Interleukin-1 signaling and CD4+ T cells control B cell recruitment to the lungs in chronic beryllium disease

Figures

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  Figure 1. IL-1R1 signaling controls BeO-induced B cell recruitment to the lungs. Wildtype (WT) and interleukin-1 receptor 1 knockout (IL-1R1-/-) mice were exposed to BeO and sacrificed on day 21. (A) Flow cytometry plots show the recruitment of adaptive immune cells in the lungs of PBS or BeO-exposed mice. Graph plots show a total number of tissue-specific B220+ B cells (B) and neutrophils (C) in the lungs of PBS and BeO exposed WT and IL-1R1-/- mice on day 21. (D) Total protein concentration in the BALF measured by BCA, and (E) CXCL13 levels in the BALF on day 21 measured by ELISA. (F) Hematoxylin and eosin (H&E) staining of BeO-exposed lung tissues from WT (top) and IL-1R1-/- (bottom) mice. (G) Cellular aggregates in the lungs of WT and IL-1R1-/- mice exposed to BeO were quantified using QuPath. Data are representative of three independent experiments having n=5 mice per group. One-way ANOVA was used to test statistical differences among the groups. P<0.05 (*) is considered statistically significant. P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

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  Figure 2. Neutralization of IL-1α and IL-1R1 abrogates BeO-induced lung inflammation and B cell recruitment. BeO exposed mice were treated with an isotype control antibody, anti-IL-1α, or Anakinra, and sacrificed on day 12. (A) Flow cytometry analysis of BALF shows CD19+ B cell and CD3+ T cell populations on day 12. (B) Total number of tissue-specific B220+ B cells in the lungs of antibody-treated mice on day 12. (C) Total number of CD3+ T cells in the lungs of antibody-treated mice on day 12. (D) Total number of neutrophils in the lungs on day 12. Data are representative of three independent experiments, 4 mice per group. One-way ANOVA was used to test statistical differences among the groups. P<0.05 (*) is considered statistically significant. P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

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  Figure 3. Adoptively transferred B cells protect against BeO-induced lung injury. μMT mice were adoptively transferred with wild-type B cells purified from the spleen or with splenocytes from μMT mice. Recipient mice were sensitized and boosted with BeO using the previously described protocol. Percent frequency (A) and total number of B cells (B) in the BAL of BeO-exposed mice as examined on day 21. (C) Protein leak in the bronchoalveolar lavage fluid (BALF) of μMT mice adoptively transferred with WT splenic B cells or μMT splenocytes and exposed to BeO on day 21. (D) Hematoxylin and eosin staining show low magnification (top) and high magnification (bottom), and (E) cellular aggregates quantified in the lungs of mice adoptively transferred with WT splenic B cells or splenocytes from μMT mice into μMT mice and exposed to BeO. Cellular aggregates were quantified using QuPath. (F) Immunohistochemical (IHC) staining of B cells low magnification (top) and high magnification (bottom) on day 21 using an anti-rabbit B cell antibody in the lungs of mice adoptively transferred with WT splenic B cells or splenocytes from μMT mice into μMT mice and exposed to BeO. Data are representative of three…

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  Figure 4. CXCR5 expression is required for B cell recruitment to the lung in response to BeO exposure. WT and CXCR5-/- mice were sensitized with seven doses of BeO (100 μg) on days 0, 1, 2, 14, 15, 18, and 19 and sacrificed on day 21. (A) Representative dot plot shows the presence of CD19+ and CD3+ T cells and (B) cumulative percent frequency (left) and total number (right) of B cells in the BAL of BeO-exposed mice. (C) Percent frequency (left) and the total number of CD3+ T cells (right) in the BAL of BeO-exposed WT and CXCR5-/- mice on day 21. (D) Hematoxylin and eosin staining and (E) immunohistochemical (IHC) staining of B cells using an anti-rabbit B cell antibody of BeO-exposed lung tissues. (F) Cellular aggregates in the lungs of WT and CXCR5-/- mice were quantified using QuPath. Data are representative of three independent experiments, 4-5 mice per group. One-way ANOVA was used to test statistical differences among the groups. P<0.05 (*) is considered statistically significant. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

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  Figure 5. Kinetics of leukocyte recruitment to the lung in response to BeO exposure. HLA-DP2 FVB/N Tg mice were sensitized with three doses of BeO (100 μg) on days 0, 1, and 2. Mice (3-4) per time point were sacrificed, and immune cells were analyzed by flow cytometry. Graph plots show the percent frequency of (A) CD3+ T cells, (B) CD19+ B cells, and (C) CXCL13 in the bronchoalveolar lavage fluid. Graph plots show the percent frequency of neutrophils (D) and protein leak (E) in the bronchoalveolar lavage fluid (BALF) of BeO exposed mice. Data are representative of two independent experiments, 3-4 mice per group. One-way ANOVA was used to test statistical differences among the groups. P<0.05 (*) is considered statistically significant. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

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  Figure 6. B cells are dispensable for CD4+ T cell recruitment and CXCL13 expression. HLA-DP2 FVB/N Tg mice were sensitized with three doses of BeO (100 μg) on days 0, 1, and 2. Mice were then randomly assigned and treated with PBS, isotype control monoclonal antibody (Iso), or an anti-CD20 monoclonal antibody one time (d-1), or weekly (w). (A) Total B cells (B) CD3+ T cells, and (C) CD4+ T cells in the lungs of BeO-exposed mice on day 21. (D) Percent neutrophils were examined in the BAL of BeO-exposed mice. Graph plots show protein leak (E) and CXCL13 (F) in the bronchoalveolar lavage fluid (BALF) of BeO exposed mice. Data are representative of two independent experiments, four mice per group. One-way ANOVA was used to test statistical differences among the groups. P<0.05 (*) is considered statistically significant. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

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  Figure 7. CD4+ T cells control optimal B cell recruitment to the lung in BeO-exposed HLA-DP2 Tg mice. HLA-DP2 FVB/N Tg mice were sensitized with BeO on days 0, 1, and 2. Mice were treated with either PBS, an isotype control antibody, or an anti-CD4 monoclonal antibody (100 μg) one day before the experiment (d-1), day 4, day 9, or day 15. (A) Total B cells in the lungs on day 21. (B) Percent neutrophils in the BAL on day 21. (C) CXCL13 levels were examined in the BALF of BeO-exposed HLA-DP2-Tg mice on day 21, treated with either PBS, an isotype control antibody, or an anti-CD4 monoclonal antibody (100 μg) one day before the experiment. Data are representative of two independent experiments, four mice per group. One-way ANOVA was used to test statistical differences among the groups. P < 0.05 (*) is considered statistically significant. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

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  Figure 8. Phenotyping of tissue-specific B cells in the lungs of BeO-exposed HLA-DP2 Tg mice. Mice were sensitized and boosted with BeO (100 μg) on days 0, 1, 2, 14, 15, 18, and 19. (A) Lung B cell-surface expression of IgD (top left), IgM (top right), CD44 (bottom left), and CD86 (bottom right) were examined on day 21. The histogram represents FMO control (solid-grey), PBS (red), and BeO (black) groups. (B) Graph plots show the mean fluorescence intensity (MFI) of various molecules on the tissue-specific B cells. Data are representative of two independent experiments containing n=3-5 mice/group. Significance was determined by the Mann-Whitney t-test. P<0.05 (*) is considered statistically significant.

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  Figure 9. RNA-sequencing of lung tissue-specific B cells from BeO exposed HLA-DP2 Tg mice identify genes involved in increased activation and antigen presentation. HLA-DP2 FVB/N Tg mice were sensitized and boosted with BeO on days 0, 1, 2, 14, 15, 18, and 19. On day 21, IVCD45- B cells were sorted by FACS. B cells were pooled from n=5 mice/per experiment, and RNA samples (1, 2, 3) were prepared. The numbers (1, 2, 3) represent individual experiments. RNA sequencing was performed and compared against control samples (4, 5, 6), prepared from CD23+ follicular B cells sorted from the spleens of WT mice. (A) Principal component analysis showing the distribution of control and BeO BeO-exposed samples. Differentially expressed genes are associated with interferon signaling (B), tight junction interactions (C), and antigen presentation (D).