susceptibility to M. tuberculosis infection in GM- function have been linked to cryptococcal disease and some cases CSF-deficient mice has been associated with an impaired T cell- of pulmonary TB28. In addition, GM-CSF treatment of mouse mediated IFN-γ-response at the site of infection27, we evaluated and human macrophages restricts intracellular mycobacterial the effects of αGM-CSF treatment on T cells in the lungs of growth in vitro25,26,29,30 and mice lacking GM-CSF are highly infected mice. We observed a slight but not significant decrease in susceptible to M. tuberculosis infection27,31. Impaired innate the percentage of CD4+ and CD8+ T cells in the lungs of infected immune responses and decreased recruitment of protective IFN- mice treated with αGM-CSF compared to Ctrl mAbs, with no γ producing CD4+ T cells into the site of infection have been effect on T cell numbers (Supplementary Fig. 1a, b). Moreover, proposed as mechanisms for the increased susceptibility fol- similar percentages and numbers of IFN-γ-producing CD4+ and lowing M. tuberculosis infection in mice deficient in GM- CD8+ T cells were detected ex vivo from both αGM-CSF and Ctrl CSF27,31. However, these studies are difficult to interpret owing Ab treated mice (Supplementary Fig. 1a, b), demonstrating that to the developmental defects in alveolar macrophages and blockade of GM-CSF did not impact IFN-γ responses in infected abnormalities in surfactant recycling in mice lacking GM-CSF mice. Blockade of GM-CSF during M. tuberculosis infection also from birth, leading to the development of a lung disease that resulted in increased lung mycobacterial loads in IFN-γ receptor- resembles human pulmonary alveolar proteinosis (PAP)33,34. deficient (IFNγR−/−) mice (Supplementary Fig. 1c) and in WT Therefore, the mechanisms underlying the pathology resulting mice co-treated with anti-IFN-γ mAbs (Supplementary Fig. 1d), from an absence of GM-CSF signalling during M. tuberculosis compared to their respective infected Ctrl Ab treated mice. Our infection remain poorly understood. findings thus reveal an additional in vivo role for GM-CSF in host To further investigate the mechanisms underlying the pathology resistance to M. tuberculosis infection that is independent of IFN-γ. and disease exacerbation resulting from an absence of GM-CSF signalling, without the confounding developmental lung pathologies resulting from gene deletion, here we instead administer blocking GM-CSF controls blood transcriptional TB signatures. To antibodies against GM-CSF during M. tuberculosis infection. We investigate pathways underlying the pathogenesis resulting upon show that increased expression of genes associated with neutrophil M. tuberculosis infection in the absence of GM-CSF signalling, we recruitment and activation, as well as type I IFN-inducible genes, in performed RNA-Seq of whole blood from uninfected and HN878- blood and lungs of M. tuberculosis-infected mice in the absence of infected mice treated with αGM-CSF or Ctrl mAbs. Principal GM-CSF signalling, accompanies enhanced mycobacterial growth component (PC) analysis depicted distinct global transcriptional and lung pathology. Disease exacerbation driven by GM-CSF signatures in blood samples from the different groups (Supple- blockade is neutrophil-dependent and associates with exacerbated mentary Fig. 2a), with PC1 explaining ~73% of the variance and neutrophil extracellular trap (NET) formation at the site of infection. segregating the uninfected from HN878-infected samples. On the 2 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 ARTICLE a b Body weight c CFU HN878 120 109 Day 0 Ctrl Ab αGM-CSF ** Days p.i. 110 % of initial weight Viable bacteria 0.3 mg αGM-CSF or 108 Day Day 100 isotype control (Ctrl Ab) **** –1 21 C57Bl/6 i.p. twice weekly WT 90 Harvest 107 1 mg αGM-CSF or isotype control (Ctrl Ab) i.p. 80 70 106 0 3 6 9 12 15 18 21 Ab SF rl -C Days post infection Ct M αG d Ctrl Ab αGM-CSF g Ctrl Ab αGM-CSF e f Intra-alveolar Lesion burden Necrosis necrotic debris 5 *** 4 *** 4 *** 4 3 Relative score Relative score 3 Relative score 3 2 2 2 1 1 1 0 0 0 Ab SF Ab SF Ab SF rl -C rl -C rl -C Ct M Ct M Ct M αG αG αG other hand, PC2 segregates the infected samples according to Data 2 and Supplementary Fig. 2c, left panel). While we observed treatment (αGM-CSF versus Ctrl Ab, ~13% of the variance negligible transcriptional changes due to αGM-CSF treatment in explained) (Supplementary Fig. 2a, b). We identified 7010 genes the blood from uninfected mice (Supplementary Fig. 2c, middle differently expressed in the blood of HN878-infected Ctrl Ab mice panel), GM-CSF blockade during infection triggered major tran- compared to uninfected Ctrl Ab treated mice (Supplementary scriptional changes in the blood, with 4479 differently expressed NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 Fig. 1 GM-CSF controls lung bacterial growth and necrotic inflammation. a Schematic of experimental design for GM-CSF blockade during M. tuberculosis infection: intraperitoneal (i.p.) injection of 1 mg anti-GM-CSF (αGM-CSF; open circles) or isotype control (labelled as Ctrl Ab; closed circles) monoclonal antibodies (mAbs) the day prior to aerosol infection with HN878 strain, followed by i.p. injections of 0.3 mg of αGM-CSF or Ctrl mAbs, twice weekly for 3 weeks. b Change in body weight at various days post-infection (n = 8 mice/group; mean ± SD). ***P < 0.0001. c Viable bacterial loads determined in the lungs at day 21 post-infection. **P = 0.0079. d Representative hematoxylin and eosin (H&E) staining of infected lung sections (n = 3 mice/group). Scale bars from top to bottom: 1 mm; 200 µm; 50 µm, 10 µm. e Relative lung lesion burden from H&E stained sections (Supplementary Data 1). 0 = no lesions, 1 = focal lesion, 2 = multiple focal lesions, 3 = one or more focal severe lesions, 4 = multiple focal lesions that are extensive and coalesce, 5 = extensive lesions that occupy the majority of the lung lobe. ***P < 0.0001. f Relative necrosis (left) and intra-alveolar necrotic debris (right) scores from H&E stained lung sections (Supplementary Data 1). 0 = not present, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. ***P < 0.0001. g Representative Ziehl–Neelsen staining of infected lung sections (n = 3 mice/group). Scale bars, 20 µm. Data representative (b–d, g) or pooled (e, f) of five biological experiments. Represented is the mean ± SD; each dot represents an individual mouse: n = 5 mice/group (c) or n = 15 mice/group (e, f). Source data are provided as a Source Data file. Statistical analysis was performed using two-way ANOVA (b) or two-tailed Mann–Whitney test (c, e, f). genes detected between αGM-CSF versus Ctrl Ab treated infected GM-CSF controls lung transcriptional TB signatures. To mice (Supplementary Data 2 and Supplementary Fig. 2c, right determine the transcriptional response at the site of infection, RNA- panel). seq data were obtained from whole lungs of the same mice used for To gain insight into the changes in gene expression caused by the blood data from Fig. 2a. Sample distribution of lung RNA-Seq GM-CSF blockade during M. tuberculosis infection, we tested our data PC analysis highly resembled blood RNA-Seq data, with the previously reported blood modular TB signature, which distin- biggest transcriptional variance explained by the infection (PC1; guishes TB patients from healthy controls35 and is recapitulated in Supplementary Fig. 4a, b) and with 11,532 genes differentially TB-susceptible strains of mice16, on our blood RNA-Seq data from expressed between the lung samples from HN878-infected Ctrl Ab infected and uninfected αGM-CSF versus Ctrl Ab treated mice treated mice versus the lung samples from uninfected Ctrl Ab (Supplementary Data 3 and Fig. 2a). We detected a weak modular treated mice (Supplementary Data 4 and Supplementary Fig. 4c, left signature in the blood from infected mice (Fig. 2a), qualitatively panel). Although with a smaller explained variance (~3%), PC2 similar to the human TB signature35. Interestingly, the blood clearly segregated the infected samples according to αGM-CSF or modular immune signature observed in αGM-CSF treated infected Ctrl Ab treatment (Supplementary Fig. 4a, b). GM-CSF blockade mice was more enriched than in Ctrl Ab treated infected mice and triggered transcriptional changes in the lungs from both uninfected better resembled the blood signature of active human TB disease and infected mice (Supplementary Data 4 and Supplementary (Fig. 2a). Myeloid and granulocyte-associated modules HB3, HB5, Fig. 4c, middle and right panel). In uninfected mice, we detected HB8, HB14 and HB23 previously described as over-abundant in 3 significantly upregulated and 327 downregulated genes between the blood from TB patients35 and mouse models of increased αGM-CSF versus Ctrl Ab groups (Supplementary Data 4 and susceptibility to TB16, showed increased over-abundance in the Supplementary Fig. 4c, middle panel). Among the genes down- blood from infected mice treated with αGM-CSF (Fig. 2a), shown regulated by GM-CSF blockade at steady-state, we identified genes quantitatively by Eigengene expression (Fig. 2b). This is in keeping associated with alveolar macrophages37 (Supplementary Data 4 and with cellular deconvolution analysis36 showing an increase in the Supplementary Fig. 4c, middle panel), in keeping with a key role for percentage of myeloid and neutrophil fractions in the blood GM-CSF in the maintenance of these cells38,39. During infection, from infected αGM-CSF compared to infected Ctrl Ab treated GM-CSF blockade affected the lung expression of 2119 genes, with mice (Supplementary Fig. 3a), which was reflected by increased alveolar macrophage-associated genes among the top down- expression of neutrophil, monocyte and macrophage-associated regulated and neutrophil-related genes among the top upregulated genes induced by GM-CSF blockade during infection (Supple- genes (Supplementary Data 4 and Supplementary Fig. 4c, right mentary Data 2 and Supplementary Fig. 3b–d), and was similar to panel). our previous report of the blood signature in human TB and TB- To identify GM-CSF driven changes in co-expressed sets of susceptible mice16. Similarly, the two IFN-associated modules, genes across the infected lungs, we next tested the previously HB12 and HB23, also previously reported as over-abundant in the published mouse lung disease modular signature36 on our lung TB blood from patients with active TB disease35 and from mouse RNA-Seq data (Supplementary Data 5 and Fig. 2c). We observed a models of increased TB susceptibility16, were over-abundant in the similar modular signature in the lungs of HN878-infected mice blood from infected Ctrl Ab treated mice and further increased in treated with Ctrl Ab (left) or αGM-CSF (right) mAbs, but with the blood from αGM-CSF treated mice (Fig. 2a, b). This is distinct pattern in the abundance of certain modules (Fig. 2c). The consistent with the presence of IFN-inducible genes among the Type I IFN/Ifit/Oas module (L5) was over-abundant in the infected genes upregulated by GM-CSF blockade during infection (Supple- lungs but slightly more enriched in infected mice treated with mentary Data 2 and Supplementary Fig. 2c, cluster II). In contrast, αGM-CSF compared to Ctrl Ab (Fig. 2c, d). This is consistent with the cell cycle module (HB13) was strongly over-abundant upon an increased expression of IFN-inducible genes resulting from infection but less so in the blood from αGM-CSF treated mice GM-CSF blockade during infection (Supplementary Data 4 and (Fig. 2a). Similarly to the human TB blood signature and that of Supplementary Fig. 4c, cluster III). Modules associated with innate TB-susceptible mice16, the T cell and B cell modules (HB2, HB4, inflammation (L10–L13) were also over-abundant in infected lungs HB15 and HB21) were slightly less abundant in the blood from but further over-abundance was observed in lungs from infected infected mice treated with αGM-CSF mAbs compared to infected mice treated with αGM-CSF mAbs compared to infected Ctrl Ab Ctrl Ab treated mice (Supplementary Data 3 and Fig. 2a). In treated mice, also shown quantitatively by Eigengene expression keeping with this, cellular deconvolution analysis36 showed a (Fig. 2c, d). The elevated expression of inflammatory genes is decrease in the percentage of B cells and CD4+ T cell fractions in consistent with the exacerbated lung pathology observed in the blood from infected αGM-CSF compared to infected Ctrl Ab HN878-infected αGM-CSF treated mice compared to infected Ctrl treated mice (Supplementary Fig. 3a), which was also reflected Ab treated mice (Fig. 1d–f and Supplementary Data 1). In contrast, by decreased expression of key T and B cell-specific genes the modules Ifng/Gbp/antigen presentation (L7) and Cytotoxic/ (Supplementary Data 2 and Supplementary Fig. 2c, cluster V). T cells/ILC/Tbx21/Eomes/B cells (L35), which have been associated 4 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 ARTICLE a Human TB blood modules c Mouse lung modules αGM-CSF αGM-CSF Ctrl Ab Ctrl Ab (Singhania et al., Nat. Commun. 2018) (Singhania et al., Nat. Commun. 2019) Tested on blood from HN878 infected mice Tested on lung from HN878 infected mice Module name Module name DNA binding HB1 Miscellaneous L1 T cells HB2 Inflammation/metabolism L2 Inflammasome/granulocyte HB3 Leukocytes/myeloid/signaling/Il10 L3 T cells HB4 Protein translation/Ubiq L4 Innate/hemopoietic mediators HB5 Type I IFN/Ifit/Oas L5 Ubiquitination HB6 Cell cycle/stem cells/chemokine receptors L6 Cell proliferation/metabolism HB7 Ifng/Gbp/antigen presentation L7 Innate immunity/PRR/C'/granulocyte HB8 Glycolysis L8 Coagulation HB9 Ribosomal functions/other L9 Th2/eosinophils/mast cells HB10 Myeloid/granulocyte function L10 Immunoglobulins HB11 IL-17 pathway/granulocytes L11 Inflammation/IL-1 signaling/myeloid cells L12 Interferon/PRR HB12 Myeloid cells/Il1b/Tnf L13 Cell cycle HB13 Myeloid cells and signaling L14 Myeloid/C'/adhesion HB14 Myeloid cells/other signaling L15 B cells HB15 Ribosomal and metabolic process L16 Hematopoiesis HB16 Miscellaneous/GCC receptor/signaling L17 Extracellular matrix/eicosanoid HB17 Diverse signaling/Mapk/cytoskeleton L18 PRR/enzymes HB18 Diverse signaling L19 Cell structures HB19 Miscellaneous signaling/other L20 Microtubule/enzymes HB20 Miscellaneous L21 NK and T cells HB21 Calcium/cytoskeleton signaling L22 Ion channels/cell cycle HB22 Membrane molecules/metabolism L23 Interferon/C'/myeloid HB23 Diverse signaling L24 Immunoglobulin h/k enriched L25 −1 0 1 Allergy L26 Diverse/TM/solute carrier/Wnt signaling L27 Ctrl Ab HN878-infected Ctrl Ab (d21pi)/uninfected Ctrl Ab Diverse intracellular signaling L28 αGM-CSF HN878-infected αGM-CSF (d21pi)/uninfected Ctrl Ab Cilium organization/signaling L29 Cilia/microtubule formation L30 Cilia/microtubule/inflammation L31 b Quantification of blood modules Diverse intracellular signaling L32 Diverse intracellular signaling L33 Inflammasome/ Interferon/PRR granulocyte (HB3) (HB12) K-channel/transporters/drug metabolism L34 0.4 Cytotoxic/T cells/ILC/Tbx21/Eomes/B cells L35 NK cells/leukocytes/cytotoxic L36 Expression 0.25 Expression 0.2 B and T cells/myeloid cells L37 0.0 Apolipoproteins/protease inhibitors L38 0.00 −0.2 −1 −0.5 0 0.5 1 −0.25 Innate/hemopoietic Interferon/C’/ d Quantification of lung modules mediators (HB5) myeloid (HB23) Type I IFN/Ifit/Oas Inflammation/IL-1 signaling/ (L5) myeloid cells (L12) 0.4 Expression 0.25 0.2 Expression 0.2 Expression 0.25 Expression 0.00 0.0 0.0 0.00 −0.2 −0.25 −0.2 −0.25 Innate immunity/PRR/ Ctrl Ab αGM-CSF Ctrl Ab αGM-CSF C’/granulocyte (HB8) Myeloid/granulocyte Myeloid cells/Il1b/Tnf function (L10) (L13) 0.4 0.50 0.25 Expression Uninfected HN878 Expression Expression 0.2 0.25 Infected 0.00 0.0 0.00 −0.25 −0.2 −0.25 Ctrl Ab αGM-CSF Ctrl Ab αGM-CSF IL-17 pathway/ granulocytes (L11) 0.4 Expression 0.2 Uninfected HN878 Infected 0.0 −0.2 with TB resistance16, were slightly less over-abundant in infected GM-CSF prevents NETosis during M. tuberculosis infection. To lungs from infected mice treated with αGM-CSF versus Ctrl Ab further investigate the immune cell subsets associated with the (Supplementary Data 5 and Fig. 2c). In all, the transcriptional exacerbated inflammation observed at the site of infection fol- profile of lungs from HN878-infected αGM-CSF treated mice lowing GM-CSF blockade (Fig. 1d–f, Supplementary Data 1 and resembled that of infected TB-susceptible mice16. Fig. 2c), we tested our RNA-Seq lung data for enrichment of NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 Fig. 2 GM-CSF controls transcriptional signatures associated with TB pathogenesis. WT mice were infected and/or treated as in Fig. 1a. Blood and lungs were collected from M. tuberculosis HN878-infected and uninfected mice for RNA-Seq analysis (n = 4–5 mice/group). a Blood modules of co-expressed genes derived using WGCNA from human TB datasets35 are shown for blood RNA-Seq datasets obtained from infected mice treated with αGM-CSF or Ctrl mAbs, compared to uninfected Ctrl Ab treated mice (Supplementary Data 3). c Lung modules of co-expressed genes derived using WGCNA from mouse lung disease modules36 are shown for lung RNA-Seq datasets obtained from infected mice treated with αGM-CSF or Ctrl mAbs, compared to uninfected Ctrl Ab treated mice (Supplementary Data 5). a, c Fold enrichment scores derived using QuSAGE are depicted, with red and blue indicating modules over- or under-abundant, compared to the controls. Colour intensity and size of the dots represents the degree of perturbation, indicated by the colour scale, with the largest dot representing the highest degree of perturbation within the plot. Only modules with fold enrichment scores with FDR P-value < 0.05 were considered significant and depicted here. Module name indicates biological processes associated with the genes within the module. b, d Boxplots depicting the module eigengene expression, i.e., the first principal component for all genes within the module, are shown for uninfected Ctrl Ab (light green) or αGM-CSF (light purple) and infected Ctrl Ab (dark green) or αGM-CSF (dark purple) treated groups (n = 4–5 mice/group), for (b) blood modules: Inflammasome/Granulocyte (HB3), Innate/hemopoietic mediators (HB5), Innate Immunity/PRR/C’/Granulocyte (HB8), Interferon/PRR (HB12) and Interferon/C’/Myeloid (HB23); and (d) lung modules: Type I IFN/Ifit/Oas (L5), Myeloid/granulocyte function (L10), IL-17 pathway/granulocytes (L11), Inflammation/IL-1 signalling/myeloid cells (L12), and Myeloid cells/Il1b/Tnf (L13). Gene expression profiles are shown using boxplots, where the upper and lower box limits show the interquartile range (limits of second and third quartiles), a thick horizontal bar within the box shows the median, whiskers show the minimum and maximum values, and each dot represents an individual mouse (n = 4–5 mice/group). C’, complement.; PRR, pathogen recognition receptor. GCC, glucocorticoid; K-channel, potassium channel; TM, transmembrane; Ubiq, ubiquitination. previously curated immune cell-type associated gene sets40. Gene colocalization of DAPI (DNA staining) and citrullinated H3 set enrichment analysis (GSEA) revealed that the neutrophil- (CitH3), a specific marker for NET formation41,42. NETs were associated gene set was the highest positively enriched set in detected in surprisingly high amounts in the lung lesions from infected lungs from αGM-CSF versus Ctrl Ab treated mice (Fig. 3a; αGM-CSF treated mice but were scarcely found in infected Ctrl normalized enrichment score (NES) = 6.8, FDR < 0.0001), in Ab treated mice, as shown quantitatively by measuring the keeping with the increased over-abundance of neutrophil- percentage of CitH3 staining areas within the MPO positive areas associated modules in the blood (HB3, HB5 and HB8; Fig. 2b) in the lung lesions (Fig. 3f). These data indicate that neutrophil- and lung transcriptional signatures (L10 and L11; Fig. 2d). The driven disease exacerbation induced by GM-CSF blockade during expression of genes associated with neutrophil recruitment and M. tuberculosis infection correlates with excessive NETosis in the activation, such as S100a6, S100a8, S100a9, Mmp8, Mmp9, Cxcr2, infected lungs. Cd177 and Lcn2, was upregulated in infected lung samples but further increased in lungs from infected mice treated with αGM- NETs are detected in TB-susceptible mice and human TB. We CSF mAbs compared to infected Ctrl Ab treated mice (Fig. 3b and have recently reported that over-abundance of neutrophil- Supplementary Data 5). Increased expression of these neutrophil- associated genes in the blood and lungs also correlated with associated genes was also observed in the blood from infected mice lung disease severity in other mouse models of increased sus- treated with αGM-CSF compared to infected Ctrl Ab mice (Sup- ceptibility to TB, such as the highly susceptible C3HeB/FeJ mice plementary Fig. 3b and Supplementary Data 3). Despite the infected with M. tuberculosis HN87816. However, the mechan- increased expression of the majority of genes in the neutrophil- isms of neutrophil-mediated pathology in these TB-susceptible associated gene set driven by GM-CSF blockade (Fig. 3b), we mice remain unknown. Neutrophil depletion by the administra- observed a modest and variable increase in the number of neu- tion of mAbs against Ly6G after one-week post-infection, and trophils and inflammatory Ly6C+ monocytes in the lungs from then every other day, resulted in reduced mycobacterial loads in infected mice treated with αGM-CSF versus Ctrl Ab (Fig. 3c, the lungs of M. tuberculosis-infected C3HeB/FeJ mice (Fig. 4a), Supplementary Fig. 5 and Supplementary Data 1), indicating that confirming the deleterious role for neutrophils in host control of the neutrophil-associated transcriptional signature was resulting mycobacterial replication in C3HeB/FeJ mice as previously from activation and not only increased numbers of neutrophils. In reported19. Given our findings of a high abundance of NETs at contrast, alveolar macrophages were consistently decreased in the the site of infection upon blockade of GM-CSF, which correlated lungs from infected mice treated with αGM-CSF mAbs compared with increased blood and lung neutrophil-associated transcrip- to infected Ctrl Ab treated mice (Fig. 3c, right panel). This is in line tional signatures and disease severity, we hypothetised that with the downregulation of genes associated with alveolar macro- NETosis also occurs in the lungs of TB-susceptible C3HeB/FeJ phages induced by GM-CSF blockade (Supplementary Data 4 and mice. We found that NETs (DAPI+CitH3+) were abundant in Supplementary Fig. 4c, right panel). To investigate the role of the neutrophilic (MPO+) lung lesions from infected C3HeB/FeJ neutrophils in mycobacterial control we treated mice with mAbs mice (Fig. 4b), showing that NET formation correlates with dis- against Ly6G from one-week post-infection. Notably, neutrophil ease severity in other models of increased susceptibility to TB depletion led to reduced mycobacterial growth in the lungs of other than in M. tuberculosis-infected mice during GM-CSF infected mice treated with αGM-CSF mAbs (Fig. 3d), confirming a blockade. role for neutrophils in the disease exacerbation observed upon Importantly, we have also reported that over-abundance and GM-CSF blockade during infection. increased expression of genes associated with activation and As neutrophils were required for disease exacerbation (Fig. 3d) recruitment of neutrophils in the blood of active TB patients and their activation profile by gene expression appeared altered correlated with the extent of lung radiographic disease16. We following GM-CSF blockade during infection (Fig. 3b), we therefore investigated the presence of NETs in lung lesions from a examined neutrophil responses in vivo by immunofluorescence cohort of patients undergoing resection for pulmonary TB, as microscopy. We found an increased accumulation of neutrophils, after completion of chemotherapy these patients had radiographic detected by myeloperoxidase staining (MPO+), in the lesions of evidence of residual disease43. Notably, in 12 out of 13 lung infected αGM-CSF treated mice compared to the lung lesions from sections examined (one section per TB patient, Supplementary infected Ctrl Ab treated mice (Fig. 3e). These findings prompted us Table 1), we detected NETs immediately bordering and within to determine whether NETs were also present by examining the areas of caseous necrosis (Fig. 5 and Supplementary Fig. 6a), but 6 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 ARTICLE a c Ly6C+ Alveolar d 1010 Cell-type enrichment Neutrophils Monocytes Macrophages ** Neutrophils 5 × 106 * 5 × 10 6 1 × 106 109 Viable bacteria Monocytes * 4 × 10 6 4 × 106 8 × 105 108 Macrophages Cell number 6 6 5 B cells 3 × 10 3 × 10 6 × 10 CD4+ T cells 107 + 2 × 106 2 × 106 4 × 105 CD8 T cells 1 × 106 106 NK cells 1 × 106 2 × 105 γδT cells 0 0 0 105 –5 0 5 10 b b e e Ab SF lA SF lA SF yp 6G yp 6G rl -C r -C r -C ot αL y ot αL y NES Ct M Ct M Ct M Is Is αG αG αG Ctrl Ab αGM-CSF b e Merged CitH3 MPO DAPI Uninfected Infected Ctrl Ab αGM-CSF Ctrl Ab αGM-CSF S100a6 Mmp9 Mmp8 Ctrl Ab S100a8 S100a9 Cxcr2 Csf3 Mmp10 Ptx3 Ltb4r1 Mmp3 Ngp Ceacam10 Orm1 αGM-CSF Orm2 Osmr Il1f9 Cxcl2 Csf3r Arg2 Trem1 Chl1 Timp1 Lcn2 Il1rn Higher magnification Il17ra Ncf2 Ccr1 Cd300lf Osm Ctrl Ab Csf1 Ccl4 Cxcl3 Il1r2 Cd177 Nfil3 Plaur Cd33 C5ar1 Alox5ap Il10 Il18rap αGM-CSF Fpr2 Fcgr3 Adam8 Mmp13 Z-core −2 −1 0 1 2 f NETosis 60 ** 40 % CitH3/MPO 20 0 Ab SF rl -C Ct M αG not outside the necrotic TB lesions. Consistent with the presence human TB necrotic lesions showing a poor response to antibiotic and potential contribution of neutrophils to the necrotizing lung therapy, in support of a role for NETs in human TB pathogenesis. lesions in human TB, analysis of protein content previously reported by Marakalala et al.44 showed that neutrophil-associated proteins were enriched in the necrotic/caseous areas compared to Type I IFN signalling induces NETosis upon GM-CSF block- the cellular periphery of human TB granulomas (Supplementary ade. To identify the factors leading to exacerbated NETosis during Fig. 6b). Our data thus demonstrate that NETs are formed in M. tuberculosis infection in the absence of GM-CSF signalling, we NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 Fig. 3 Disease exacerbation upon GM-CSF blockade is neutrophil-dependent and correlates with NETosis. a GSEA of immune cell-type associated gene sets40 showing normalized enrichment scores (NES) for lung RNA-Seq data from infected αGM-CSF versus infected Ctrl Ab treated mice. b Heatmap showing relative expression of genes in the neutrophil-associated gene set for individual lung samples from uninfected Ctrl Ab (light green) or αGM-CSF (light purple) and infected Ctrl Ab (dark green) or αGM-CSF (dark purple) treated mice (n = 4–5 mice/group). Gene expression values were averaged and scaled across the row to indicate the number of standard deviations above (red) or below (blue) the mean, denoted as row Z-score. The dendrogram shows unsupervised hierarchical clustering of genes. c WT mice were infected and treated with Ctrl Ab (closed circles) or αGM-CSF (open circles) as in Fig. 1a. Lung cell suspensions were prepared and stained for the detection of myeloid cells (Supplementary Fig. 5). Cell numbers for neutrophils (CD11b+ Ly6G+), Ly6C+ monocytes (CD11b+Ly6G−Ly6C+) and alveolar macrophages (CD11blowCD11c+) are shown. *P = 0.0317. d WT mice were infected and treated with Ctrl Ab (closed symbols) or αGM-CSF (open symbols) as in Fig. 1a with the exception that from day 7 post-infection, mice also received 0.2 mg of anti-Ly6G (αLy6G; squares) or isotype control (labelled as Isotype; circles) mAbs every other day by i.p. injection. Lung viable bacterial loads were determined. **P = 0.0079. e Representative images of lung sections stained with citrullinated histone H3 (citH3; red), MPO (green), and DAPI (blue). NETs are visualized by colocalization of citH3 and DAPI staining (merged images; n = 3 mice/group). Bottom rows show detail from the top rows for each group. Scale bars, 50 µm. f Percentage of NET area in the lung normalized to MPO positive signal. **P = 0.0019. Data representative of one (a, b), two (d) or five (c, e) biological experiments; or data pooled of three independent experiments (f). Represented is the mean ± SD (c, f) or mean ± SEM (d); each dot represents an individual mouse: n = 5 mice/group (c, d) or n = 9 mice/group (f). Source data are provided as a Source Data file. Statistical analysis was performed using two-tailed Mann–Whitney test. a 1010 b HN878 infected C3HeB/FeJ mice ** 109 Merged CitH3 MPO DAPI Viable bacteria 108 107 106 105 e yp 6G ot αL y Is Fig. 4 NETs are detected in lung lesions of TB-susceptible C3HeB/FeJ mice. a C3HeB/FeJ mice were aerosol infected with M. tuberculosis HN878 treated by i.p. injection with 0.2 mg of anti-Ly6G (αLy6G; open circles) or isotype control (labelled as Isotype; closed circles) mAbs every other day, from day 7 post-infection until harvest on day 26 post-infection. Lung viable bacterial loads were determined. **P = 0.0079. Represented is the mean ± SD; each dot represents an individual mouse: n = 5 mice/group. Source data are provided as a Source Data file. Statistical analysis was performed using two-tailed Mann–Whitney test. b C3HeB/FeJ mice were aerosol infected with M. tuberculosis HN878 and their lungs harvested at day 35 post-infection. Formalin- fixed paraffin-embedded lung sections were stained with citrullinated histone H3 (citH3; red), MPO (green), and DAPI (blue). NETs are visualized by colocalization of citH3 and DAPI staining (merged image; n = 3 mice/group). Scale bars, 50 μm. Data from one experiment. analysed the global transcriptional changes at the site of infection type I IFN signalling (Fig. 6b–d and Supplementary Data 1). We caused by αGM-CSF treatment. GSEA revealed significant observed less severe lesions, with reduced lung lesion burden enrichment of several hallmark gene sets in the lungs from αGM- (Fig. 6b, c) and reduced necrosis and intra-alveolar necrotic debris CSF compared to infected Ctrl Ab treated mice (Supplementary (Fig. 6d), which also correlated with decreased neutrophil infil- Fig. 7a). Top positively enriched gene sets included biological tration (Supplementary Data 1), in the lungs from infected processes associated with host resistance to M. tuberculosis Ifnar−/− mice compared to WT mice treated with αGM-CSF infection4,21,22,24, such as TNF, IFN-γ and IL-6 signalling (Sup- mAbs. Of note, we also observed reduced lung necrosis (Fig. 6d) plementary Fig. 7a), which in excess can themselves be associated and neutrophil infiltration (Supplementary Data 1) in the absence with pathology45–48. Interestingly, we also found genes associated of IFNAR in the infected Ctrl Ab groups, indicating that type I with the IFN-α response among the top 5 positively enriched gene IFN signalling promotes neutrophil recruitment and necrosis sets (Supplementary Fig. 7a–c), consistent with the increased over- during M. tuberculosis infection even in the setting of intact abundance of the Type I IFN/Ifit/Oas module (L5) observed in the GM-CSF signalling. In contrast to WT mice, lesions containing lungs from αGM-CSF treated compared to infected Ctrl Ab multi acid-fast bacilli were not detected in the infected lungs from treated mice (Fig. 2c, d). Since increased and sustained levels of Ifnar−/− mice treated with αGM-CSF mAbs (Fig. 6e), in keeping type I IFN signalling have been shown to exacerbate TB in several with the reduced lung mycobacterial loads observed in the absence settings10–15, we then tested whether the increased type I IFN- of IFNAR signalling during GM-CSF blockade in M. tuberculosis- response, induced by GM-CSF blockade during M. tuberculosis infected mice (Fig. 6a). NETs were detected in abundance in the infection, promoted disease exacerbation. Confirming this lung lesions from infected WT mice treated with αGM-CSF mAbs hypothesis, type I IFN receptor-deficient (Ifnar−/−) mice infected but practically non-existent in the lung lesions from Ifnar−/− with HN878 and treated with αGM-CSF mAbs exhibited reduced mice, as shown quantitatively (Fig. 7a, b). These findings lung mycobacterial loads compared to infected αGM-CSF treated demonstrate that elevated levels of type I IFN signalling are WT mice (Fig. 6a). This reduced mycobacterial load was brought required for exacerbated NETosis in the lung and disease severity to the level detected in infected control WT and Ifnar−/− mice in triggered by GM-CSF blockade during M. tuberculosis infection. the absence of αGM-CSF mAbs, indicating that type I IFN sig- nalling is responsible for disease exacerbation upon GM-CSF blockade. Consistently, the exacerbated lung pathology prompted Neutrophil over-activation by type I IFN promotes TB. We by αGM-CSF blockade during infection was also dependent on have previously shown that neutrophils isolated from patients 8 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 ARTICLE TB patient Merged CitH3 MPO DAPI #1 #2 #3 #4 #5 #6 Fig. 5 NETs are detected in necrotic lung lesions of human pulmonary TB. Formalin-fixed paraffin-embedded lung sections from a total of thirteen patients with pulmonary TB (1 section per TB patient; Supplementary Table 1) were labelled with antibodies specific for citrullinated histone H3 (citH3; red) MPO (green) and DAPI (blue). NETs, visualized by colocalization of citH3 and DAPI staining (merged images, left), are shown for 6 TB patients. Scale bars, 50 µm. (All thirteen slides were stained and scanned together. See also Supplementary Fig. 6a). with active TB over-express type I IFN-inducible genes6,49, sug- over-activation of neutrophils by type I IFN impairs control of gesting that over-activation of neutrophils by type I IFN during mycobacterial replication and promotes disease during M. infection may contribute to disease progression. To determine tuberculosis infection, supporting a role for type I IFN signalling whether type I IFN acts directly on neutrophils to promote dis- in neutrophil-drivenTB pathogenesis. ease severity during M. tuberculosis infection, we crossed Ifnarfl/fl mice with MRP8 (also known as S100a8) Cre mice to delete Ifnar in neutrophils50,51. Supporting our hypothesis, we observed IFNAR signalling induces NETosis in TB-susceptible mice. reduced mycobacterial loads in the lungs from M. tuberculosis- Next, we examined whether the dependence on type I IFN sig- infected Ifnarfl/fl-MRP8-Crepos mice, which lack Ifnar in neu- nalling for exacerbated NET formation and neutrophil-driven trophils, compared to Ifnarfl/fl-MRP8-Creneg mice, both receiving disease was more generally observed in models of increased sus- treatment with αGM-CSF mAbs (Fig. 8a). Similar lung myco- ceptibility to TB that do not result from GM-CSF blockade. To bacterial loads were detected between Ifnarfl/fl-MRP8-Crepos and address this, C3HeB/FeJ mice were treated with anti-IFNAR Ifnarfl/fl-MRP8-Creneg infected mice treated with Ctrl Ab (αIFNAR) or isotype control (labelled as Ctrl Ab) mAbs the day (Fig. 8a). NETs were detected in the lung lesions from infected prior to M. tuberculosis HN878 infection, and during the course of Ifnarfl/fl-MRP8-Creneg and Ifnarfl/fl-MRP8-Crepos mice treated infection (Fig. 9a). Blockade of IFNAR signalling in susceptible with αGM-CSF mAbs, but could barely be detected in infected C3HeB/FeJ mice infected with M. tuberculosis resulted in sig- mice treated with Ctrl Ab (Fig. 8b, c). A similar percentage of nificantly reduced lung mycobacterial loads (Fig. 9b) and pathology normalised NET area was quantified in the lung lesions from both (Fig. 9c), as compared to infected Ctrl Ab treated mice. Scoring of groups of infected mice treated with αGM-CSF mAbs (Fig. 8c), histological sections showed a significant reduction in lung lesion suggesting that type I IFN is acting via other cells to promote burden (Fig. 9d), necrosis and intra-alveolar necrotic debris NET-formation in neutrophils. However, the observation that (Fig. 9e), accompanied by fewer neutrophils (Supplementary lung neutrophil infiltration was found to be reduced in infected Data 1), in infected C3HeB/FeJ mice treated with αIFNAR mAbs Ifnarfl/fl-MRP8-Crepos mice compared to Ifnarfl/fl-MRP8-Creneg compared to Ctrl Ab treated mice. A reduced number of acid-fast mice treated with αGM-CSF mAbs (Fig. 8b), suggests that type I bacilli were also detected in the lungs of infected C3HeB/FeJ mice IFN signalling in neutrophils is promoting their recruitment upon IFNAR blockade (Fig. 9f). Notably, blockade of IFNAR sig- to the site of infection. Collectively, these findings suggest that nalling in susceptible C3HeB/FeJ mice abrogated NET formation at NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 a CFU b WT Ifnar–/– 9 10 ** Ctrl Ab αGM-CSF Ctrl Ab αGM-CSF Viable bacteria 108 107 106 Ab SF Ab SF rl -C rl -C Ct M Ct M αG αG WT Ifnar–/– c Lesion burden *** 5 *** *** 4 Relative score 3 2 1 0 Ab SF Ab SF rl -C rl -C Ct M Ct M αG αG WT Ifnar–/– d Necrosis e WT Ifnar–/– *** * *** *** Ctrl Ab Ctrl Ab 4 Relative score 3 2 1 0 Ab SF Ab SF rl -C rl -C Ct M Ct M αG αG WT Ifnar–/– Intra-alveolar necrotic debris αGM-CSF αGM-CSF *** *** *** 4 Relative score 3 2 1 0 Ab SF Ab SF rl -C rl -C Ct M Ct M αG αG WT Ifnar–/– the site of infection, with significantly fewer NET-forming neu- Discussion trophils detected in the lung lesions from αIFNAR as compared to GM-CSF has been increasingly appreciated as an essential med- Ctrl Ab treated mice, as shown quantitatively (Fig. 9g, h). These iator of protective immunity against TB25–31. However, the findings show that uncontrolled mycobacterial replication, exa- mechanisms underlying the pathology observed in the absence of cerbated NETosis and pulmonary necrosis are also dependent on GM-CSF signalling during M. tuberculosis infection remain type I IFN signalling in TB-susceptible C3HeB/FeJ mice, showing poorly understood, particularly since past studies performed in that this is a generalised mechanism underlying TB pathogenesis. GM-CSF-deficient mice had confounding lung pathologies33,34, 10 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 ARTICLE Fig. 6 IFNAR deletion abrogates disease exacerbation prompted by GM-CSF blockade during infection. WT (circles) and Ifnar-/- (triangles) mice were infected and treated with Ctrl Ab (closed symbols) or αGM-CSF (open symbols) as in Fig. 1a. a Viable bacterial loads determined in the lungs at day 21 post-infection. **P < 0.0079. Represented is the mean ± SEM; each dot represents an individual mouse: n = 5 mice/group. Source data are provided as a Source Data file. Data representative of two biological experiments. Statistical analysis was performed using the two-tailed Mann–Whitney test. b Representative H&E staining of infected lung sections (n = 2–4 mice/group). Scale bars from top to bottom: 1 mm; 200 µm; 50 µm; 10 µm. c Relative lung lesion burden from H&E stained sections (Supplementary Data 1). 0 = no lesions, 1 = focal lesion, 2 = multiple focal lesions, 3 = one or more focal severe lesions, 4 = multiple focal lesions that are extensive and coalesce, 5 = extensive lesions that occupy the majority of the lung lobe. ***P < 0.0001. d Relative necrosis and intra-alveolar necrotic debris scores from H&E stained lung sections (Supplementary Data 1). 0 = not present, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. ***P < 0.0001; *P = 0.0294. c, d Data pooled from four biological experiments. Represented is the mean ± SD; each dot represents an individual mouse: n = 11–13 mice/group. Source data are provided as a Source Data file. Statistical analysis was performed using one-way ANOVA Tukey’s multiple comparisons test. e Representative Ziehl–Neelsen staining of infected lung sections (n = 2–4 mice/group). Scale bars, 20 µm. Data representative of four biological experiments (b, e). a WT Ifnar –/– Ctrl Ab αGM-CSF Ctrl Ab αGM-CSF Merged CitH3 MPO DAPI b NETosis 100 ** 80 % CitH3/MPO 60 40 20 0 Ab SF Ab SF rl -C rl -C Ct M Ct M αG αG WT Ifnar –/– Fig. 7 IFNAR deletion abrogates NET formation prompted by GM-CSF blockade during infection. WT (circles) and Ifnar−/− (triangles) mice were infected and treated with Ctrl Ab (closed symbols) or αGM-CSF (open symbols) as in Fig. 1a. a Representative images of lung sections stained with citrullinated histone H3 (citH3; red), MPO (green), and DAPI (blue). NETs are visualized by colocalization of citH3 and DAPI staining (merged images; n = 3 mice/group). Scale bars, 50 µm. Data representative of two biological experiments. b Percentage of NET area in the lung normalized to MPO positive signal. **P = 0.0022. Data pooled from two biological experiments. Represented is the mean ± SD; each dot represents an individual mouse: n = 6 mice/ group. Source data are provided as a Source Data file. Statistical analysis was performed using the two-tailed Mann–Whitney test. NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 a Ifnar fl/fl MRP8-Cre mice: b Ctrl Ab αGM-CSF pos neg CFU Creneg Cre Cre Crepos 10 9 ** Merged Viable bacteria 108 107 CitH3 106 Creneg Crepos Creneg Crepos Ctrl Ab αGM-CSF c 80 NETosis MPO 60 % CitH3/MPO 40 DAPI 20 0 Creneg Crepos Creneg Crepos Ctrl Ab αGM-CSF Fig. 8 Type I IFN activates neutrophils to promote bacterial growth. Ifnarfl/fl MRP8-Cre negative (Creneg; circles) or positive (Crepos; squares) mice were infected and treated with Ctrl Ab (closed symbols) or αGM-CSF (open symbols) as in Fig. 1a. a Viable bacterial loads determined in the lungs at day 21 post-infection. **P < 0.0022. Data representative of two biological experiments. Represented is the mean ± SEM; each dot represents an individual mouse: n = 4–6 mice/group/experiment. b Representative images of lung sections stained with citrullinated histone H3 (citH3; red), MPO (green), and DAPI (blue). NETs are visualized by colocalization of citH3 and DAPI staining (merged images). Scale bars, 50 µm. Data representative of two biological experiments (n = 3–4 mice/group/experiment). c Percentage of NET area in the lung normalized to MPO positive signal. Data pooled from two biological experiments. Represented is the mean ± SD; each dot represents an individual mouse: n = 7–8 mice/group. Source data are provided as a Source Data file. Statistical analysis was performed using two-tailed Mann–Whitney test. hampering interpretation of the findings. Here, using blocking M. tuberculosis infection. Conversely, our findings showed that mAbs, we describe an IFN-γ-independent mechanism of patho- GM-CSF blockade during infection did not compromise IFN-γ- genesis in the absence of GM-CSF signalling during M. tuber- producing T cell numbers and instead resulted in increased culosis infection. Transcriptomic analyses revealed that blockade transcriptional IFN-γ responses in infected lungs. Moreover, of GM-CSF during M. tuberculosis infection results in increased impaired control of mycobacterial growth driven by GM-CSF expression of genes associated with neutrophil activation and a blockade during infection was also observed in the absence of type I IFN response, which have been previously associated with IFN-γ signalling, revealing an IFN-γ-independent mechanism for disease severity in mouse models and human TB16. Confirming the resulting disease exacerbation during M. tuberculosis infection their role in TB pathogenesis, we show that disease exacerbation in the absence of GM-CSF. Developmental defects in both driven by either GM-CSF blockade during M. tuberculosis alveolar macrophages and other immune cells such as dendritic infection or during infection of susceptible C3HeB/FeJ mice was cells52,53, reported in GM-CSF-deficient mice, may account for dependent on neutrophil over-activation by type I IFN leading to the differences in T cell recruitment to the infected lungs exacerbated NETosis at the site of infection. NETs were also observed in the GM-CSF-deficient mice27,31, but not in the cur- found in necrotic lung lesions from patients with nonresolving rent scenario of lungs of adult mice where GM-CSF was only pulmonary TB, further supporting a role for NETosis in TB blocked during infection. pathogenesis. In contrast to the small impact of GM-CSF blockade on the Previous studies addressing the in vivo effects of GM-CSF on numbers of T cells, neutrophils and monocytes, we found that the immune response against M. tuberculosis infection have been GM-CSF blockade reduced the numbers of alveolar macrophages performed using GM-CSF-deficient mice27,31. However, in the in the infected lungs, in keeping with the importance of GM-CSF steady-state, these mice display dramatic defects on alveolar in the maintenance and function of these cells38,39. It has been macrophages and lung function, including features of human shown that the depletion of alveolar macrophages reduced M. PAP33,34,38,39 and deficiencies in other immune cells52,53. This tuberculosis burden in vivo54, suggesting that alveolar macro- makes it difficult to distinguish the mechanisms of pathogenesis phages are permissive to mycobacterial growth. Therefore, the resulting from M. tuberculosis infection in the absence of GM- reduced numbers of alveolar macrophages observed may not CSF from the developmental defects reported in the GM-CSF- explain the increased M. tuberculosis burden observed upon GM- deficient mice. Studies using GM-CSF-deficient mice27,31 have CSF blockade. suggested that GM-CSF is required for the recruitment of pro- We found that GM-CSF blockade perturbed the expression of tective IFN-γ-producing T cells into the site of infection following different networks of genes associated with protective versus 12 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 ARTICLE a c Ctrl Ab αIFNAR HN878 Day0 Days p.i. 0.5 mg αIFNAR or isotype Day Day-1 control (Ctrl Ab) i.p. 3×/week 26 C3HeB/FeJ 0.5 mg αIFNAR or Harvest isotype control (Ctrl Ab) i.p. b CFU d Lesion burden 109 ** 5 * 4 Viable bacteria Relative score 108 3 2 107 1 106 0 Ctrl Ab αIFNAR Ctrl Ab αIFNAR e Intra-alveolar Necrosis necrotic debris 4 ** 4 ** 3 3 Relative score 2 2 1 1 g Ctrl Ab αIFNAR 0 0 Merged Ctrl Ab αIFNAR Ctrl Ab αIFNAR f Ctrl Ab CitH3 MPO αIFNAR DAPI h NETosis 100 ** 80 % CitH3/MPO 60 40 20 0 Ctrl Ab αIFNAR damaging immunity in the lungs and blood during M. tubercu- we show that GM-CSF blockade boosted transcriptional losis infection. We have recently shown that transcriptional responses reflective of type I IFN-inducible signalling and neu- dominance of a type I IFN response and neutrophil activation, trophil activation during M. tuberculosis infection, promoting together with a loss of B cell, NK and T cell effector responses disease exacerbation. The effect of GM-CSF blockade to increase contribute to the pathogenesis of M. tuberculosis infection16. bacterial load was abrogated in the absence of IFNAR on neu- Using distinct but complementary transcriptomic analyses, here trophils, providing an explanation to the link between type I IFN NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 Fig. 9 IFNAR signalling is required for severity of lung NETosis in C3HeB/FeJ mice. a Schematic of experimental design: C3HeB/FeJ mice were treated by intraperitoneal (i.p.) injection of 0.5 mg of anti-IFNAR (αIFNAR; open circles) or isotype control (labelled as Ctrl Ab; closed circles) mAbs the day prior low dose HN878 aerosol infection, followed by i.p. injections of 0.5 mg of αIFNAR or Ctrl mAbs, three times per weekly until harvest on day 26 post- infection. b Viable bacterial loads were determined. **P = 0.0079. c Representative hematoxylin and eosin (H&E) staining of infected lung sections (n = 3 mice/group). Scale bars from top to bottom: 1 mm; 200 µm; 50 µm, 10 µm. d Relative lung lesion burden from H&E stained sections (Supplementary Data 1). 0 = no lesions, 1 = focal lesion, 2 = multiple focal lesions, 3 = one or more focal severe lesions, 4 = multiple focal lesions that are extensive and coalesce, 5 = extensive lesions that occupy the majority of the lung lobe. *P = 0.0152. e Relative necrosis (left) and intra-alveolar necrotic debris (right) scores from H&E stained lung sections (Supplementary Data 1). 0 = not present, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. **P = 0.0023. f Representative Ziehl–Neelsen staining of infected lung sections. Scale bars, 20 µm. g Representative images of lung sections labelled with citrullinated histone H3 (citH3; red), MPO (green), and DAPI (blue). NETs are visualized by colocalization of citH3 and DAPI staining (merged images). Scale bars, 50 μm. h Percentage of NET area in the lung normalized to MPO positive signal. **P < 0.0012. b, c, f, g Data representative of two biological replicate experiments. d, e, h Data pooled from two biological experiments. Represented is the mean ± SD; each dot represents an individual mouse: n = 5 mice/ group (b) or n = 6–7 mice/group (d, e, h). Source data are provided as a Source Data file. Statistical analysis was performed using the two-tailed Mann–Whitney test. and neutrophil-driven transcriptional signatures. In contrast to antibiotics to access the infected site. Although future mechanistic the increased type I IFN and neutrophil-driven transcriptional studies are required to fully decipher the contribution of NETosis signatures, GM-CSF blockade during infection, decreased the to disease progression, these observations suggest that inhibiting abundance of modules associated with NK, T and B cells, simi- NET release in conjunction with antibiotic therapy could be larly to the decrease in these signatures seen in the blood of TB clinically beneficial and prevent long-term morbidity due to lung patients which correlated with advanced disease16. We also destruction in TB patients68, particularly in cases of MDR or observed a decreased over-abundance in the blood Cell cycle XDR TB where current anti-mycobacterial drugs are of limited module upon GM-CSF blockade during infection, which may be use. linked to the increased type I IFN signalling and the anti- Our data are consistent with a critical role for type I IFN in the proliferative effects of type I IFN55. regulation of NETosis, and in turn exacerbated NETosis serving Neutrophils have been emerging as key players of TB patho- as a leading effector of type I IFN-induced pathology. In our genesis and their influx into the site of infection is associated with infection models of increased susceptibility to TB, NETosis was poor outcomes16–20. However, the mechanisms linking neu- completely abrogated in the absence of type I IFN signalling, trophils and increased TB susceptibilty are still unclear56. We which correlated with better control of infection and less tissue observed enhanced neutrophil recruitment to the site of infection damage. Similarly, previous in vitro studies have reported that and NETosis following M. tuberculosis infection in the absence of neutrophils produce NETs when exposed to plasma samples from GM-CSF signalling, in agreement with previous reports of patients with autoimmune disorders showing elevated levels of increased neutrophilic infiltration into the lungs of GM-CSF- type I IFN or exogeneous IFN-α69,70. These data suggested that deficient mice following bacterial infection57. Transcriptional type I IFN primes neutrophils for the formation of NETs. Several analysis revealed that the expression of genes associated with mechanisms have been proposed for the role of sustained and neutrophil recruitment but also activation was increased upon increased levels of type I IFN in exacerbating M. tuberculosis GM-CSF blockade in the blood and lungs from infected mice, infection7,10,11,13–15,55,71,72. Our data indicate that this mechan- suggesting that GM-CSF blockade during M. tuberculosis infec- ism, that was originally identified in autoimmune and inflam- tion affects not only the recruitment but also the activation status matory conditions, is also critically relevant in certain infection of neutrophils in circulation and at the site of infection, similarly scenarios. Type I IFN appear to be key regulators of neutrophil to what was observed during infection of TB-susceptible C3HeB/ antimicrobial strategy decisions in both sterile disease and FeJ mice16. We found that neutrophils were required for disease infection, although the mechanisms may vary given that type I exacerbation induced in TB resistant C57Bl/6 mice by GM-CSF IFN signalling acts directly on neutrophils in autoimmune con- blockade during M. tuberculosis infection but additionally in TB- ditions. Although the contribution of type I IFN signalling in susceptible C3HeB/FeJ mice, and that neutrophil-driven disease other cells to TB disease exacerbation has been previously exacerbation correlated with excessive NETosis at the site of reported6,11,71,72, we now demonstrated that increased type I IFN infection in both models. NETs were originally described as a signalling also promotes TB pathogenesis through direct stimu- host antimicrobial defence mechanism against pathogens41, but lation of neutrophils in vivo during infection, leading to uncon- recent studies reported that NETs can modulate immune trolled mycobacterial growth at the site of infection. The responses and may also induce damaging inflammation in var- selectivity of NET release in response to different microbes sug- ious infections and other inflammatory diseases41,42,58–62. NETs gests that NETosis does not always result in an antimicrobial have been shown to be released in vitro by M. tuberculosis- response, such as phagocytosis, to control certain pathogens41, infected mouse63 and human64,65 neutrophils, however, NETs did but that in other infections such as TB contributes to disease not prevent M. tuberculosis replication in vitro63. Consistently, exacerbation. the presence of the NET component citH3 in induced sputum65 Together, our data from TB patients and mouse models of and neutrophil-related inflammatory proteins associated with increased susceptibility to TB by infection of susceptible C3HeB/ NETs in sputum65, serum18,66,67 and lung biopsies18,65 have been FeJ mice, or resistant C57Bl/6 mice where GM-CSF signalling is shown to reflect active TB and TB-related lung damage. We now abrogated during infection, indicate that excessive formation of show unequivocally that NETs are induced in human TB lung NETs at the site of infection correlates with poor disease out- lesions showing a poor resolution after antibiotic therapy, further comes. Our findings provide an insight as to how uncontrolled supporting a role for NETs in human TB pathogenesis and neutrophil infiltration and activation may exert a detrimental suggesting that although initially perceived as antimicrobials, effect during M. tuberculosis infection. Moreover, we show that NETs may promote TB infection and may limit the ability of this process is controlled by type I IFN signalling as demonstrated 14 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 ARTICLE by reduced NETosis and mycobacterial replication when IFNAR Antibody-mediated neutrophil depletion. Anti-mouse-Ly6G (αLy6G) mAb activity was abrogated by using IFNAR-deficient mice or anti- (clone 1A8, Lot 695418J3) and isotype control (rat IgG2a, clone 2A3, Lot 71671801; labelled as Isotype in Figs. 3d and 4a) were purchased from BioXCell. M. tuber- IFNAR mAbs during infection of TB-susceptible mice. These culosis-infected mice received a total of 7 i.p. injections containing 0.2 mg of αLy6G findings provide a mechanism to explain how the reported type I or isotype control mAbs every other day from day 7 post-infection. IFN-induced neutrophil signatures in human TB promote disease exacerbation. Antibody-mediated IFNAR blockade. Anti-mouse-IFNAR1 (αIFNAR) mAb (clone MAR1-5A3, Lot 0419L555) was purchased from Leinco Technologies, Inc. Isotype control (rat IgG1, clone GL113, Lot A2022916; labelled as Ctrl Ab in Fig. 9) Methods was a gift from DNAX (now Merck, USA). Mice were injected i.p. with 200 μL Human samples. The human samples used for this study came from the collection containing 0.5 mg of either αIFNAR or isotype control (Ctrl Ab) mAbs the day obtained within the SH-TBL project (Supplementary Table 1; https://doi.org/ prior to M. tuberculosis infection and then three times per week after infection for 10.17632/knhvdbjv3r.1), led by the Experimental Tuberculosis Unit (UTE) and the course of the experiment. conducted in collaboration with the National Center for Tuberculosis and Lung Diseases of Georgia (NCTLD); and registered at the ClinicalTrials.gov database under code NCT02715271. The project was reviewed and approved by both the Flow cytometry. Single-cell homogenates prepared from harvested lungs from Ethics Committee of the NCTLD (IRB00007705 NCTLD Georgia #1, infected mice were stained according to manufacturer’s instructions to exclude dead IORG0006411) and the Germans Trias I Pujol Hospital (IGTP) ethics committee cells using a Live/Dead fixable red dead cell stain kit (Invitrogen). Cells were pre- (EC: PI-16-171). Written informed consent was obtained for the collection of treated for 10 min with anti-FcγRI/FcγRII (anti-CD16/CD32; clone 24G2, Harlan; biological material and data from all study participants before being enroled. 1:100) Ab and then stained with Abs against specific extracellular markers to identify myeloid cells or lymphocytes. Myeloid cell panel included Thy1.2 (clone 53- 2.1, eBioscience; 1:400), Ly6G (clone 1A8, BD; 1:100), Ly6C (clone HK1.4, Experimental animals and ethics. C3HeB/FeJ, C57BL/6 wild-type (WT), IFN-γ eBioscience; 1:200), CD11c (clone HL3, BD; 1:100), CD11b (clone M1/70, BD; receptor-deficient (Ifngr1tm1Agt; IFNγR−/−) and type I IFN receptor-deficient 1:100). Lymphoid cell panel included Thy1.2 (clone 53-2.1, eBioscience; 1:400), CD3 (Ifnar1tm1Agt; Ifnar−/−) mice were bred and housed in specific pathogen-free (clone 145-2C11, eBioscience; 1:200), CD4 (clone RM4-5, eBioscience/BD; 1:200) facilities at The Francis Crick Institute. Ifnarflox/flox (Ifnar1tm1Uka; Ifnarfl/fl) and CD8 (clone 53-6.7, BD; clone Ly-2, eBioscience; 1:400). For detection of IFN-γ mice73,74, which have loxP sites flanking exon 10 of the Ifnar1 gene, were crossed production, cells were restimulated ex vivo with M. tuberculosis tuberculin purified with MRP8-Cre (MRP8 also known as S100a8; Tg(S100A8-cre,-EGFP)1Ilw) mice50,51, to protein derivative (PPD; 20 μg/ml; Statens Serum Institute) and anti-CD28 (2 μg/ml; delete Ifnar in neutrophils. Ifnarfl/fl-MRP8-Crepos mice and Ifnarfl/fl-MRP8-Creneg clone 37.51, Harlan) for 20 h. Brefeldin A (10 μg/ml; Sigma-Aldrich) was added littermate control mice were used in experiments. All protocols for breeding were during the last 4 h. After extracellular staining, cells were fixed and treated with performed in accordance with Home Office (U.K.) requirements and the Animal permeabilization buffer (BD) according to manufacturer’s instructions and stained Scientific Procedures Act, 1986. Age-matched females were used in experiments. with anti-IFN-γ (clone XMG1.2, eBioscience; 1:100) or isotype control (clone Experiments were performed in accordance with Home Office (U.K.) requirements eBRG1, eBioscience; 1:100) Abs. All stained samples were fixed with stabilizing and the Animal Scientific Procedures Act, 1986, and with recommendations of the fixative (BD) and refrigerated in the dark overnight before being acquired on a European Union Directive 2010/63/EU and approved by Portuguese National CyAN ADP analyser (Dako, Ely, U.K.) using Summit software v4.4.0 (Cytomation). Authority for Animal Health – Direção Geral de Alimentação e Veterinária Data were analysed using FlowJo software v10.3 (Tree Star). Myeloid cell popula- (DGAV-Ref.0421/000/000/2016). Mice were kept under specific-pathogen-free tions were analysed after exclusion of dead cells and Thy1.2+ cells; neutrophils were conditions, with controlled temperature (20–24 °C), humidity (45–65%) and light gated as CD11b+Ly6G+, Ly6C+ monocytes as CD11b+Ly6G−Ly6C+ and alveolar cycle (12-h light/dark). Mice were kept with food and water ad libitum and macrophages as CD11blowCD11c+. T cell populations were analysed after the humanely euthanized by CO2 asphyxiation. exclusion of dead cells and gating on Thy1.2+CD3+ cells. Histopathological analysis of lung samples. Lung tissues from M. tuberculosis- Experimental infection. M. tuberculosis experiments were performed under ABSL- infected mice were perfused and fixed in 10% neutral-buffered formalin followed 3 conditions. M. tuberculosis HN878 bacilli (clinical isolate) were grown to midlog by 70% ethanol, processed and embedded in paraffin, sectioned at 4 µm and phase in Middlebrook 7H9 broth supplemented with 10% oleic acid albumin stained with hematoxylin and eosin (H&E) or Ziehl-Neelsen (ZN). A single dextrose complex (Difco), 0.05% Tween 80, and 0.5% glycerol before being section from all lung lobes was viewed and scored as a consensus by three board- quantified on 7H11 agar plates and stored in aliquots at −80 °C. Aliquots frozen at certified veterinary pathologists (S.L.P., E.H. and A.S-B.) blinded to the groups −80 °C were then thawed (6 aliquots) and quantified, to determine the con- (Supplementary Data 1, which contains data from all original experiments centration of the stored inocula. Mice were infected via the aerosol route using an contributing to the study). The relative lesion burden scoring (0–5 points) was inhalation exposure system, calibrated to deliver ∼200 CFUs to the lung. The determined using the following scale: 0 = no lesions, 1 = focal lesion, 2 = mul- infection dose was confirmed by determining the number of viable bacteria in the tiple focal lesions, 3 = one or more focal severe lesions, 4 = multiple focal lesions lungs of five mice immediately after the aerosol infection. Infected mice were that are extensive and coalesce, and 5 = extensive lesions that occupy the monitored regularly for signs of illness such as wasting, piloerection and hunching. majority of the lung lobe. A semi-quantitative scoring (0–4 points) method was Mice were euthanized by CO2 inhalation and blood and/or lung samples from each devised to assess the following histological features: inflammatory cells (neu- group were collected from individual mice at day 21 post M. tuberculosis infection, trophils, lymphocytes, macrophages), necrosis, and pleuritis; using the following unless otherwise indicated in the figure legend. Blood and/or lung samples from scale: 0 = not present, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked age-matched uninfected mice were collected at the same time and used as controls. changes. The presence of M. tuberculosis bacilli detected by ZN positive staining Determination of lung bacterial load was performed by plating serial dilution of the was scored as paucibacillary or multibacillary according to their abundance in organ homogenate on Middlebrook 7H11 agar supplemented with 10% oleic acid the tissue. Representative images of each group were acquired on an Olympus albumin dextrose complex plus PANTA to prevent contamination with other BX43 microscope using an Olympus SC50 camera and cellSens Entry software bacteria. CFUs were counted after 3 weeks of incubation at 37 °C, and the bacterial (Ver. 1.18). load per lung was calculated. Immunofluorescence detection of NETs. To identify NETs from lung tissues, Antibody-mediated GM-CSF blockade. Anti-mouse-GM-CSF monoclonal anti- sections from M. tuberculosis-infected mice or from TB patients were de-waxed body (αGM-CSF mAb: clone MP1-22E9, Lot A1102882) and isotype control mAb and re-hydrated before staining. Sections were treated with Dako Target Retrieval (rat IgG2a, clone GL117, Lot A1102882; labelled as Ctrl Ab throughout the Solution, pH 9 Ready-to-Use (Agilent) at 97 oC for 45 min for antigen retrieval, manuscript and Figs. 1–3 and 6–8) were gifts from DNAX (now Merck, USA). and permeabilized in PBS 0.5% triton x-100. Samples were incubated with a Mice were injected intraperitoneally (i.p.) with 300 μl containing 1 mg of either blocking buffer (PBS with 2% BSA and 2% of donkey serum (Sigma-Aldrich)) for αGM-CSF or isotype control mAbs diluted in PBS the day prior to M. tuberculosis 1 h at room temperature (RT) and stained with rabbit anti-mouse antibodies infection. After infection, mice received twice weekly i.p. injections of 200 μL directed against citrullinated histone H3 (ab5103 from Abcam; 1:500) and goat containing 0.3 mg αGM-CSF or isotype control mAbs for the course of the anti-mouse antibodies directed against MPO (AF3667, R&D systems; 1:40) over- experiment. Similar results were obtained with mAbs purchased from BioXCell night at 4 oC. After washing samples with PBS, sections were incubated with sec- (αGM-CSF mAb: clone MP1-22E9, Lot 683718A1; isotype control mAb: rat IgG2a, ondary donkey anti-rabbit IgG antibodies conjugated with Alexa Fluor 568 clone 2A3, Lot 686318A2). (A10042, Invitrogen; 1:200), donkey anti-goat IgG antibodies conjugated with For IFN-γ blockade experiments, mice received 0.5 mg of either anti-mouse- Alexa Fluor 488 (A11045, Invitrogen; 1:200) and DAPI (Invitrogen; 1:1000) for 2 h IFN-γ (αIFN-γ) mAb (clone XMG1.2, Lot 5543-3/5543/0715; from BioXCell) or in the dark at RT. Glass coverslips were mounted onto the slides using Prolong isotype control mAb (rat IgG1, clone GL113, Lot A2022916; gift from DNAX; Gold (Invitrogen). Controls were stained with secondary antibody only, and labelled as Isotype in Supplementary Fig. 1d) together with αGM-CSF or isotype nonspecific fluorescent staining was not detected when secondary antibodies were control mAbs for the course of the experiment. tested alone. Slides were analysed using Olympus VS120-L100 Slide Scanner and NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications 15 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19412-6 the OLYMPUS OlyVIA software (v2.9). Representative images for each group were Reporting summary. Further information on research design is available in the Nature exported using QuPath software (v0.2.0-m5)75. Research Reporting Summary linked to this article. The percentage of NET area normalized to MPO signal was calculated by exporting three regions of interest of 2000 × 2000 μm representative of each lung sections from Qupath (version 0.2.0) and analysed with FIJI (v 2.0.0). The images Data availability were split into the different channels; the DAPI channel was subjected to the RNA sequence data have been deposited in the NCBI Gene Expression Omnibus (GEO) threshold Triangle, a mask was generated to account for the total amount of tissue database under the primary accession code GSE141207. Data regarding the sorted cells present in the region of interest. Default and Triangle thresholding was used with from Immunological Genome Project were accessed from GEO under the accession code the MPO and the CitH3 channel respectively. The percentage of the area was GSE109125. All other data that support the findings of this study are available in the calculated for the different channels based on the generated masks. The measured article and Supplementary Information files or from the corresponding author upon percentage of CitH3 signal was normalized to the measured percentage of MPO request. Source data are provided with this paper. signal and the % CitH3/MPO values obtained from the three different regions were averaged and plotted. Statistical analysis of the plots was obtained by performing a nonparametric Mann–Whitney test. Received: 3 February 2020; Accepted: 12 October 2020; RNA extraction and pre-processing for RNA-Seq. Blood was collected in Tempus reagent (Life Technologies) at 1:2 ratio. Total RNA was extracted using the Tempus™ Spin RNA isolation kit (Thermo Fisher Scientific). Globin RNA was depleted from total RNA (1.5–2 µg) using the GLOBINclear™-Mouse/Rat Globin References mRNA removal kit (Thermo Fisher Scientific). Lungs were collected in TRI- 1. WHO. Global Tuberculosis Report 2019. (World Health Organization, 2019). Reagent (Sigma-Aldrich). 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S.H. contributed with experiments involving IFNγR−/− mice and anti-IFN-γ expression including gene-gene correlations. Nucleic Acids Res. 41, e170 mAb treatment. J.S., K.L.F. and M.S. performed initial M. tuberculosis infection (2013). experiments with Ifnar−/− mice. P.C. analysed RNA-Seq data. E.H., S.L.P. and A.S-B. performed histopathological analysis and interpretation. M.I. and Q.W. carried out initial NETs staining and, together with V.P., contributed to the data interpretation. Acknowledgements I.V.A performed quantification analysis and interpretation of NET-forming neutrophils We thank Xuemei Wu (Laboratory of Immunoregulation and Infection, The Francis in lung sections. S.V., P.R-M. and C.V. provided human lung tissue samples. All co- Crick Institute) for her help in coordinating all the breeding and maintenance of the mice authors have read, reviewed, and approved the manuscript. used in this study. We thank Jean Langhorne (Malaria Immunology Laboratory, The Francis Crick Institute) and Ilaria Malanchi (Tumour-Host Interaction Laboratory, The Competing interests Francis Crick Institute) for the Ifnarfl/fl and the MRP8-Cre mice, respectively. We thank The authors declare no competing interests. Akul Singhania and Olivier Tabone (Laboratory of Immunoregulation and Infection, The Francis Crick Institute) for critical discussion and input regarding RNA-Seq analyses. We thank Alan Sher (National Institute of Allergy and Infectious Diseases, NIH) and Paulo Additional information Vieira (Institut Pasteur) for discussion and critical reading of the manuscript. We thank Supplementary information is available for this paper at https://doi.org/10.1038/s41467- The Francis Crick Institute Biological Services for animal husbandry and technical 020-19412-6. support; Advanced Sequencing Facility and Bioinformatics and Biostatistics Science Technology Platforms for helping with sequence sample processing and analyses; and Correspondence and requests for materials should be addressed to L.M.-T. Experimental Histopathology for their work in preparing lung sections for histological analyses. This study was funded by The Francis Crick Institute which receives its core Peer review information Nature Communications thanks the anonymous reviewers for funding from Cancer Research UK (FC001126, FC001999, FC001129), the UK Medical their contribution to the peer review of this work. Peer review reports are available. Research Council (FC001126, FC001999, FC001129), and the Wellcome Trust (FC001126, FC001999, FC001129); before that by the UK Medical Research Council Reprints and permission information is available at http://www.nature.com/reprints (MRC U117565642); and by the European Research Council (294682-TB-PATH). The collection of human lung tissue samples for this study was funded by the Spanish Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in Government-FEDER Funds through CP13/00174, CPII18/00031 and PI16/01511 grants, published maps and institutional affiliations. and the CIBER Enfermedades Respiratorias Network; and by the Spanish Society of Pneumology and Thoracic Surgery (SEPAR) through grant 16/023. A.O’G., L.M-T., P.J.S., E.S. and S.H. were supported by The Francis Crick Institute which receives its core Open Access This article is licensed under a Creative Commons funding from Cancer Research UK (FC001126), the UK Medical Research Council Attribution 4.0 International License, which permits use, sharing, (FC001126), and the Wellcome Trust (FC001126); before that by the UK Medical adaptation, distribution and reproduction in any medium or format, as long as you give Research Council (MRC U117565642). S.L.P., A.S-B., and E.H. were funded by the Royal appropriate credit to the original author(s) and the source, provide a link to the Creative Veterinary College and The Francis Crick Institute. M.S. was funded by grants POCI-01- Commons license, and indicate if changes were made. The images or other third party 0145-FEDER-028955, and by FCT through Estimulo Individual ao Emprego Científico. material in this article are included in the article’s Creative Commons license, unless K.L.F. was funded by FCT PhD scholarship SFRH/BD/114405/2016. indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from Author contributions the copyright holder. To view a copy of this license, visit http://creativecommons.org/ L.M-T. and A.O’G. conceived and designed the experiments. L.M-T and A.O’G. wrote licenses/by/4.0/. the manuscript. M.S. managed some of the experiments in the study, and V.P. provided advice and technical help on neutrophil studies, and both provided feedback on data analysis and quantitation and helpful discussion for the project. L.M-T. performed © The Author(s) 2020 most experiments, compiled the data and prepared the figures. E.S. assisted in all 18 NATURE COMMUNICATIONS | (2020)11:5566 | https://doi.org/10.1038/s41467-020-19412-6 | www.nature.com/naturecommunications