Extended Data Fig. 2: ATG5 functions independent of autophagy to suppress NET release during M. tuberculosis infection in vivo and in vitro.

(A) Representative confocal immunofluorescence images of lung sections at 60X magnification from GFP-Mtb infected mice at 14 dpi that were probed with antibodies to detect citrullinated histone H3 (H3Cit; red), Ly6G (neutrophil marker; cyan), and DNA (Hoechst; blue). GFP-Mtb is also shown. Scale bar is 50μm. (B) The % of H3Cit⁺ pixel area per field and (C) the percent of Ly6G⁺ pixel area that was H3Cit⁺ per lung section in confocal immunofluorescence images of lung sections from GFP-Mtb infected mice at 14 dpi. (B-C) Each data point is from one field of view from 4 mice from at least two separate infection experiments. (D) The proportion of H3Cit⁺ neutrophils in the lungs of GFP-Mtb infected mice at 21 dpi that are GFP-Mtb^- (bystander) and GFP-Mtb⁺ determined by flow cytometry. Each data point is from one mouse, compiled from 2 independent experiments. (E) Representative SEM images of mouse neutrophils infected with GFP-Mtb in vitro at 3000x magnification. Yellow arrows indicate NETs being released from cells. Scale bar is 10μm. (F) The number of NETs released per 10 cells quantified from SEM images at 1000X magnification of neutrophils infected with GFP-Mtb for 4 or 18 hpi. Each datapoint represents one field with 3 fields containing 10-40 cells/field compiled from 3 independent experiments. (G) Proportion of neutrophils infected in vitro with GFP-Mtb that are GFP-Mtb⁺ at 4 hpi, and (H) GFP-Mtb MFI in GFP-Mtb⁺ neutrophils at 4 hpi measured by flow cytometry. (I) The proportion of GFP-Mtb^- (bystander) and GFP-Mtb⁺ neutrophils from GFP-Mtb infected neutrophil cultures at 4 hpi that are H3Cit⁺ as determined by flow cytometry. (J) Representative confocal immunofluorescence microscopy images of neutrophils mock treated in vitro for 4 or 18 h and stained for H3Cit (red), neutrophil marker myeloperoxidase (MPO), and DNA (Hoechst, blue) (60X magnification). Scale bar is 50μm. (K) The proportion of Hoechst⁺ cells that were H3Cit⁺ and (L) the number of extracellular H3Cit⁺ NETs released per 100 cells after mock-infection of neutrophils for 4 or 18 h, as quantified from fluorescence microscopy experiments. (M) Representative confocal immunofluorescence microscopy images of neutrophils infected in vitro with GFP-Mtb for 4 or 18 h and stained for H3Cit (red) and DNA (Hoechst, blue) (60X magnification). Scale bar is 50μm. (N) The proportion of Hoechst⁺ cells that were H3Cit⁺ and (O) the number of extracellular H3Cit⁺ NETs released per 100 cells after GFP-Mtb infection of neutrophils for 4 or 18 h, as quantified from fluorescence microscopy experiments. (P-R) Assessment of single copy LysM-Cre mediated Cre-Lox recombination efficiency of Atg5, Becn1 and Atg16l1 in bone marrow neutrophils from 3 biological replicates reported as percent recombination by calculating the percentage of the deletion PCR product intensity over the combined intensity of the floxed product and the deleted product. (P) Samples are from Atg5^fl/fl and Atg5^fl/fl-LysM-Cre mice, where Atg5^fl/fl-LysM-Cre neutrophils exhibit 96.30%, 94.91% and 97.05% recombination efficiency. (Q) Samples are from Becn1^fl/fl and Becn1^fl/fl-LysM-Cre mice, where Becn1^fl/fl-LysM-Cre neutrophils exhibit 94.54%, 90.02% and 95.32% recombination efficiency. (R) Samples are from Atg16l1^fl/fl and Atg16l1^fl/fl-LysM-Cre mice, where Atg16l1^fl/fl-LysM-Cre neutrophils exhibit 94.07%, 93.33% and 92.93% recombination efficiency. (S) Mtb CFU recovered from cultures of neutrophils infected with GFP-Mtb in vitro for 4 or 18 hpi, reported as a ratio to 0 hr CFU. Each datapoint is from an independent well of infected cells compiled from at least 2 independent experiments. (T) Kaplan Meier curve of survival proportions during Mtb infection of mice. The median survival time for each genotype is reported. (U) Log transformed graph of the colony forming units (CFU) in the right lung of mice at 21 dpi. (V) Proportion of CD45⁺ cells that are neutrophils (Ly6G⁺ CD11b⁺) in the lungs of mice at 21 dpi. (W) Proportion of neutrophils (CD45⁺ Ly6G⁺ CD11b⁺) infected in vitro with GFP-Mtb at 4 hpi that are positive for surface staining for the primary granule marker CD63, and (X) the MFI of CD63 on CD63⁺ neutrophils at 4 hpi determined by flow cytometry. Each datapoint represents one biological replicate compiled from 2 independent experiments. All graphs report the mean ± SD. Statistical differences were determined by an unpaired two-tailed student t-test (B-C, F-H, K-L, W-X), one-way ANOVA and Šídák multiple comparison test (I, N-O, S, U-V) or a log-rank Mantel-Cox test (T). * P < 0.05, ** P < 0.01, and **** P < 0.0001. Differences that are not statistically significant are designated as ns. Complete statistical analysis including the number of samples used is in Supplementary Table 4.