IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung

Abstract

IL-23 is required for the IL-17 response to infection with Mycobacterium tuberculosis, but is not required for the early control of bacterial growth. However, mice deficient for the p19 component of IL-23 (Il23a(-/-)) exhibit increased bacterial growth late in infection that is temporally associated with smaller B cell follicles in the lungs. Cxcl13 is required for B cell follicle formation and immunity during tuberculosis. The absence of IL-23 results in decreased expression of Cxcl13 within M. tuberculosis-induced lymphocyte follicles in the lungs, and this deficiency was associated with increased cuffing of T cells around the vessels in the lungs of these mice. Il23a(-/-) mice also poorly expressed IL-17A and IL-22 mRNA. These cytokines were able to induce Cxcl13 in mouse primary lung fibroblasts, suggesting that these cytokines are likely involved in B cell follicle formation. Indeed, IL-17RA-deficient mice generated smaller B cell follicles early in the response, whereas IL-22-deficient mice had smaller B cell follicles at an intermediate time postinfection; however, only Il23a(-/-) mice had a sustained deficiency in B cell follicle formation and reduced immunity. We propose that in the absence of IL-23, expression of long-term immunity to tuberculosis is compromised due to reduced expression of Cxcl13 in B cell follicles and reduced ability of T cells to migrate from the vessels and into the lesion. Further, although IL-17 and IL-22 can both contribute to Cxcl13 production and B cell follicle formation, it is IL-23 that is critical in this regard.

Figure 1. Mice lacking IL-23 are less able to control bacterial growth when chronically infected with Mtb

(A) B6 (closed circles) and Il23a^−/− (open circles) were infected via the aerosol route with 100 Mtb H37Rv and the number of bacteria in the lungs was determined. The graph shows the combined data of three experiments all of which showed a significant difference between B6 and Il23a^−/− in bacterial burden after day 120. Data points are the mean ± standard deviation for an n of 12–15 mice per group. (B) The number of B cell follicles in the lungs of B6 (closed circles) and Il23a^−/−(open circles) mice infected for 200 days was determined. Data points are from an n of 4 mice per group and represent one experiment of two total. The significance was determined by the Student’s t test with * P ≤ 0.05, ** P ≤ 0.001 or *** P ≤ 0.0001. (C) B6 mice and Il23a^−/− mice were infected as in panel A for 200 days and representative sections stained with hematoxylin and eosin are shown (top panels). Sections were probed with antibodies to the B cell marker, B220 (green), the T cell marker, CD3 (red) and macrophage marker iNOS (also red) (bottom panels).

Figure 2. Mice lacking IL-23 maintain expression of IFN-γ and LRG-47 but not IL-17 or IL-22

(A) B6 (filled circles) and Il23a^−/− (open circles) were infected as for Fig 1 and induction of mRNA for IFN-γ, Lrg-47, IL-17 and IL-22 relative to uninfected control mice determined by RT-PCR. One of two representative experiments is shown. (B) A single cell suspension from infected mice was analyzed by flow cytometry (left panel) or ELISpot (right panel, Day 238) for IFN-γ production. Data points are the mean ±standard deviation for an n of 3–4 mice per group. Significance was determined by the Student’s t test with * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Figure 3. Mice lacking IL-17R or IL-22 are unaffected in their ability to control Mtb

(A) B6 (closed circles) and Il17ra^−/− (open squares) mice were infected as in Fig 1 and the number of bacteria in the lungs was determined. One of two representative experiments is shown. (B) B6 (closed circles) and Il22^−/− (open triangles) mice were infected. One of two representative experiments is shown. (C) B6 mice were infected and treated with isotype control antibody (closed circles) or with anti-IL-22 antibody (open diamonds) for 30 days prior to each day of harvest. One of two representative experiments is shown. For A–C data points are the mean ±standard deviation for an n of 4–5 mice per group. (D) Il17ra^−/− mice were infected and treated with anti-IL-22 antibody for 30 days prior to harvest on day 150. (E) Il22^−/− mice were infected and treated with anti-IL-17 antibody for 30 days prior to harvest on day 150. For both D and E data represent one experiment total with a n=4. There were no significant differences.

Figure 4. Mice lacking IL-23, IL-22, IL-17R have altered development of B cell follicles over time

B6 (closed circles), Il23a^−/− (open circles), Il17ra^−/− (open squares), B6 treated with anti-IL-22 (open diamonds) and Il22^−/− (open triangles) were infected as in Fig 1 and the area of B cell follicles measured on days 50 (A), 80 (B), 150 (C) and 200 (D). The data points are the individual values for each follicle in the caudal lobe section for each mouse with an n of 3–5 mice per group. (E) Lymphocyte accumulations were characterized in (i) the lungs of B6 (top panel) and Il17ra^−/− (bottom panel) mice infected for 50 days and (ii) the lungs of B6 (left panel) and Il22−/− (upper right panel) and anti-IL-22-treated (lower right) mice infected for 80 days. The lymphocyte accumulations were characterized as in Fig 1C.

Figure 5. Mice lacking IL-23a are unable to express CXCL13 in B cell follicles induced by Mtb infection

(A) B6 (i) and Il23a^−/− (ii) mice were infected with Mtb as described for Fig 1 and lung sections stained for the CXCL13 protein shown in green with the B220 positive cells shown in red (n=2, images representative of all tissues examined are shown). (B) B6 (i, and ii) and Il23a^−/− (iii) mice were infected with Mtb and lung sections subjected to in situ hybridization with (ii, iii) the antisense probe for Cxcl13 mRNA (ii, iii) or the sense control probe (i) n=4, images representative of all tissues examined are shown, positive staining is highlighted by the arrows. (C) Formalin fixed sections from B6 (closed circles), isotype control (crosses), Il23a^−/− (open circles), Il17ra^−/−(open squares) and anti-IL-22 treated mice (open diamonds) were also stained for CD3 and the tendency for T cell cuffing to occur was measured as area of CD3 positive cells around blood vessels at day 200 post infection. (D) Fibroblasts from mouse lungs were exposed to IL-17 and/or IL-22 and the production of CXCL13 measured by bead array. The data points are the mean of triplicate wells pooled from two mice, one experiment of two showing an effect of IL-17 and IL-22. For F,G, significance was determined by the ANOVA with * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001