Type 1 immune responses are similar but myeloid cell activation is different between C57BL/6 and AID^−/−µS^−/− Mtb-infected mice.

<p>(A–F) C57BL/6 (filled symbols) and AID^−/−µS^−/− (opened symbols) mice were infected as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a>. (A) Lung cells were cultured with ESAT_1–20 peptide to determine the number of IFN-γ-producing ESAT_1–20-specific cells. The number of CD3⁺CD4⁺IFN-γ⁺ or CD3⁺CD8⁺IFN-γ⁺ was determined by flow cytometry. (B) The presence of mRNA for <i>ifng</i> in the lungs of infected mice was determined by real-time RT-PCR. (B) IFN-γ protein was determined in lung homogenates by Luminex. (C) MHC-II expression on CD11c+ cells (expressed by alveolar macrophages and dendritic cells) derived from the lungs of uninfected (shaded curve) or infected C57BL/6 (solid line) or AID^−/−µS^−/− mice (dashed line) was determined by flow cytometry. (A–C) One experiment representative of 3 independent experiments is shown. <i>n</i> = 4 mice per group. *, p<0.05; **, p<0.01; ***, <i>p</i><0.001 by Student’s <i>t</i> test. (D) At specific time points after infection, the caudal lobe of the lung from each mouse was processed for histologic analysis and stained using H&E. Sections were screened and scored for inflammation, PMN infiltration and necrosis by a pathologist in a blinded manner (0: absent; 1: minimal; 2: mild; 3: moderate; 4: marked). One experiment representative of 2 independent experiments is shown. <i>n</i> = 4 mice per group. (E) At different time points after infection, RNA was extracted from lung tissue and analyzed by real-time PCR for the expression of <i>nos2, irgm1, fizz1, fizz2, arg1 and arg2</i>. (F) At day 60 post-infection, arginase activity was determined in the lungs of C57BL/6 (filled symbols) and AID^−/−µS^−/− (open symbols). One experiment representative of 2 independent experiments is shown. <i>n</i> = 4–5 mice per group. *, p<0.05; **, p<0.01; ***, <i>p</i><0.001 by Student’s <i>t</i> test.</p

B cell depletion in Mtb-infected AID^−/−µS^−/− mice allows for improved control of bacterial burdens in the spleen, but not in the lung.

<p>C57BL/6 (closed symbols) and AID^−/−µS^−/− (open symbols) mice were infected as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> and then treated every other week starting either day 0 or day 30 with an isotype control antibody (circles) or with an anti-mouse CD20 antibody (αCD20, squares) and (A) flow cytometry was used to determine depletion of CD19⁺CD3⁻ cells at day 30 (depletion was maintained through day 60 - not shown). (B) The bacterial burden was determined by plating the lung and spleen on agar and counting viable colony forming units. (C) Radiation bone marrow chimeras were made with bone marrow from C57BL/6 (closed circles) AID^−/−µS^−/− (open circles) or a mixture of the two (half filled circles) and then infected as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> and the bacterial burden in the spleen measured. (D) C57BL/6 (closed symbols) and AID^−/−µS^−/− (open symbols) mice were infected as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> and then treated every three days starting at day 15 with normal mouse serum (triangles) or were left untreated (circles). Bacterial burden was assessed at day 60. Data from two separate experiments showing the same results independently have been combined in A–D (<i>n</i> = 8–10 mice per group). **, p<0.01; ***, <i>p</i><0.001 by ANOVA with Tukey’s multiple comparisons post-test.</p

AID^−/−µS^−/− mice have an altered B cell population in the lung and are more susceptible to aerosol infection with Mtb.

<p>C57BL/6 (filled circles) and AID^−/−µS^−/− mice (opened circles) were infected with Mtb H37Rv via the aerosol route. (A–C) At day 30 of infection, B cells (gated on live lymphocytes and CD19 expression) were analyzed for the expression of IgM and IgD by flow cytometry. The frequency (A) and total number of CD19⁺ cells, CD19⁺IgD⁻IgM⁺ cells and CD19⁺IgD⁺IgM⁺ cells in the lung (B) and spleen (C) was calculated. (D) The bacterial burden was determined in lungs and spleen over time (<i>n</i> = 4). (E) Survival of infected mice was determined over the course of the experimental infection (<i>n</i> = 5). (A–E) One experiment representative of at least three independent experiments is shown *, p<0.05**, p<0.01; ***, <i>p</i><0.001 by Student’s <i>t</i> test. (F–H) C57BL/6 (filled circles), AID<i>^−/−</i> (F), µS^−/− (G) and µMT (H) mice (opened circles) were infected, and the bacterial burden was determined in the lungs and spleen over time (<i>n</i> = 4–8). Data sets from a total of two experiments were combined. *, p<0.05; ***, p<0.001 by Student’s <i>t</i> test.</p

Blocking of IL-10R improves the ability of AID^−/−µS^−/− mice to control Mtb.

<p>C57BL/6 and AID^−/−µS<i>^−/−</i> mice were infected as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a>. (A) The concentration of IL-10, IL-6 and G-CSF in lung homogenates of Mtb-infected mice was determined by Luminex. One experiment representative of 2 independent experiments is shown (<i>n</i> = 4). *, p<0.05; **, p<0.01 ***, <i>p</i><0.001 by Student’s <i>t</i> test. (B) Lung sections from mice infected for 60 days as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> were stained for B220 (red) and IL-10 (green), white dashed line represents edge of B cell follicle area (bar represents 50 microns). Representative sections shown for C57BL/6 (left) and AID^−/−µS^−/− (right panel), reproduced in five other mice and in 2 separate experiments. (C) C57BL/6 (filled symbols) and AID^−/−µS^−/− (open symbols) mice were infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a>. At day 0 and once a week thereafter, mice were treated with anti-IL-10R antibody (squares) or with the same concentration of an isotype control antibody (circles). Lung and spleen bacterial burden was determined at day 60 post-infection. One experiment representative of two independent experiments is shown (<i>n</i> = 5). *, p<0.05; ***, <i>p</i><0.001 by one-way ANOVA with Tukey’s multiple comparisons post-test.</p