Type I IFN signaling mediates protection in the absence of IL-1 signaling in rpoB-H445Y Mtb infection

IL-1 signaling is essential in controlling wt Mtb infection [15,18]. However, this signaling pathway is dispensable when mice are infected with rpoB-H445Y Mtb [24], a drug-resistant but non-isogenic Mtb strain derived from wt Mtb [25], suggesting that there are alternate signaling pathways mediating protection. To determine if type I IFN signaling was protective in the absence of IL-1 signaling against rpoB-H445Y Mtb in vivo, we generated mice lacking both IL-1 and type I IFN receptors and evaluated their susceptibility to wt or rpoB-H445Y Mtb infection. As expected, Il1r1-/- mice were unable to control wt Mtb infection, with elevated lung burden at 14- and 30-days post-infection (dpi), relative to C57BL/6 (B6) mice (Fig 1A), whereas loss of Ifnar1 alone has no impact on Mtb burden. As before, following wt Mtb infection [18,20], the increase in lung burden caused by loss of Il1r1 was reversed in mice lacking both Ifnar1 and Il1r1 [double knockout (DKO)] (Fig 1A). An increase in the susceptibility of Il1r1^-/- mice was accompanied by an increase in inflammatory damage in the lungs of wt Mtb infected (S1A Fig), but this inflammatory damage did not significantly decrease in the DKO mice where Mtb control was evident. We also observed increased numbers of recruited macrophages (RMs) expressing high levels of Major Histocompatibility Complex Class II (MHCII^hi), a surrogate marker for activation, of neutrophils, and of MHCII^hi monocytes in the lungs of wt Mtb-infected Il1r1^-/- mice (S1B Fig). In the infected DKO mice where susceptibility was reversed, only the numbers of neutrophils were significantly decreased. We found no differences in the numbers of cytokine-producing, activated (CD44^hi) T lymphocyte populations between the infected groups (S1C and S1D Fig). A different pattern of susceptibility was apparent when we infected the mice with rpoB-H445Y Mtb. We observed higher bacterial burdens only in the DKO mice at 30 dpi (Fig 1B), while loss of either IL1 or type I IFN signaling alone did not impact lung rpoB-H445Y Mtb burden. The increased bacterial load in the DKO rpoB-H445Y Mtb-infected lungs also coincided with increased lung inflammation (Fig 1C) and increased numbers of lung MHCII^hi RMs and MHCII^hi myeloid dendritic cells (mDCs) in the DKO mice at 30 dpi (Fig 1D). However, there were no differences in the numbers of neutrophils, MHCII^hi monocytes, MHCII^hi alveolar macrophages (AMs), and cytokine-producing, CD44^hi T lymphocyte populations between the DKO mice and B6 and Il1r1^-/- mice (S2A–S2E Fig). As previously noted [25], loss of Ifnar1 alone was sufficient to drive an increase in the numbers of these cellular populations and the amount of inflammation during rpoB-H445Y Mtb infection. Thus, while type I IFN signaling is dispensable globally for control of wt and rpoB-H445Y Mtb infection, in the absence of IL-1 signaling, type I IFN signaling is necessary for immunity against rpoB-H445Y Mtb.

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Fig 1. Type I interferon signaling is protective in the absence of interleukin-1 signaling in rpoB-H445Y Mycobacterium tuberculosis infection.

C57Bl/6 (B6), Ifnar1^-/-, Il1r1^-/-, and Ifnar1^-/- Il1r1^-/- [double knockout (DKO)] mice were aerosol infected with a low dose of wt or rpoB-H445Y Mtb. Lung Mtb burden was evaluated at 14- and 30-days following A) wt or B) rpoB-H445Y Mtb infection. C) Formalin-fixed, paraffin-embedded (FFPE) lung sections were stained with hematoxylin and eosin (H&E), and the total inflammatory areas were quantified after rpoB-H445Y Mtb infection at 30 days post-infection (dpi). D) Using flow cytometry, the total numbers of lung MHCII^hi RMs and mDCs were determined at 30 dpi after infection with rpoB-H445Y Mtb. The data shown represent the means ± SD of seven to ten mice per experiment. The data were evaluated for normality using the Shapiro-Wilk Test and passed (p-value > 0.05). Two-way ANOVA was used for A and B, and One-way ANOVA was used for C and D, with Tukey’s multiple comparisons test. Significant differences are indicated with asterisks (*, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001; ****, p-value ≤ 0.0001) by appropriate statistical tests. One of three independent experiments shown.

https://doi.org/10.1371/journal.ppat.1012137.g001

Similar type I IFN transcriptional pathways are induced during wt and rpoB-H445Y Mtb infections but result in divergent infection outcomes

Type I IFN signaling has been thought to be detrimental during wt Mtb infection based upon its association with the progression of TB patients towards active disease and its ability to antagonize the protective IL-1 signaling pathway [18,20,22]. To discern the roles of type I IFN signaling during both Mtb infections, we determined the lung transcriptional landscape in response to either infection at 14 days post-infection, an early timepoint before significant differences in lung rpoB-H445Y Mtb burden were apparent in the gene-deficient mice. We identified the most differentially expressed genes (DEGs) across groups and categorized them by pathways (Fig 2). In the lungs of mice infected with wt Mtb, genes related to IFN signaling were upregulated in the B6 and Il1r1^-/- mice compared with Ifnar1^-/- and DKO mice (Fig 2A). Specific analysis of the type I IFN transcriptional signature revealed downregulation of genes in this pathway in the Ifnar1^-/- and DKO mice when compared to the B6 and Il1r1^-/- mice during wt Mtb infection (S3A Fig). Additionally, genes involved in antigen presentation via MHC I, such as H2-q4 and H2-t22, were induced by type I IFN signaling during wt Mtb infection at this early timepoint. We also observed induction of genes involved in branched chain amino acid catabolism (Aldh6a1 and Ivd), phagocytosis [26] (Snap23 and Stx4a), endoplasmic reticulum (ER) stress [27,28] (Hspa5 and Calr), and cellular response to heat stress (Hsp90aa1 and St13) specifically in the B6 mice infected with wt Mtb relative to the single knockout and DKO mice (Fig 2A). We observed enrichment of DEGs in pathways involved in type I IFN signaling during wt Mtb infection in both B6- and Il1r1^-/—infected mouse groups, although they differed in their ability to control infection. Together, our data suggest that decreased co-expression of type I IFN-induced genes with genes involved in metabolism and stress could drive detrimental does not determine the outcomes of wt Mtb infection.

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Fig 2. Type I IFN signaling signatures are common across wt and rpoB-H445Y Mtb infections.

C57Bl/6 (B6), Ifnar1^-/-, Il1r1^-/-, and Ifnar^-/- Il1r1^-/- [double knockout (DKO)] mice were aerosol infected with a low dose of wt or rpoB-H445Y Mtb and sacrificed at 14 dpi. RNA was extracted from homogenized lung tissue and sequenced. Heatmaps displaying average expression levels of top differentially expressed genes (DEGs) across mouse genotypes after A) wt or B) rpoB-H445Y Mtb infection with annotated pathways (four to six mice per group). Significant DEGs related to the corresponding reactome pathways are visualized using heatmaps(|log₂FC| >0, adjusted p-value < 0.05). Average expression (normalized) of selected genes on log₂ scale are shown.

https://doi.org/10.1371/journal.ppat.1012137.g002

In the lungs of mice infected with rpoB-H445Y Mtb, we also observed that DEGs related to IFN signaling were upregulated in B6 and Il1r1^-/- mice, whereas these pathways were downregulated in the Ifnar1^-/- and DKO mice (Fig 2B). Indeed, the type I IFN transcriptional signature was downregulated in both the Ifnar1^-/- and DKO mouse groups when compared with either wt or Il1r1^-/- mice following rpoB-H445Y Mtb infection (S3B Fig). Interestingly, expression of some interferon-stimulated genes (ISGs), such as Abce1 and Ptpn11, was higher in the lungs of Il1r1^-/- mice than in B6 mice after rpoB-H445Y Mtb infection. Another IFN-related gene, Ube2l6, decreased in expression in the rpoB-H445Y Mtb infected Il1r1^-/- mice lungs relative to infected B6 counterparts (Fig 2B). These findings suggest that IL-1 signaling plays a complex role in the regulation of IFN-related genes. Coincident with the upregulation of IFN-related genes in B6 and Il1r1^-/- rpoB-H445Y Mtb-infected mice, genes involved in metabolic pathways, such as fatty acid metabolism and cholesterol biosynthesis were upregulated. In contrast, mice lacking IL-1 signaling exhibited decreased expression of genes, such as Slc2a3 and Slc4a1, that are part of the solute carrier (SLC)-mediated transmembrane transport family. Therefore, our data indicate that the loss of rpoB-H445Y Mtb control observed in DKO mice was associated with the downregulation of various cellular processes that could be triggered by either type I IFN or IL-1 signaling. Many IFN-related genes, such as Isg15, Ifnar1, and Mx2, were induced following either wt or rpoB-H445Y Mtb infection. In contrast, genes such as Abce1 and Ptpn11 were only upregulated following rpoB-H445Y Mtb infection, whereas Uba7 was upregulated only during wt Mtb infection. Furthermore, Ube2l6, which exhibited differential regulation in Il1r1^-/- and B6 mice during rpoB-H445Y Mtb infection, was upregulated in both groups of mice during wt Mtb infection. So, while we observed broad induction of many IFN signaling-related genes during both Mtb infections, some IFN-related genes were differentially induced across wt and rpoB-H445Y Mtb infections.

CD11b⁺ myeloid cells produce type I IFN, while LysM⁺ cells respond to type I IFN following rpoB-H445Y Mtb infection

Type I IFN is rapidly produced by many cell types in response to infection [21,29]. We used IFNβ-YFP reporter mouse strain, in which cells that produce IFN β also co-express YFP and are readily detectable [30–32] (Fig 3A), and found that type I IFN production was induced in various myeloid lung populations following infection with rpoB-H445Y Mtb (Fig 3B). As early as 7 dpi, rpoB-H445Y Mtb infection lead to significantly higher accumulation of IFN-producing lung monocytes and RMs as compared with uninfected controls. No discernable production of type I IFN in AMs, neutrophils, or any non-immune (CD45^-) population was detected at this timepoint. Plasmacytoid dendritic cells (pDCs), a major producer of type I IFN during bacterial or viral infection [14,32], were not readily detected in our analysis. We then tracked type I IFN production in rpoB-H445Y Mtb infection at later timepoints (Fig 3B). Lung monocytes were also the primary cell type expressing type I IFN production at 14 dpi, and RMs were the major cellular population that was producing type I IFNs at 28 dpi. Taken together, our data indicate that the main sources of type I IFN production in rpoB-H445Y Mtb infection are CD11b⁺ myeloid cells, namely monocytes and RMs.

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Fig 3. Type I IFN producers are CD11b⁺ myeloid cells, while LysM⁺ cells respond to type I IFN following rpoB-H445Y Mtb infection.

IFNβ-YFP mice were aerosol infected with rpoB-H445Y Mtb and sacrificed at the indicated timepoints along with uninfected controls. A) Representative histograms for various subsets [Monocytes (left), Recruited Macrophages (RMs) (middle left), Neutrophils (middle right), and Alveolar Macrophages (AMs) (right)] depicting YFP expression by intracellular antibody staining (green) compared to controls [uninfected IFNβ-YFP mice stained with intracellular antibody (orange), infected IFNβ-YFP mice stained with intracellular isotype antibody (blue), and infected B6 non-reporter mice stained with intracellular antibody (red)]. B) The frequencies of YFP⁺ cells were determined by flow cytometry following sacrifice of rpoB-H445Y Mtb-infected IFNβ-YFP mice at 7, 14, and 28 dpi, along with uninfected controls. C) Experimental scheme for the LysM-Cre Ifnar1^fl/fl mice with the purple arrow indicating aerosol infection with rpoB-H445Y Mtb and the pink arrow indicating administration of a-IL1R or isotype antibodies i.p. D) Lung Mtb burden was determined at 28 dpi. E) LysM-Cre⁺ Ifnar1^fl/fl (semi-closed green circles) and Cd11c-Cre⁺ Ifnar1^fl/fl mice (semi-closed orange circles), along with littermate controls (open blue circles), were infected with rpoB-H445Y Mtb, and bacterial burden was determined in the lungs at 28 dpi. The data shown represent the means ± SD of three to eight mice per experiment. The data were evaluated for normality using the Shapiro-Wilk Test and passed (p-value > 0.05). Two-way ANOVA and One-way ANOVA, with Tukey’s multiple comparisons tests, were used for B and D-E, respectively. Significant differences are indicated with asterisks (*, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001; ****, p-value ≤ 0.0001) by appropriate statistical tests. One-way ANOVA. Two independent experiments shown.

https://doi.org/10.1371/journal.ppat.1012137.g003

Loss of control of rpoB-H445Y Mtb infection in mice lacking type I IFN and IL-1 signaling was accompanied by an influx of inflammatory myeloid cells, and the primary producers of type I IFN throughout rpoB-H445Y Mtb infection were myeloid cells. To test whether myeloid cells were also responding to type I IFN in rpoB-H445Y Mtb infection in the absence of IL-1 signaling, we generated LysM-Cre Ifnar1^fl/fl mice (deletion of the type I IFN receptor on LysM-expressing myeloid cells [33]), infected these mice with rpoB-H445Y Mtb, and also treated them with IL-1 blocking antibodies or isotype controls (Fig 3C). As we observed in the rpoB-H445Y Mtb-infected Il1r1^-/- mice, blocking of IL-1 signaling with antibody did not impact lung bacterial burden in the Ifnar1^fl/fl mice (Fig 3D). But, similar to the rpoB-H445Y Mtb-infected DKO mice, loss of the type I IFN receptor in LysM-Cre⁺ Ifnar1^fl/fl mice treated with IL-1 signaling blocking antibody led to an increase in lung Mtb burden. Unexpectedly, loss of the type I IFN receptor in the LysM-expressing cells alone led to increased rpoB-H445Y Mtb lung burden (Fig 3D). The increased susceptibility in LysM-Cre⁺ mice infected with rpoB-H445Y Mtb correlated with increased pulmonary inflammation (S4A Fig) and increased accumulation of lung MHCII^hi RMs and mDCs (S4B Fig). Together, our results suggest that type I IFN signaling is necessary in LysM⁺ cells to mediate control of rpoB-H445Y Mtb infection but dispensable when deleted globally as in the Ifnar1^-/- mice. To better understand this apparent discrepancy, we assessed the impact of the loss of type I IFN signaling in CD11c-expressing cells, which include AMs and DCs (Fig 3E). Cd11c-Cre⁺ Ifnar1^fl/fl mice better controlled rpoB-H445Y Mtb infection relative to littermate controls. Thus, during rpoB-H445Y Mtb infection, type I IFN signaling is protective in LysM-expressing cells, especially in the absence of IL-1 signaling, but detrimental in CD11c-expressing cells.

NO production restricts intracellular rpoB-H445Y Mtb growth in murine macrophages

To address the importance of macrophages in producing and responding to type I IFN signals, we utilized bone marrow-derived macrophages (BMDMs) to elucidate the mechanistic role of type I IFN signaling in the absence of IL-1 signaling during rpoB-H445Y Mtb infection. As before [24], BMDMs lacking IL-1 signaling were more susceptible to wt Mtb infection at 6 dpi (Fig 4A). Additional loss of type I IFN signaling rescued this defect in the DKO BMDMs. In contrast, during rpoB-H445Y Mtb infection, loss of either type I IFN or IL-1 signaling led to an increase in intracellular Mtb growth, with loss of both (DKO) causing a further increase in Mtb growth (Fig 4B). We also assessed cell death dynamics during the BMDM infections (S5 Fig). Across the four genotypes, we find that BMDMs lacking only Il1r1 exhibited increased cell death as seen with increased frequencies of cells at apoptotic (AnnexinV^hi 7AAD^lo) and necrotic (AnnexinV^hi 7AAD^hi) stages. While wt Mtb drove high levels of necrotic cell death early in infection, the rpoB Mtb mutant induced high levels of necrosis later during infection. Similarly, high levels of apoptosis were observed in Il1r1^-/- BMDMs only later during rpoB mutant Mtb infection. As both infections induced high levels of cell death in the Il1r1^-/- BMDMs, but not in the DKO BMDMs, these responses could be a general response to Mtb infection that is mediated by type I IFNs [34]. While these increased frequency of cell death in Il1r1^-/- BMDMs coincided with their inability to control wt Mtb infection, the same does not appear to be the case during rpoB Mtb mutant infection, suggesting a role for other host cellular processes to mediate protection against rpoB-H445Y Mtb.

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Fig 4. NO production restricts intracellular rpoB-H445Y Mtb growth in macrophages.

Bone marrow-derived macrophages (BMDMs) from B6, Ifnar1^-/-, Il1r1^-/-, DKO mice were infected with either A) wt Mtb or B) rpoB-H445Y Mtb, and Mtb CFU was determined at 6 dpi. C) The concentration of nitrite in the supernatants of wt or rpoB-H445Y Mtb infected BMDMs was determined, along with uninfected controls. D) B6, Il1r1^-/-, Ifnar^-/- and DKO BMDMs were infected with rpoB-H445Y Mtb and then treated with the indicated concentrations of an NO scavenger, Carboxy-PTIO (C-PTIO). Bacterial burden was determined on 6 dpi. Fold change in Mtb growth was determined relative to untreated and infected BMDMs. E) B6 and Nos2^-/- BMDMs were infected with rpoB-H445Y Mtb, and Mtb CFU was determined at 6 dpi. The data shown represent the means ± SD of three to four biological replicates per experiment. The data were evaluated for normality using the Shapiro-Wilk Test and passed (p-value > 0.05). One-way ANOVA and Two-way ANOVA, with Tukey’s multiple comparisons tests, were used for A-B and C-D, respectively. Unpaired two-tailed student’s t-test was used for E. Significant differences are indicated with asterisks (*, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001; ****, p-value ≤ 0.0001) by appropriate statistical tests. One of three independent experiments shown.

https://doi.org/10.1371/journal.ppat.1012137.g004

Type I IFN signaling can drive nitric oxide (NO) production as part of its anti-bacterial functions [29]. Thus, we assessed the abundance of nitrite, the byproduct of NO generation, in the supernatants of wt or rpoB-H445Y Mtb infected BMDMs (Fig 4C). BMDMs from mice with functional type I IFN and IL-1 signaling produced increased NO in response to rpoB-H445Y Mtb infection when compared with other infected BMDMs. While there was a reduction in NO production in rpoB-H445Y Mtb infected BMDMs lacking either IL-1 or type I IFN signaling relative to infected B6 BMDMs, BMDMs from these two groups still produced more NO than their infected DKO counterparts. The stepwise decrease in NO production and the stepwise increase in CFU between B6 BMDMs to Ifnar1^-/- and Il1r1^-/- BMDMs to DKO BMDMs support the idea that NO production is associated with protection against rpoB-H445Y Mtb infection. To assess the role of NO in controlling rpoB-H445Y Mtb infection, we used carboxy-PTIO (C-PTIO), an NO scavenger, at varying concentrations (Fig 4D). Relative to untreated controls, the addition of 100 uM C-PTIO resulted in a loss of control in B6 BMDMs during rpoB-H445Y Mtb infection. In contrast, the addition of 1 uM and 10 uM of C-PTIO was sufficient to increase the susceptibility of Ifnar1^-/- and Il1r1^-/- BMDMs, respectively, while adding any amount of C-PTIO did not impact the course of infection in DKO BMDMs. This “minimum susceptibility” analysis suggests a stoichiometric role for NO in limiting rpoB-H445Y Mtb during infection. To more specifically assess the role of NO production in controlling rpoB-H445Y Mtb infection, we generated BMDMs from Nos2^-/- bone marrow and compared their ability to control bacterial replication relative to B6 BMDMs (Fig 4E). Similar to B6 BMDMs where NO production was inhibited by C-PTIO addition, BMDMs that were unable to produce NO due to lack of the inducible nitric oxide synthase were unable to control mycobacterial growth. Collectively, our data show that the protective mechanisms underlying type I IFN signaling are partially through NO production to restrict intracellular rpoB-H445Y Mtb replication.

Ethics statement

Protocols involving the use of animals were approved by the Washington University in St. Louis or The University of Chicago Institutional Animal Care and Use Committee (IACUC) guidelines. All of the experiments were performed in accordance with the protocols.

Mice

C57BL/6J (B6), Ifnar1^−/−, Il1r1^-/-, Nos2^-/-, and Ifnb^mob mice on the B6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Ifnar1^-/- Il1r1^-/- mice were generated by crossing Ifnar1^−/− and Il1r1^-/- mice. LysM-Cre Ifnar1^fl/fl and Cd11c-Cre Ifnar1^fl/fl mice were generously provided by the Baldridge Lab (Washington University in St. Louis). In some experiments, LysM-Cre⁺ and LysM-Cre^- Ifnar1^fl/fl mice were administered sterile PBS or a-IL1R blocking antibody (BioXCell) at 100 ug/mouse in 200uL every other day intraperitoneally (IP) starting at -1 dpi. All mice were maintained in the animal facility at Washington University in St. Louis and bred in house. Experimental mice were age and sex matched and infected between the ages of 6 and 8 weeks. All mice were maintained and used in accordance with the approved Washington University in St. Louis Institutional Animal Care and Use Committee guidelines.

Generation of BMDMs

Bone marrow derived macrophages (BMDMs) were generated as previously described [11,24]. Briefly, bone marrow cells were collected from the tibia and femur of B6 mice and cultured in complete Dulbecco’s modified eagle’s medium (cDMEM) with 20 ng/mL of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Cells were cultured at 37°C in 5% CO₂ and supplemented with media on day 3. On day 7, floating cells were taken up and discarded, and adherent cells were collected as BMDMs.

Mtb infections

Mtb strain HN878 (wt) was obtained from BEI resources (Manassas, VA) under National Institutes of Health contract AI-75320. Independent rifampicin resistant Mtb colonies (biological replicates) were selected from rifampicin (2 μg/ml) containing 7H11 agar plates [3]. The sequences of rpoB were confirmed by Sanger sequencing (Genewiz), and Mtb stocks were created for further experimentation. The whole genomes of all Mtb strains were previously sequenced, with all SNPs between both, wt and rpoB-H445Y, Mtb strains are published [25]. All Mtb strains were cultured in Proskauer Beck medium supplemented with 0.05% Tween 80 and frozen at −80°C while in mid-log phase prior to use in mouse and macrophage infections. Colony forming units (CFU) of the bacterial stocks were determined through serial dilutions on 7H11 agar plates. Mice were aerosol infected with low doses (~100 CFU) of indicated Mtb strains in sterile PBS using a Glas-col nebulizer [71]. Mtb bacterial burden/organ was quantitated by plating serial dilutions of homogenized lung tissue on 7H11 agar plates (BD Biosciences, Franklin Lakes, NJ). Plates were incubated for 2–3 weeks at 37°C, and the number of colonies were counted. In vitro infections were carried out at a multiplicity of infection (MOI) of 1 in antibiotic free media for six days. In some experiments, macrophages were treated with indicated amounts of carboxy-PTIO (C-PTIO) four hours after the start of infection. Bacterial burden in each well was determined through serial plating of supernatant and lysed macrophages after six days post infection.

Flow cytometry

Lungs were perfused with heparin in PBS, minced, digested with DNAse/collagenase, lysed for red blood cells, and pressed through a 70 μm filter to generate a single cell suspension [71]. For quantification of cytokine responses, single cell suspensions were stimulated ex vivo with purified Mtb antigens ESAT6 and Ag85B (10 μg/mL) overnight followed by a five hour stimulation with brefeldin and monensin (BD Biosciences) as previously described [9,10]. Cells were treated with Fc Block (CD16/CD32,2.4G2, Tonbo Biosciences) and stained with appropriate fluorochrome-labeled specific antibodies or isotype control antibodies. Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences). Mouse antibodies used include CD11b (M1/70; Tonbo Biosciences), CD11c (HL3; BD Biosciences), Gr-1 (RB6–8C5, eBioscience), SiglecF (E50-2440; BD Biosciences), CD64 (X54-5/7.1; Biolegend), GFP polyclonal antibody (Cat#A21311; Thermo Fisher) or rabbit IgG isotype control (Catalog # 11-4614-80), CD3 (500A2; BioLegend), CD4 (RM4–5; BD Biosciences), CD44 (IM7; Tonbo Biosciences), IFN-γ (XMG1.2; Tonbo Biosciences) or isotype control IgG1, k (A85-1; BD Biosciences), CD8 (53–6.7; BD Biosciences), and MHC Class II (M5/114.15.2; Tonbo Biosciences).

Cells were processed with the Becton Dickinson (BD) Fortessa X-20 flow cytometer using FACS Diva software, or the BD FACSJazz flow cytometer using FACS software (BD). Flow cytometry experiments were analyzed using FlowJo (Tree Star Inc.). As before [11,24,72], neutrophils were defined as CD11b⁺CD11c^-Gr-1^hi, monocytes were defined as CD11b⁺CD11c^-Gr-1^med cells, and recruited macrophages (RMs) were defined as CD11b⁺CD11c^-Gr-1^low cells. Myeloid dendritic cells (mDCs) were defined as CD11b⁺CD11c⁺ cells and alveolar macrophages (AMs) were defined as CD11b^-CD11c⁺SiglecF^hiCD64^hi. T cells were defined as CD3⁺CD4⁺ or CD3⁺CD8⁺ cells. Total numbers of cells within each gate were back-calculated based on cell counts/individual sample.

For the determination of frequencies of apoptotic and necrotic cells, BMDMs were collected at the indicated timepoints and stained with Annexin V (PE) and 7AAD as per the instructions of the PE Annexin V Apoptosis Detection Kit (559763, BD Pharmigen), prior to processing on the X-20 flow cytometer.

Histology

Lung lobes were perfused with 10% neutral buffered formalin and embedded in paraffin. Formalin-fixed paraffin-embedded (FFPE) lung sections were stained with hematoxylin and eosin (H&E) and inflammatory features were evaluated by light microscopy. Inflammatory lesions were outlined with the automated tool of the Zeiss Axioplan 2 microscope (Carl Zeiss) and the total inflammatory area in each lung lobe was measured.

Protein determination

BMDMs were either stimulated with Mtb antigens for 1 day or left unstimulated, lysed in RIPA Lysis Buffer (Santa Cruz Biotechnology), and heat denatured in reducing sample buffer (Thermo Fisher Scientific). Proteins were separated in 4% to 20% polyacrylamide gradient gels (Bio-Rad) and transferred onto PVDF membranes. Membranes were probed with primary antibodies against IFNAR1 (R&D, Cat#AF3039), and GAPDH (Santa Cruz Biotechnology, Cat# SC-25778) followed by incubation with HRP-conjugated anti–rabbit IgG secondary antibody (Santa Cruz Biotechnology) and developed by Clarity Western ECL Substrate (Bio-Rad).

RNA isolation, sequencing, and processing

In brief, lung tissue was homogenized in RLT buffer, and total RNA was extracted with RNeasy Mini Kit (Qiagen) as previously described [11]. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12–15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. RNA-seq reads were then aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3–8 software (GTAC MGI).

The read count data of each gene were generated using featureCounts from Rsubread package [73] with default parameter settings. Significantly differentially expressed genes across different groups were identified using DESeq2, version 1.30.1[74], with default settings and a minimum p-value significance threshold of 0.05 (after FDR correction for the number of tests)[75]. Pathway enrichment analysis among differentially expressed gene sets of interest was performed for Reactome pathways using ReactomePA [76] with p-value ≤ 0.05 after FDR correction. Gene set enrichment analysis was also performed among differentially expressed gene sets of interest with hallmark gene sets for Mus musculus using fgsea [77] with eps set to zero to estimate p-value more accurately and p-value ≤ 0.05 after FDR correction. Additionally, several R packages were used including stringr [78], pheatmap [79], ggplot2[80], and org.Hs.eg.db [81] for data processing and generate figures.

Statistical analysis

All datasets were evaluated for normality using the Shapiro-Wilk Test. Differences between the means of more than two groups were analyzed using One-way ANOVA or Two-way ANOVA, where applicable, with Tukey’s post-tests for normally distributed distributions or Kruskal Wallis Test with Dunn’s multiple comparisons tests. Differences between the means of two groups were analyzed by the unpaired two-tailed student’s t-test. All statistical analyses were done in GraphPad Prism 9. A p-value < 0.05 was considered significant. The data points across the figures represent the mean (±SD or +SEM) of values as noted. *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001; ns-not significant (p>0.05). All experiments were replicated for reproducibility.