STZ-NA induced diabetes models Type 2 Diabetes Mellitus (DM) and its associated complications.

(A) Schematic representation of the experimental plan.

(B) Body weight change in DM and healthy mice during the course of the experiment. Healthy, n = 10; DM, n= 35. Error bars represent standard deviation.

(C) Fasting blood glucose in DM mice and healthy controls during the experiment. Healthy, n = 10; DM, n= 35. Error bars represent standard deviation.

(D) Insulin tolerance test (ITT) in DM and healthy controls. Healthy, n = 5; DM, n= 10. Error bars represent standard deviation.

(E) Intraperitoneal glucose tolerance test (iPGTT) in DM and healthy controls. Healthy, n = 9; DM, n= 12. Error bars represent standard deviation.

(F-H) Serum triglyceride, cholesterol, free fatty acid and insulin levels in DM mice and healthy controls. Healthy, n = 7; DM, n= 8. Error bars represent standard deviation.

(I) Serum insulin levels in DM mice and healthy controls. Healthy, n = 5; DM, n= 10. Error bars represent standard deviation.

Statistics: (B-I) unpaired t-test; ****: p < 0.0001, ***: p < 0.0005, **: p < 0.005, *: p < 0.05. A non-significant p-value was not indicated in the plots.

At 21 days post-Mtb H37Rv infection, hyperglycemic (DM) mice and DM-TB mice display comparable tissue bacterial loads to Mtb infected (TB) group with distinct serum cytokine and lung immune cell profiles.

(A) Schematic representation of the experimental plan.

(B, C) Gross lung and spleen pathology in Mtb H37Rv infected mice at 21 days post-infection (d.p.i.).

(D) Hematoxylin and Eosin-stained lung tissue sections show histopathology differences and lung inflammation scores between groups. TB, n = 3; DM-TB, n = 3. Error bars represent standard deviation.

(E) Spleen weight at 21 d.p.i. in DM-TB and TB mice. TB, n = 6; DM-TB, n = 6. Error bars represent standard deviation.

(F) Lung and spleen bacterial burden at 21 d.p.i. in DM-TB and TB mice. TB, n = 6; DM-TB, n= 5 for lung CFU. TB, n = 4; DM-TB, n = 5 for spleen CFU. Error bars represent standard deviation.

(G) Serum IFN-γ, Il-6, TNF-α and Il-17A levels (pg/mL) measured via LEGENDplex assay in healthy, DM, TB and DM-TB groups at 21 d.p.i. Healthy, n = 7; DM, n = 8; TB, n = 8; DM-TB, n = 8 for IFN-γ and IL-6. Healthy, n = 7; DM, n = 8; TB, n = 8; DM-TB, n = 7 for TNF-α and Il-17A. Error bars represent standard deviation.

(H) The gating strategy was used to sort lung CD3+ and CD11C+ cells from all study groups. A representative sample from the TB group is used.

(I) Frequency (%) CD3+ and (B) CD11C+ cells in healthy, DM, TB and DM-TB mice at 21 d.p.i. Healthy, n = 9; DM, n = 10; TB, n = 8; DM-TB, n = 11. Error bars represent standard deviation.

Statistics: (D-I) unpaired t-test; ****: p < 0.0001, ***: p < 0.0005, **: p < 0.005, *: p < 0.05. A non-significant p-value was not indicated in the plots.

scRNA-seq analysis shows marked alterations in the lung immune cell profiles of diabetic mice.

(A) Schematic representation of the experimental plan.

(B) Uniform Manifold Approximation and Projection (UMAP) visualization of the major cell types representing lymphoid/myeloid cells in dataset.

(C) Annotated UMAP showing lung immune cell subtypes in DM and Healthy mice.

(D) Dot plot indicating expression of key genes detected for each cell subtype.

(E) A stacked bar plot shows the percentage of cell subtypes in each group.

Altered functional, transcriptional, and intercellular communication profiles in lung immune cells of diabetic mice.

(A) Volcano plot showing significantly up-/down-regulated genes in naïve CD4+ T-cells between DM and healthy mice. Significantly deregulated genes: p-value ≤ 0.05, log2fold change ≥ ± 1.

(B) Heat map showing a subset of differentially activated regulon for cell clusters in hyperglycemic (DM) and euglycemic (healthy) groups. An unpaired Wilcoxon rank-sum test was performed to compare the activity of each regulon between datasets (healthy and DM). p-value ≤ 0.05 was selected for finding the significant difference in regulon activity.

(C) CellChat circle plot showing signaling networks in DM versus healthy groups. The thickness of the edge represents the strength of signaling, and the red and blue colour represent upregulated and downregulated interaction, respectively, in the DM group compared to the healthy control.

(D) A stacked bar plot showing the relative information flow between the ligand-receptor pairs in healthy and DM groups. A paired Wilcoxon test was performed to determine whether there was a significant difference in the signaling information flow between healthy and DM groups. The top signaling pathways coloured green are enriched in DM, and those coloured blue are enriched in the healthy group. p-value ≤ 0.05 was selected for finding the significant difference in regulon activity.

(E) CellChat circle plot showing IL-16 signaling network in DM and healthy groups. The thickness of the edge represents the strength of signaling.

Impact of Mtb H37Rv infection on lung immune cell landscape of diabetic mice.

(A) Schematic representation of the experimental plan.

(B) Uniform Manifold Approximation and Projection (UMAP) visualization of the major cell types representing lymphoid/myeloid cells in the dataset.

(C) Annotated UMAP showing lung immune cell subtypes in DM-TB and TB mice.

(D) Dot plot indicating expression of key genes detected for each cell subtype.

(E) A stacked bar plot shows the percentage of cell subtypes in each group.

Impaired anti-tuberculosis responses in diabetic mice following Mtb H37Rv infection.

(A) Volcano plot showing significantly up-/down-regulated genes in M1 macrophages between DM-TB and TB mice. Significantly deregulated genes: p-value ≤ 0.05, log2fold change ≥ ± 1.

(B) Heat map showing a subset differentially activated regulon for cell clusters in the DM-TB and TB groups. An unpaired Wilcoxon rank-sum test was performed to compare the activity of each regulon between datasets (TB and DM-TB). Significant difference in regulon activity: p-value ≤ 0.05.

(C) CellChat circle plot showing signaling networks in DM-TB and TB groups. The thickness of the edge represents the strength of signaling, and the red and blue colours represent upregulated and downregulated interaction, respectively, in the DM-TB group compared to the TB control.

(D) A stacked bar plot showing relative information flow between ligand-receptor pairs in the DM-TB and TB groups. Paired Wilcoxon test was performed to determine whether there was a significant difference in the signaling information flow between healthy and DM groups. The top signaling pathways coloured yellow are enriched in DM-TB, and those coloured pink are enriched in the TB group. Significant difference in information flow: p-value ≤ 0.05.

(E) CellChat circle plot showing IFN-II signaling network in DM-TB and TB groups. The thickness of the edge represents the strength of signaling.

Chronic Mtb H37Rv infection in diabetic mice increases bacterial burden and worsens tissue pathology.

A) Schematic representation of the experimental plan.

(B) Body weight change in DM and healthy mice during the course of the experiment. Healthy, n = 20; DM, n= 11. Error bars represent standard deviation.

(C) Fasting blood glucose levels in diabetic mellitus (DM) mice and healthy controls during the experiment. Healthy, n = 15; DM, n= 21. Error bars represent standard deviation.

(D) Spleen and lung weight in TB and DM-TB groups at 3 weeks post-infection. TB, n = 5; DM-TB, n= 5. Error bars represent standard deviation.

(E) Lung mycobacterial burden in TB and DM-TB groups at 3 weeks post-infection. TB, n = 5; DM-TB, n= 5. Error bars represent standard deviation.

(F) Spleen and lung weight in TB and DM-TB groups at 8 weeks post-infection. TB, n = 5; DM-TB, n= 6. Error bars represent standard deviation.

(G) Lung mycobacterial burden in TB and DM-TB groups at 8 weeks post-infection. TB, n = 9; DM-TB, n= 6. Error bars represent standard deviation.

(H, I) Hematoxylin and Eosin-stained lung tissue sections with inflammation and cellular infiltration scores of TB and DM-TB groups at 8 weeks post-infection.

Statistics: (D-I) unpaired t-test; **: p < 0.005., ns: p > 0.05 at 95% confidence.