Fig 1.
Comparative Analysis of Survival, Weight Gain, and Pulmonary Pathology in B6 vs. B6.Sst1S Mice Infected with M. tuberculosis.
A. Survival curves of mice infected with M. tuberculosis. Survival of B6 (n = 12) and B6.Sst1S (n = 37) following hock infection with 106 CFU of Mtb Erdman(SSB-GFP, smyc′::mCherry). Survival curves were estimated using the Kaplan–Meier method. Differences between groups were assessed using the Mantel-Cox (Log-rank) test. B. Percent weight in mice infected with M. tuberculosis. Percent weight of B6 (n = 12) and B6.Sst1S (n = 37) following hock infection with 106 CFU of Mtb Erdman(SSB-GFP, smyc′::mCherry). The statistical significance was performed by two-way ANOVA using Tukey’s multiple comparison test. C. Quantification of lung inflammation in B6 and B6.Sst1S mice infected with Mtb Erdman at 11 (B6, n = 4, B6.Sst1S, n = 14) and 20 weeks (B6, n = 5, B6.Sst1S, n = 15) post-infection. Orange dots represent B6.Sst1S mice displaying necrosuppurative pneumonia. D. Representative low-magnification (1X) histopathology images of lung sections from B6 and B6.Sst1S mice following Mtb infection, showing the early disease stage observed in B6 mice and multiple stages of disease progression in B6.Sst1S mice. E. Representative histopathology and AFB staining of lung sections from B6 and B6.Sst1S mice following Mtb infection, illustrating the early disease stage in B6 mice (top left) and various stages of disease progression in B6.Sst1S mice: stage I (top right), stage II (bottom left), and stage III (bottom right). F. Histopathological scores representing M. tuberculosis loads in various organs of B6 and B6.Sst1S mice. Each dot represents a single animal. Sample sizes for B6 mice were: lungs (n = 12), spleen (n = 7), popliteal lymph node (n = 6), and gastrointestinal tract (n = 3). For B6.Sst1S mice, samples included lungs (n = 32), spleen (n = 23), popliteal lymph node (n = 11), gastrointestinal tract (n = 3), and liver (n = 6). Statistical significance was assessed using a two-tailed unpaired t-test. The infection experiment comparing B6 and B6.Sst1S mice was performed twice, while B6.Sst1S mice were infected an additional three times, for a total of five infection experiments involving B6.Sst1S. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 2.
B cell follicles in early TB lesions.
A. Paucibacillary lesions in B6 and B6.Sst1S mice at 11 weeks post-infection (wpi) (H&E staining, 8X, 200X and 800X original magnification). Plasma cells in intralesional lymphoid follicles are characterized by polarized nuclei, strongly basophilic cytoplasm, and an eosinophilic perinuclear Golgi apparatus (arrows). B. Fluorescent multiplex immunohistochemistry (fmIHC) of TB lung lesions in B6 (left column) and B6.Sst1S (right column) mice, shown at low and high magnifications, highlights prominent lymphoid follicles.
Fig 3.
Comparative Cellular and Spatial Transcriptomic Analysis of Paucibacillary and Multibacillary pulmonary TB Lesions of B6.Sst1S Mice.
A. Representative images of paucibacillary and multibacillary lesions from B6.Sst1S lung: H&E (top row), acid-fast bacilli (AFB) (middle row) and CD11b immunohistochemistry (bottom row) at 14wpi. The M lesions had areas of micronecrosis, neutrophilic influx, and AFB. B. Quantification of Mtb load and myeloid cell populations in paucibacillary (P) versus multibacillary (M) lesions using the HALO area quantification algorithm. Lung sections of a total of eight B6.Sst1S mice were analyzed. The number of lesions analyzed: for Mtb load P (n = 20) and M (n = 13); for CD11b+ cells, P (n = 19) and M (n = 14); and for iNOS+ cells, P (n = 16) and M (n = 12). Statistical significance was determined by two-tailed unpaired t-test. C. Representative fluorescent multiplex immunohistochemistry (fmIHC) of paucibacillary (left) and multi-bacillary (right) lesions. Ionized calcium-binding adaptor molecule 1 (Iba1) - blue; CD19 - green; CD3 epsilon (CD3ε) – red and DAPI - grey. 200x total magnification. D. Comparisons of pauci- and multibacillary pulmonary TB lesions of B6 and B6.Sst1S mice. B cell and T cell densities, and Iba1 ⁺ cell area were quantified using HALO algorithm. A total of 5 mice per group were analyzed. Total number of B6 paucibacillary lesions analyzed: for: B cells (n = 7), T cells (n = 16), and Iba1 ⁺ cells (n = 13); for B6.Sst1S paucibacillary lesions: B cells (n = 13), T cells (n = 28), and Iba1 ⁺ cells (n = 22); for B6.Sst1S multibacillary lesions: B cells (n = 24), T cells (n = 31), and Iba1 ⁺ cells (n = 21). Statistical significance was determined using ordinary one-way ANOVA with Bonferroni’s multiple comparison test. E. Heatmaps of GeoMx spatial transciptomic analysis comparing uninvolved lung (U, n = 4), paucibacillary (P, n = 8) and multibacillary (M, n = 8) lung lesions from B6.Sst1S mice. Lung lobes were selected from 2 mice with paucibacilarly lesions and 2 mice with multibacillary lesions with micronecrotic areas. The four lobes were assembled on one slide and processed in parallel. Slides were stained with CD45-, pan-keratin-specific antibodies and DAPI. Gene expression, Red-High and Blue-Low. F. Cellular composition of pulmonary TB lesions analyzed using GeoMx spatial transcriptomics (panel 3E) were deconvoluted using CYBERsort algorithm. Statistical analysis was performed using Kruskal-Wallis nonparametric ANOVA for multiple comparisons. G and H. Nuclear HIF1α staining of paucibacillary (P) vs multibacillary (M) lesions. Representative immunohistochemistry images (G) and percent lesion area quantification by HALO algorithm (H) of hypoxia-inducible factor 1-alpha (HIF-1α) expression in P (n = 12) and M (n = 10) TB lesions. 200x total magnification. The statistical significance was performed by two-tailed unpaired t test. The p value <0.05 was considered statistically significant. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 4.
Characterization of Arg1 + cell in pulmonary TB lesions of B6.Sst1S mice.
A. Representative fluorescent multiplex immunohistochemistry (fmIHC) of paucibacillary (left) and multi-bacillary (right) lesions at low and high magnifications. Arginase 1 (Arg1) - yellow; inducible nitric oxide (iNOS) - teal; ionized calcium-binding adaptor molecule 1 (Iba1) - red and DAPI - grey. B. Quantitative analysis of iNOS + Arg1-, iNOS-Arg1+ and iNOS + Arg1 + cells in P (n = 5) and M (n = 5) lung lesions of B6.Sst1S mice. C. High magnification of interacting Arg1 - (yellow) and iNOS (teal) – expressing macrophages. T cells – red, Iba1 + iNOS-Arg1- macrophages – blue. D. FmIHC images showing proliferation of Arg1+ (Top panel) and iNOS+ (lower panel) cells (marked as circle) in TB lesion area. The proliferation was observed and quantified using staining of tissues with Ki67 markers. E. Quantification of Ki67 + macrophage populations. Left panel – area quantification of iNos + Ki67+ and Arg1 + K67 + cells within multibacillary PTB lesions (n = 38). Right panel - percentage of Ki67 ⁺ cells that are either iNOS⁺ or Arg1⁺ within individual lesions (n = 60). Total 6 mice were analyzed. Statistical significance was determined using the two-tailed Mann–Whitney test. F and G. Quantification of Arg1 + macrophages co-expressing CD11b, CD206, or CD163, representing recruited and resident myeloid cell populations in naïve lung (n = 4), Mtb infected lung uninvolved area (n = 9) and multibacillary PTB lesion area (n = 12). (F). Representative fmIHC images showing Arg1 + , Arg1 + CD11b + , Arg1 + CD206+ and Arg1 + CD163 + populations (G). In the images, CD11b (green) marks recruited myeloid cells, while CD206 (yellow) and CD163 (teal) indicate resident myeloid cells. Arg1 + cells are shown in orange. A total of 5 mice were analyzed from Mtb infected groups. The statistical significance was performed by ordinary one-way Anova using Sidak’s multiple comparison test. The p value <0.05 was considered statistically significant. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 5.
M2 polarization of circulating myeloid cells.
A. Heatmaps depicting the differentially expressed genes (DEGs) in the blood of mice, categorized as uninfected controls or mice with either P or M lung lesions. Gene expression levels were quantified by blood transcriptomics, highlighting the distinct transcriptional profiles between these groups. B. Volcano plots showing differentially expressed genes in blood samples from M. tuberculosis-infected mice with multibacillary lung lesions compared to non-infected controls. Each point represents a single gene, with the x-axis indicating log2 fold change and the y-axis showing -log10(p-value). Significantly downregulated and upregulated genes are highlighted in red (left and right, respectively), while non-significant genes are shown in gray. Significance thresholds are set at p < 0.05 and fold change > ±1.5. C. Expression levels of Arg1, Chil3, Ifnb1, Rsad2, Il6, and Chac1 mRNA in the blood of Mtb infected mice with paucibacillary and multibacillary lung lesions, normalized to non-infected control mice. The expression of Arg1, Chil3 and Il6 is shown for both paucibacillary and multibacillary lung lesions, while the expression of the other genes are presented for mice with multibacillary lung lesions. Each dot represents a single animal. The statistical significance was performed by two-tailed unpaired t test. D. Blood transcriptome polarization indices calculated using MacSpecrum for comparisons of mice with paucibacillary (P), multibacillary (M) PTB lesions and uninfected control mice (UI). E. Heatmaps showing the expression levels of M1 and M2 macrophage-specific genes in blood samples from mice with multibacillary lesions compared to paucibacillary. F and G. Expansion of Arg1 expressing progenitor cells in the bone marrow during Mtb infection. After the infection, the bone marrow cells were collected and analyzed by flowcytometry for Arg1 expression. F. The dot plot of CD45 + Arg1 + cells for CD11b and Ly6C expression (Left panel). The bar graph showing the increase in subsets of Arg1 expressing CD11b+Ly6C+ and CD11b-Ly6C- cells upon Mtb infection (Right panel). G. The CD11b-Ly6- cells were further analyzed for Sca1 expression. The dot plot showing that CD11b-Ly6- cells express Sca1. The dot plots of five individual mouse per group were merged into one dot plot. The total numbers of cells from five mice of uninfected or infected groups were shown in the gates. The dots in the bar graph indicate individual animals. The statistical significance was performed by two-way ANOVA using Tukey’s multiple comparison test. The p value <0.05 was considered statistically significant. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 6.
Mechanisms of lung-specific TB progression.
A. 3D confocal fluorescent multiplex immunohistochemistry (fmIHC) images of 50 μm thick lung sections stained with p63 (teal) Krt17 (yellow), Krt8 (red). Scale bar:100 μm. B. 3D confocal images of cleared thick lung sections (50 μm) of the TB lesion of B6.Sst1S,Ifnb1-YFP mice. Endogenous YFP is shown in green, Nkx2.1 staining - in magenta, and reporter Mtb (smyc’:: mCherry) in red. Left image - all the stacks in 3D, on the right – individual images of Nkx2.1 + cells expressing YFP (denoted by white boxes on the left panel). Scale bar- 100 µm. C. 3D confocal images of cleared thick lung sections (50 μm) of the TB lesion of B6.Sst1S,Ifnb1-YFP mice. Endogenous YFP is shown in green, iNOS staining - in teal, and reporter Mtb (smyc’:: mCherry) in red. Left image - all the stacks in 3D, on the right – individual images of iNOS+ cells expressing YFP (denoted by white box on the left panel). Scale bar- 50 µm.D. Adjacent lung (left) and spleen (right) implants under the mouse skin. Left image - H&E. Original magnification - 100X. Right image – fmIHC revealing lung epithelial cells (Nkx2.1+) exclusively in lung implants and Iba1 + macrophages in both spleen and lung implants. Organized necrotic granuloma in the lung implant is marked by star.E. Representative hematoxylin and eosin (H&E) and acid-fast bacilli (AFB) staining of lung implant. Lung implant with a focal caseating granuloma surrounded by collagen (top) with innumerable AFB (bottom). 200x total magnification. A total of 10 implants were processed and analyzed. F and G. fmIHC image showing presence of Arg1+ and iNOS+ cells in lung implant but not in spleen implant.
Fig 7.
Mtb traffics to and proliferates in human lung xenografts.
Human lung tissue was engrafted by suturing fragments into the muscle fascia in the dorsal subcutaneous space. A. Representative AFB of lung in HIS mouse demonstrating abundant bacilli in a solid, developing lesion (left, top) and a necrotic granuloma (right, top). B. Human lung explants demonstrating Mtb bacilli and areas of proliferation in human lung xenografts. Representative H&E showcasing lung explant characteristics (left, top) and areas of inflammation with abundant AFB (right, top). High power images (bottom) demonstrating proliferation in inflammatory areas adjacent to blood vessels and alveoli (left, bottom) and inflammatory foci in parenchyma (right, bottom). Bottom panels, 40X.