Figure 1.
Infection of human PBMC with Mycobacterium tuberculosis resulted in the formation of microscopic granulomas.
(A) Infected PBMCs, (B) uninfected PBMCs, (C) H & E staining showing micro granulomas, (D) H & E staining showing multinucleated giant cells. (E) Fluorescent staining of granulomas sections with DAPI (nuclear stain), CD68 (macrophage marker-shown in red) and CD3 (T cells-shown in green) monoclonal antibodies.
Figure 2.
Mtb infection resulted in a decrease in macrophage and CD4+ T cell number.
Granuloma samples were harvested and host cells were analyzed for expression of cellular markers by flow cytometry as described in methods. Graphs depict 1 wk and 2 wk post-infection profiles of uninfected and infected granuloma samples for (A) CD4+3+ T cells, (B) CD8+3+ T cells, (C) CD19+ B cells, (D) CD4+25+ T cells, (E) CD14+11c+ macrophages. Data are represented as mean +/− SEM from 3 experiments.* p<0.05 for CD4+25+ cells at 2 wk time point for 1∶0.1 MOI-infected group compared to the uninfected group.
Figure 3.
Infection of human PBMCs with Mtb induces inflammatory Cytokines.
Supernatants from granuloma samples or control uninfected samples were saved at 8-day post-infection and cytokine/chemokine profile determined by multiplex cytokine analysis as described in methods. Profiles for prominent cytokines and chemokine IL-1α, IL-12p40, IFN-γ, RANTES and TNFα are shown in Figure 3A, whereas Cytokines and chemokines IL-1β, IL-8, IL-6, MCP-1, IP-10, MIP-1α are shown in Figure 3B. Data are represented as mean +/− SEM from 3 experiments.
Figure 4.
Mtb within the granuloma goes into a dormant state.
(A) Auramine-O and Nile red staining of granuloma sections shows Nile Red positive Mtb cells within the granuloma (figure 4A lower left panel, insert in 4A shows lipid bodies). The number of Auramine-O and Nile red positive Mtb cells were counted from multiple fields, and percentage of Auramine-O (green) and Nile red (red) positive Mtb cells are presented graphically (Figure 4A lower right panel) (B) Mtb from 8 day granuloma was resistant to Rif when compared to 0 day in vitro grown Mtb. Data are represented as mean +/− SEM from 3 experiments. P values were calculated using students t test. * p<0.05 for Rif resistance of Mtb from 8day granuloma compared to Mtb from 0day in vitro grown Mtb.
Figure 5.
Addition of anti-TNF α monoclonal antibody caused reactivation of dormant Mtb.
(A) Auramine-O and Nile red staining of WT Mtb from IgG and anti-TNF α antibody treated granulomas. (B) Alamar Blue readouts at different time intervals when WT Mtb cells from granulomas treated with anti-TNF-α antibody or control IgG, were incubated with Alamar blue dye. (data of one representative data set has been displayed.) (C) WT Mtb cells from granulomas treated with anti-TNF-α antibody had a lower antibiotic resistance as compared to WT Mtb cells from control IgG treated granulomas. (D) Mtb cells from granulomas treated with anti-TNF α antibody catabolized fatty acids at a higher rate than Mtb from control IgG treated granuloma. P values were calculated using students t test. p<0.05 for metabolism of oleic and palmitic acid for Mtb from anti-TNF-αantibody treated granuloma compared to Mtb from IgG treated granuloma. (E) Infection of hPBMCs resulted in Apoptosis of these cells. Data are presented as mean +/− SEM from 3 experiments. P values were calculated using students t test. * p<0.05.
Figure 6.
Gene expression profile of Mtb cells from in vitro granuloma treated with IgG anti-TNFα-mAb.
Relative gene expression values (fold changes of day 0) in IgG and anti-TNFα mAb treated Mtb cells are represented for a selected set of genes that are known to be involved in dormancy and resuscitation conditions. Real-time Taqman RT-PCR measurement was performed to measure relative abundance of transcripts. Relative quantitation method (ddCt) was used to determine the fold change in transcripts level. Gene transcript level is expressed as fold change in log2 scale relative to the sample from in vitro grown starter culture used for infection of PBMC. Samples of starter culture (day 0) was used as calibrator and 16S rRNA gene was used as the endogenous control to normalize the expression values. tgs1, triacylglycerol synthase1; lipY, lipase Y; icl, isocitrate lyase; hspX, heat-shock protein X; dosR, dormancy response regulator; gltA1, citrate synthase 1; citA, citrate synthase II; rpf, resuscitation promoting factor (A, B, and C); rpo, RNA polymerase (A and B); atp, ATP synthase (A and B subunits); nuo, NADH dehydrogenase (A, B and E subunits).
Figure 7.
tgs1 deletion compromised the ability of the Mtb to go into a dormant state.
(A) Auramine-O and Nile red staining of WT and Δ-tgs1 mutant and Δ-tgs1 complemented strain (Δ-tgs1C+) population in in vitro granuloma. (B) Alamar Blue readouts at different time intervals when WT and Δ-tgs1 mutant and Δ-tgs1 complemented strain from granulomas treated with anti-TNF-α antibody or IgG, were incubated with Alamar blue dye. (data of one representative data set has been displayed.) (C) Δ-tgs1 mutant from granulomas treated with IgG or anti-TNF-α antibody had a lower antibiotic resistance as compared to WT Mtb cells from IgG treated granulomas. Data are presented as mean +/− SEM from 3 experiments. P values were calculated using students t test. *p<0.05.
Figure 8.
Deletion of lipY compromised the ability of Mtb to reactivate upon treatment with anti-TNFα mAb.
(A) Auramine-O and Nile red staining of WT and Δ-lip Y mutant and Δ-lip Y complemented strain (Δ-lip Y C+) population in in vitro grown granuloma. (B) Alamar Blue readouts at different time intervals when WT and Δ-lip Y, and Δ-lip Y C+ Mtb cells from granulomas treated with anti-TNF-α antibody or control IgG, were incubated with Alamar blue dye. (data of one representative data set has been displayed.) (C) Δ-lip Y mutant from granulomas treated with IgG or anti-TNF-α antibody had a higher antibiotic resistance as compared to WT Mtb cells from anti-TNF-α antibody treated granulomas. Data are presented as mean +/− SEM from 3 experiments. P values were calculated using students t test. *p<0.05.