Fig 2: Depletion of either Oxgr1 or Irg1 disrupts the clearance of respiratory bacteria.(A and B) Anesthetized mice of the indicated genotype (n = 4–10 per group) were intranasally administrated the P. aeruginosa strain PAO1 (1 × 107 CFU). BAL and lung tissue were collected 12 hours after infection. Representative images of PAO1 colonies on CTAB-selective plates after overnight culturing of BAL and lung from infected mice (dilution: 104-fold) (A) and quantification of CFU (B) are shown. *P < 0.05 and **P < 0.01, by 1-way ANOVA. Data indicate the mean ± SEM. (C and D) Anesthetized mice of the indicated genotype (n = 4–10 per group) were intranasally administrated 20 μL saline containing PAO1 (1 × 107 CFU) with or without 1 mM ITA. The CFU in BAL (C) and lung tissues (D) were quantified. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA. Data indicate the mean ± SEM.

Fig 3: Irg1 KO macrophages exhibit strong antigen presentation capability.A. The levels of pro-inflammatory cytokines IL-6, TNFα, MCP-1 and IL1β in bone marrow derived macrophages (BMDM) of Wt and Irg1 KO upon LPS stimulation for 6hrs (N=5). B. Bar graph represents mRNA expression of IL-6, TNFα and MCP-1 in BMDM of Wt and Irg1 KO upon LPS stimulation for 6 hrs (N=5). C. Flow cytometry plots (left) of Class II+ and CD86+ populations in BMDM of Wt and KO upon IFNγ stimulation for 24hrs and quantifications of Class II+ and CD86+ cells with histogram overlay (right) of Class II+ and CD86+ cells between Wt and Irg1 KO (n=5). D. Flow cytometry plot gated (left) on CD45hi+CD11b+F4/80+ (infiltrated macrophages) showing Class II+ populations in Wt and Irg1 KO and quantifications (right) of Class II+ cells with histogram overlay of Class II+ cells between Wt and Irg1 KO (N=5). E. Representative flow cytometry tSNE plot (left) of BILs gated on CD45+ cells demonstrating Class II+ populations in Wt and Irg1 KO mice and bar graph at right side showing populations of Class II expressing myeloid cells CD11b+, F4/80+, CD11c+ and Ly6C+ cells (N=5). F. Based on cell surface markers, bar graph represents populations of Class II expressing inflammatory DCs (CD45+CD11c+Class II+), monocytic DCs (CD45+CD11b+CD11c+Class II+), infiltrated monocytes (CD45+CD11b+Ly6C+Class II+) and microglia cells (CD45lowCD11b+Class II+) (N=5). **p < 0.01, and ***p < 0.001 compared to Irg1 KO group using student’s t test, one-way ANOVA.

Fig 4: Loss of Irg1 pushes CD4+ Cells towards more pathogenic Th17 subsets.A. Representation of ELISA analysis of cytokine IL17a level in pathogenic and non-pathogenic Th17 cells isolated from Wt and Irg1 KO mice (N=5). B. Representative flow cytometry plots (left) of IL17a+ populations in npTh17 & pTh17 cells and data is presented as bar graph (right) showing IL17a+ cells (MFI) in npTh17 and pTh17 cells (n=5). C. The heatmap showing mRNA expression of Th17 pathogenic and non-pathogenic signature genes in Wt and KO mice (N=3). D. An immunoblot data of RORγt (normalized with β-actin) in npTh17 and pTh17 cells of Wt and Irg1 KO. Data is representative of three independent experiments. E. Chip assay was performed to determine binding of RORγt on IL17a promoter. Data presented as a percentage fold of RORγt binding on IL17 promoter with respect to total input. Data is representative of three independent experiments. ***p < 0.001 compared to Irg1 KO using student’s t test, one-way ANOVA.

Fig 5: Irg1 KO macrophages are more potent in pushing Wt CD4 T cells towards pTh17.A. Schematic overview of co-culture study using Wt CD4 cells with Wt and Irg1 KO macrophages. B. Bar graph represents data from ELISA assay demonstrating levels of pro-inflammatory cytokines released by Wt CD4 upon interactions with Wt and Irg1 KO macrophages (N=5). C. Flow cytometry data (left) gated on CD4+ cells demonstrating the populations of double positive (IL17a+GM-CSF+) upon coculture of Wt CD4 with Wt and Irg1 KO macrophages and MFI values of positive populations of IL17a+, GMCSF+ and double positive (IL17a+GM-CSF+) are presented as a bar graph (right) (N=5). ***p < 0.001 compared to Irg1 KO group using student’s t test, one-way ANOVA.