Presence of Mtb in human pulmonary tuberculosis patients:

(A) Hematoxylin and eosin (H and E) staining of the human autopsied liver tissue sections showing the presence of granuloma-like structure in the Mtb infected liver. (B) Acid-fast staining showing the presence of Mtb in the liver section of pulmonary TB patients. (arrows point to the presence of Mtb in the enlarged image) (C-D) Auramine O-Rhodamine B (A-O) staining and fluorescence in-situ hybridization (FISH) with Mtb specific 16s rRNA primer further confirms the presence of Mtb in human liver tissue sections. Data shown here is a representative of 5 autopsied specimens having Mtb infection. (E). Dual staining of β-actin (green) and Ag85B (red) using respective antibodies shows the presence of Mtb in hepatocytes of human biopsied liver sections.

Involvement of the liver and hepatocytes in a mouse aerosol model of Mtb infection.

(A). Schematic denoting the flow of experimental set up of studying the liver at tissue level and cellular level (generated by Biorender.com). (B) C57BL/6 mice were infected with 200 CFU of H37Rv through the aerosol route and the bacterial burden of the lung and liver was enumerated at different time points post-infection in lung and spleen (C). Alexa Fluor 488 Phalloidin and DAPI staining of uninfected and infected lungs at 8 weeks post-infection. Images were taken in 20X, and 40 X magnification as mentioned (scale bar is 20 μm). (D). Immunofluorescence staining of Alexa Fluor 488 Phalloidin, DAPI, and Ag85B in the infected mice liver at 8 weeks post-infection shows the presence of Ag85B signals within hepatocytes (magnified image) (scale bar is 20 μm). (E). Isolated primary hepatocytes stained with phalloidin green and DAPI show the typical polygonal shape with binucleated morphology. (F). Anti-asialoglycoprotein receptor (ASGR1) antibody staining specifically stains primary hepatocytes isolated from infected mice as validated by the contour plot in flow cytometry. (G). Anti-asialoglycoprotein receptor (ASGR1) antibody staining specifically stains primary hepatocytes isolated from mice as visualized through confocal microscopy. (H). Primary hepatocytes were isolated from the infected mice, lysed and CFU enumeration was done at the mentioned time points. (I). Ag85B staining of cultured primary hepatocytes, isolated from mice at 8 weeks post Mtb infection. (scale bar is 20 μm). (G). Animal litter size (n)= 6-7 mice in each group.

Mtb uses hepatocytes as a replicative niche:

(A and B) Representative microscopic images showing the infection of primary hepatocytes (PHCs), AML-12, HepG2, and Huh-7 with labelled Mtb-H37Rv strains and subsequent quantification of percentage infectivity in the respective cell types. RAW 264.7, THP-1 and murine BMDMs were used as macrophage controls. The scale bar in all images is 10 µm except in HepG2, which is 5 µm (C) CFU enumeration of Mtb-H37Rv in different hepatic cell lines. (D and E) Representative confocal microscopy images of Mtb-GFP-H37Rv infected PHCs at the respective time points post-infection and bar blot depicting mean fluorescent intensity (MFI) / cell at the respective time points. (F and G) Representative confocal microscopy images of Mtb-H37Rv-GFP infected HepG2 at the respective time points post-infection and bar blot depicting MFI/cell at the respective time points, n= 4 independent experiments, with each dot representing 5 fields of 30-60 cells. Scale bar 10 µm. Growth kinetics of Mtb-H37Rv in (H) Fold change of growth rate of Mtb within HepG2, PHCs, RAW 264.7, THP-1 and BMDMs.

RNA sequence analysis of infected and sorted hepatocytes at 0 hours and 48 hours post-infection

(A) Experimental setup for infection of HepG2 with Mb-H37Rv-mCherry at 10 MOI with histogram of mCherry signals at 0- and 48-hours post-infection(Schematic depicting experimental set up generated with Biorender.com) (B) Principal component analysis (PCA) plot illustrating the HepG2 transcriptome, identified through global transcriptomic analysis of 0 hours uninfected and infected and 48 hours uninfected and infected. (C) GO pathway enrichment analysis was done for DEGs with adjusted p-value < 0.05 at 0- and 48-hours post-infection. The bubble plot depicts the enrichment of pathways on the mentioned time points post-infection, where the coordinate on the x-axis represents the gene ratio, the bubble size represents the gene count and colour represents the p-value. (D and E) Volcano plot depicting the fold change of different genes in 0 hours and 48 hours post-infection, the red dots represent the significantly upregulated and downregulated genes.

Increased fatty acid biogenesis drives Mtb growth in hepatocytes:

(A) Increased number of LDs/cells in HepG2 and PHCs post-Mtb infection (B). Quantification of the number of lipid droplets in the infected HepG2 and PHCs with their uninfected control, 50-70 cells were analyzed from 3 independent experiments. (C). Increase in the BODIPY intensity at different days post-infection in infected hepatocytes (D). A high degree of colocalization of Mtb-H37Rv-GFP (green) with lipid droplets (red) within PHCs (E) Mass spectrometric quantification of the different species of DAGs, TAGs, and cholesterol esters in the Mtb infected hepatocytes. (F). Confocal images showing puncta of fluorescently labelled fatty acid in Mtb derived from metabolically labelled (with BODIPY 558/568 C12) HepG2. Relative CFU of Mtb under the administration of different inhibitors of the lipid metabolic pathway in (G) PHCs (H) HepG2 and (I) THP-1. Representative data from 3 independent experiments. Data were analyzed using the two-tailed unpaired Student’s t-test in B and one-way ANOVA in C, F, G, and H.*p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. ns=non-significant.

PPARγ driven lipid biogenesis drives Mtb growth in hepatocytes:

(A) Heat map showing the fold change of the genes involved in the lipid biosynthesis and LD biogenesis in liver across weeks 2, 4 and 8, post infection. (B) Kinetic increase in the gene expression of Pparγ transcript levels across different weeks post infection (C) Immunoblot showing increased PPARγ in PHCs (MOI: 10) at 5, 24 and 48 hours post-infection. (D) Bar plot showing the increased band intensity of PPARγ in the infected PHCs at the mentioned time points. (E) Representative confocal microscopy images showing HepG2 infected with Mtb-mCherry-H37Rv and treated with GW9662 and rosiglitazone (F) MFI/cell of in Mtb-mCherry-H37Rv DMSO treated, GW9662 and rosiglitazone treated HepG2 cells. (G) Representative confocal images of uninfected, infected, GW9662 and rosiglitazone treated infected HepG2 showing changes in the number of LDs. (H) Plot depicting the quantification of LDs/cell in the mentioned conditions. Representative data from n=4 biological replicates. Data were analysed by using the two tailed unpaired Student’s t test in D and by one way ANOVA in B, F and H. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, ns=non-significant

Hepatocytes provide a drug-tolerant niche to Mtb:

(A). Experimental scheme of deducting the percentage drug tolerance in hepatocytes (generated with Biorender.com) (B). Percentage tolerance of Mtb-H37Rv against Rifampicin within RAW 264.7, PHCs and HepG2 at different time points post-infection. (C). Percentage tolerance of Mtb-H37Rv against Isoniazid within RAW 264.7, PHCs, and HepG2 at different time points post-infection. Representative data from 3 independent experiments. (D). Percentage tolerance of Mtb-H37Rv against rifampicin within BMDMs, PHCs, and HepG2 as measured microscopically (E). Percentage tolerance of Mtb-H37Rv against isoniazid within BMDMs, PHCs, and HepG2 measured microscopically. Representative data from 3 independent experiments. Each dot represents a single field having more than 4 infected cells. 20 such fields were analyzed (F). Transcript levels of the various DMEs involved in Rifampicin and Isoniazid metabolism in mice liver, 8 weeks post-infection (n=4 mice), the fold change has been calculated by considering the expression in the uninfected mice to be 1. Data were analyzed by using 2-way ANOVA in (B) and (C), one-way ANOVA in (D) and (E) and the two-tailed unpaired Student’s t-test in (F) and Representative data from n=4 biological replicates *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0. 0001.ns=non-significant.