Pneumocystis-Driven Inducible Bronchus-Associated Lymphoid Tissue Formation Requires Th2 and Th17 Immunity

Abstract

Inducible bronchus-associated lymphoid tissue (iBALT) is an ectopic lymphoid structure composed of highly organized T cell and B cell zones that forms in the lung in response to infectious or inflammatory stimuli. Here, we develop a model for fungal-mediated iBALT formation, using infection with Pneumocystis that induces development of pulmonary lymphoid follicles. Pneumocystis-dependent iBALT structure formation and organization required CXCL13 signaling. Cxcl13 expression was regulated by interleukin (IL)-17 family members, as Il17ra^-/-, Il17rb^-/-, and Il17rc^-/- mice failed to develop iBALT. Interestingly, Il17rb^-/- mice have intact Th17 responses, but failed to generate an anti-Pneumocystis Th2 response. Given a role for Th2 and Th17 immunity in iBALT formation, we demonstrated that primary pulmonary fibroblasts synergistically upregulated Cxcl13 transcription following dual stimulation with IL-13 and IL-17A in a STAT3/GATA3-dependent manner. Together, these findings uncover a role for Th2/Th17 cells in regulating Cxcl13 expression and provide an experimental model for fungal-driven iBALT formation.

Keywords: Cxcl13; Pneumocystis; Th17; Th2; fungus; iBALT; immunity; lymphoid tissue.

Figure 1. Inducible bronchus associated lymphoid tissue (iBALT) forms following Pneumocystis infection and exposure

A.) iBALT development over the course of Pneumocystis infection. B.) Immunofluorescent staining of an iBALT structure 14 days post-infection with Pneumocystis demonstrating the presence of PNAd⁺ high endothelial venules and Lyve- 1⁺ lymphatic vessels. C.) C57BL/6 mice co-housed for two weeks with a Pneumocystis infected Rag2^−/−Il2rg^−/− mouse have iBALT structures four weeks following separation, while similarly co-housed Rag1^−/− mice do not. D.) C57BL/6 and Rag1^−/− mice co-housed for two weeks with a Pneumocystis infected Rag2^−/−Il2rg^−/− mouse have detectable Pneumocystis burden. E.) Immunofluorescent staining of rapidly dividing B-cells (PCNA⁺B220⁺) cells and CD3⁺ T-cells in the lung of a C57BL/6 mouse 14 days after Pneumocystis infection. F.) iBALT structures are reduced following CD4⁺ T-cell depletion with GK1.5 monoclonal antibody. G.) iBALT structures are reduced following CD20⁺ B-cell depletion with 5D2 monoclonal antibody. Scale bars represent 100 μm. H.) Quantification of iBALT structures in C57BL/6 mice, C57BL/6 mice treated with GK1.5, and C57BL/6 mice treated with 5D2 (n=4 per group). Data are shown as mean ± standard error of the mean. * p<0.05, ** p<0.01, **** p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons.

Figure 2. CD4⁺ T-cells from the spleen, mediastinal lymph node, and iBALT structures are protective against Pneumocystis infection

A.) Schematic representation of isolation of spleen, mediastinal lymph node (mLN), and iBALT CD4⁺ T-cells at day 14 post-Pneumocystis infection, followed by adoptive transfer into a previously infected Rag1^−/− mouse. B.) Transfer of splenic, mLN, and iBALT CD4⁺ T-cells reduces Pneumocystis burden compared to no transfer (PBS) control. C.) Expression of Ifng, Il13, and Il17a in whole lung following transfer of CD4⁺ T-cells from spleen, mLN, and iBALT demonstrating preserved effector function in each population. Data are shown as mean ± standard error of the mean. * p<0.05, ** p<0.01, *** p<0.001 by one-way ANOVA with Tukey’s multiple comparisons.

Figure 3. Lymphotoxin-alpha is required for iBALT organization and germinal center development following Pneumocystis infection

A.) Immunofluorescent staining of C57BL/6 and Lta^−/− mice 14 days following Pneumocystis infection demonstrating a lack of PCNA⁺B220^lo germinal center B-cells in Lta^−/− mice compared to C57BL/6 mice. Scale bars represent 100 μm. B.) Immunofluorescent staining of C57BL/6 and Lta^−/− mice demonstrating intrafollicular CD3⁺ T-cells. Scale bars represent 100 μm. C.) Quantification of size, number, and area occupied by iBALT structures (n=7–8). *** p<0.001, **** p<0.0001 by student’s T-test. D.) Expression of Cxcl13 is similar between C57BL/6 and Lta^−/− mice in whole lung (n=7). E.) Lta^−/− mice have deficient production of serum anti-Pneumocystis specific IgG compared to C57BL/6 mice (n=4–6). F.) Lta^−/− mice sufficiently clear Pneumocystis infection comparable to that of C57BL/6 controls (n=3–7). Data are shown as mean ± standard error of the mean. ** p<0.01, *** p<0.001, **** p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons.

Figure 4. CXCR5 signaling is required for Pneumocystis iBALT formation

A.) Cxcl13 is induced over the course of Pneumocystis infection in C57BL/6 mice in a CD4⁺ T-cell dependent manner. Ccl19 and Ccl21 expression levels are unchanged throughout Pneumocystis infection. * p<0.05 by multiple t-tests, (n=5 per group). B.) C57BL/6 mice co-housed with a Pneumocystis infected Rag2^−/−Il2rg^−/− mouse for two weeks, followed by four weeks of separation, upregulate Cxcl13 but not Ccl19 or Ccl21 (n=3–6 per group). * p<0.05, ** p<0.01, *** p<0.001 by one-way ANOVA with Tukey’s multiple comparisons. C–D.) Immunofluorescent staining of C57BL/6 and Cxcr5^−/− mice 14 days post-Pneumocystis infection demonstrating iBALT formation in C57BL/6 and disorganized follicle structure in Cxcr5^−/− mice. Scale bars represent 100 μm. E.) Quantification of size, number, and area occupied by lymphocytic infiltrates (n=7–8). F.). C57BL/6 (n=10) and Cxcr5^−/− (n=4) control Pneumocystis burden at 14 days post-infection compared to CD4-depleted mice (n=6). Data are shown as mean ± standard error of the mean. *** p<0.001, **** p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons.

Figure 5. IL-17R family members are required for the development of Pneumocystis iBALT

A.) Representative immunofluorescent staining of C57BL/6, Il17ra^−/−, Il17rb^−/−, and Il17rc^−/− mice, respectively, 14 days following Pneumocystis infection demonstrates decreased iBALT formation (top). Il17ra^−/−, Il17rb^−/−, and Il17rc^−/− mice all have reduced proliferating B-cells (middle) and disorganized T-cell and B-cell zones (bottom). Scale bars represent 100μm. B.) Quantification of size, number, and area occupied by iBALT structures. C.) Cxcl13 expression is reduced in whole lung tissue of Il17ra^−/−, Il17rb^−/−, and Il17rc^−/− mice (n=8–16). D.) Eosinophil recruitment as measured by Prg2 expression is diminished in Il17ra^−/− and Il17rb^−/− mice (n=8–16). E.) Il17ra^−/− mice have increased Pneumocystis burden comparable to that of CD4-depleted animals, while Il17rb^−/− and Il17rc^−/− have a trend towards increased burden (n=4–12). Data are shown as mean ± standard error of the mean. * p<0.05, ** p<0.01, **** p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons.

Figure 6. IL-17A and IL-13 synergistically regulate Cxcl13 expression in pulmonary fibroblasts

A.) Expression of Cxcl13 in sorted lung populations, including CD45⁻ lung blood endothelial cells (CD31⁺Podoplanin⁻), triple negative SLO cells (CD31⁻Podoplanin⁻), lymphatic endothelial cells (CD31⁺Podoplanin⁺), pulmonary fibroblasts (CD31⁻Podoplanin⁺) and CD45⁺ cells at day 14 post Pneumocystis infection (n=4). Cxcl13 is induced in the CD45⁻ cell populations, with CD31⁻Podoplanin⁺ fibroblasts representing the largest relative contributor. B.) The receptors for IL-13 (Il4r and Il13ra1) and IL-17A (Il17ra and Il17rc) are expressed in each cell population. * p<0.05, ** p<0.01, *** p<0.001 by student’s T-test. C.) Immunofluorescent staining demonstrates CXCL13 protein within the lymphoid follicle 14 days post-infection with Pneumocystis. Scale bars represent 100 μm. D.) Pulmonary fibroblasts upregulate Cxcl1 and Ccl11 following a six hour stimulation with 100 ng of IL-17A and various doses of IL-13 (in ng), respectively. Cxcl13 expression is unchanged in all conditions at six hours post-stimulation (n=4). E.) Cxcl1 expression is reduced at 24 hours post-stimulation, while Ccl11 expression persists. Stimulation with both IL-17A and IL-13 leads to upregulation of Cxcl13 at 24 hours post-stimulation (n=2–4). Data are shown as mean ± standard error of the mean. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons.

Figure 7. Cxcl13 expression is dependent on STAT3 and GATA3 activation

A.) Expression of Il6 is upregulated in pulmonary fibroblasts following a six-hour stimulation with IL-17A (n=4). B.) Cxcl13 is induced in pulmonary fibroblast by 24-hour stimulation with IL-13 in combination with either IL-6 or IL-17A (n=4–10). C.) Cxcl13 upregulation is partially blocked by treatment with an anti-gp130 antibody (n=7–9). D.) Cxcl13 expression is attenuated in pulmonary fibroblasts from Stat3^fl/fl mice following in vitro treatment with adenovirus-Cre (n=4–12). E.) Stat6^−/− fibroblasts fail to upregulate Cxcl13 following stimulation with IL-17A and IL-13 (n=3–6). F.) Cxcl13 expression is attenuated in pulmonary fibroblasts from Gata3^fl/fl mice following in vitro treatment with adenovirus-Cre (n=3–4). Data are shown as mean ± standard error of the mean. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons.