Analysis of innate immune cell kinetics of mice infected with Mtb with different virulence levels.

<p>Single-cell suspensions prepared from lung tissues of mice infected with Mtb strains at days 2, 5, 7, 14, 56 and 112 days post-infection were stained with the indicated antibodies and analyzed by flow cytometry (<i>n</i> = 4 per group per designated time point). (A) Gating strategy for the analysis of innate immune cells present in the in lungs. All surface-stained samples were primarily gated on forward scatter (FSC)^mid/^high and side scatter (SSC)^mid/^high and secondarily gated on F4/80^-/⁺ and CD11c^low. The cells were analyzed according to the expression of Gr-1 versus CD11b. F4/80^-/CD11c^-/CD11b^high/Gr-1^high cells were designated as neutrophils (a). The lower population was designated as CD11c^-/CD11b⁺/Gr-1^int cells (b). Next, dot-blots of lung cells were primarily gated on FSC^mid/^high and SSC^mid/^high and secondarily gated on F4/80^- and F4/80⁺. The cells were analyzed according to the expression of CD11b versus CD11c. F4/80⁺/CD11c⁺/CD11b^- cells (c), F4/80⁺/CD11c^-/CD11b⁺ cells (d), F4/80^-/CD11c^int/CD11b^int cells (e), F4/80^-/CD11c⁺/CD11b⁺ cells (f) and F4/80^-/CD11c⁺/CD11b^-/Siglec-H⁺/PDCA-1⁺ cells (g) were designated as alveolar macrophages, CD11b⁺ macrophages, monocytes, CD11b^high DCs and pDCs, respectively. (B) The line graphs display the absolute numbers of cells in (A) at various time points during Mtb lung infection. *<i>p</i> < 0.05, ***<i>p</i> < 0.001, Mtb K-infected <i>vs</i>. Mtb H37Rv-infected groups. (C) Bar graphs shows the percentage of infiltrated cells in the lungs. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, non-infected <i>vs</i>. Mtb-infected groups. The data are presented as the mean (± SD) or four mice per group at each time point from one representative experiment out of two independent experiments. NI: Non-infected; Ra: H37Ra-infected lung; Rv: H37Rv-infected lung; K: Beijing-K-infected lung.</p

Histopathological analysis of lungs infected with different strains of virulent Mtb.

<p>(A) Inflammatory scores of H&E-stained sections of lungs during Mtb infection. At 28, 56, and 112 days post-infection, mice were sacrificed, and lung sections were stained with H&E (<i>n</i> = 5 per group per designated time point). Ten pictures from each group were randomly selected and analyzed (2 pictures per mouse × 5 mice in each group). Lung inflammation scores are presented as the median percentage (± IQR) of inflammation for each mouse. Lung sections were stained with H&E (bar, 500 μm). A <i>p</i>-value ≤ 0.05 was considered significant: *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001. (B) Representative histopathology and gross pathology of mouse lungs infected with Mtb strains with different virulence levels at 112 days post-infection.</p

Analysis of T cell kinetics, subtypes, and polarization in Mtb-infected mice.

<p>Single-cell suspensions prepared from lung tissues of mice infected with Mtb strains at 2, 5, 7, 14, 28, 56 and 112 days post-infection. A representative gating strategy (at 112 days post-infection) for the assessment of CD4 T cells, CD8 T cells, Th1, Th2, Th17, and Treg cells is shown in the left panel of (A), (B), and (C). CD4⁺ and CD8⁺ T cells were stained with the indicated surface and transcription factor antibodies and analyzed by flow cytometry. (D) Bar graphs display the absolute numbers of CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells in the lungs at 2, 5, 7, 14, 28, 56 and 112 days post-infection. (E) Using CD3⁺CD4⁺ T cells as the parent gate, specific staining for the transcription factors T-bet, GATA-3, RORγt and Foxp3 in lung cells from Mtb-infected mice is shown at various time points. Line graphs show the expression of T-bet (Th1 cells), GATA-3 (Th2 cells), RORγt (Th17 cells) and Foxp3 (Tregs) in CD3⁺/CD4⁺ T cell populations at the indicated time points. *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001, Mtb K-infected group <i>vs</i>. Mtb H37Rv-infected group. One representative plot out of two independent experiments is shown.</p

PPD-specific cytokine response in lung cells from Mtb-infected mice.

<p>The amount of IFN-γ, IL-5, IL-10, IL-17A (A) and IFN-α (B) produced by lung cells (14, 28, 56 and 112 days post-infection) in response to PPD (10 μg/ml) stimulation for 24 h was measured by ELISA. All data are expressed as the mean ± SD (<i>n</i> = 4 per group per designated time point) of one representative experiment out of two independent experiments.</p

Analysis of T cell proliferation and polarization by CD11b^high DCs sorted from the lungs of Mtb-infected mice.

<p>(A) Specific populations of CD11b^high DCs were sorted from the lungs of mice infected with Mtb strains at 7 days post-infection. (B, C, D) CD11b^high DCs sorted from Mtb-infected mice were treated with OVA peptides (1 μg/ml), such as OVA_323-339 or OVA_257-264, for 1 h. The OVA-treated CD11b^high DCs were co-cultured with OVA-specific CD4⁺ and CD8⁺ T cells for 3 days at a sorted DC-to-T cell ratio of 1:10. (B) After 3 days of co-culture, the proliferation of OVA-specific CD4⁺ and CD8⁺ T cells was assessed by flow cytometry. (C) IFN-γ, IL-2, IL-5, and IL-17A were analyzed by ELISA. (D) CD25⁺Foxp3⁺ Treg cells were analyzed after co-culture with CD4⁺ T cells. The T cell data are shown as the mean ± SD (<i>n</i> = 5 samples). One representative plot out of two independent experiments is shown. *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared with T cell/OVA-pulsed DCs sorted from non-infected mice. NI-DC: DC from non-infected mice; Ra-DC: DC from H37Ra-infected mice; Rv-DC: DC from H37Rv-infected mice; K-DC: DC from K-infected mice.</p

Analysis of T cell proliferation and cytokine generation induced by pDCs and Gr-1^int cells sorted from the lungs of Mtb-infected mice.

<p>Details regarding this experiment are provided in the materials and methods section. (A) Specific populations of pDCs and Gr-1^int cells were sorted from the lung cells of mice infected with Mtb strains at 28 days post-infection. T cell proliferation (B, C) and cytokine production (D-G) in response to pDCs and CD11c^-/CD11b⁺/Gr-1^int cells with (yellow bars) and without (white bars) BMDCs were analyzed by flow cytometry after 3 days of co-culture with CFSE-labeled T cells. One representative plot out of two independent experiments is shown. *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared with the T cell only or the BMDC + T cell group. Ra-pDC: pDC from H37Ra-infected mice; Rv-pDC: pDC from H37Rv-infected mice; K-pDC: pDC from K-infected mice; Ra-Gr-1^int: Gr-1^int cells from H37Ra-infected mice; Rv-Gr-1^int: Gr-1^int cells from H37Rv-infected mice; K-Gr-1^int: Gr-1^int cells from K-infected mice.</p

Comparative growth profiles of tested Mtb strains in the lungs during a 112 day-infection.

<p>(A) Early change in Mtb growth during the first 14 days of infection in C57BL/6 mice. (B) The overall growth pattern of tested Mtb strains in C57BL/6 mice. Mice (<i>n</i> = 5 per group at each designated time point) were aerosol challenged with approximately 450 CFU of Mtb H37Ra or with approximately 200 CFU of the H37Rv or the Beijing-K strain. The bacterial burden of their lungs was determined at various time points post-infection. Data are presented as the median log₁₀ CFU of five mice (± IQR) for each time point. A <i>p</i>-value ≤ 0.05 was considered significant: **<i>p</i> < 0.01 (Mtb K <i>vs</i>. Mtb H37Ra) and ^#<i>p</i> < 0.05 (Mtb K <i>vs</i>. Mtb H37Rv).</p