CFTR regulates B cell activation and lymphoid follicle development

Abstract

Background: Cystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear.

Methods: The number of LFs, BAFF⁺, TLR4⁺ and proliferation marker Ki67⁺ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr ^-/-) and wild type controls. The number of lung LFs, B cells and BAFF⁺, CXCR4⁺, immunoglobulin G⁺ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr ^-/- and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS).

Results: There was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr ^-/- mice had increased lung LFs and BAFF⁺ and CXCR4⁺ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr ^-/- mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr ^-/- mice produced increased IL-6 when stimulated with LPS compared to wild type controls.

Conclusions: These data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.

Keywords: B lymphocyte; BAFF; Cystic fibrosis; Lymphoid follicles.

Fig. 1

Lung lymphoid follicles, B cells, BAFF⁺ and Ki67⁺ B cells were increased in CF patients. a Representative confocal images of triple-color immunofluorescence staining of representative pulmonary lymphoid follicles (LFs) from subjects with CF (top panels) and normal controls (Ctrl, bottom panels). B cells are identified by staining with red fluorophore for CD20. BAFF is identified by staining with green fluorophore. Ki67 positive cells have a gray color. 4′6-diamidino-2-phenylindole (blue) was used to counterstain the nuclei. The final panel in each row is a merged file of CD20, BAFF and Ki67 staining. In the CF images (top row), the magnification is 20X. In the control images (bottom row), the magnification is 60X. However, for the control images digital zooming was used to better visualize the CD20⁺ cells. The images shown are representative of LFs in CF subjects (n = 27) and normal controls (n = 10). b Representative low power images of lung tissue from a subject with CF (left panel) and normal control (right panel). Lymphoid follicles are identified by staining with red fluorophore for CD20. 4′6-diamidino-2-phenylindole (blue) was used to counterstain the nuclei

Fig. 2

B cells and BAFF⁺ and Ki67⁺ B cells were increased in patients with CF. The number of a lymphoid follicles, b B cells, c BAFF⁺ and d Ki67⁺ B cells were quantified in lung tissue from CF subjects (n = 27) and normal controls (n = 10). There was significant increase in LFs, B cells identified by CD20 staining, BAFF⁺ and Ki67⁺ B cells in subjects with CF compared to controls. B cells, BAFF⁺ B cells and lymphoid follicles (LFs) with organized germinal centers strongly correlated with total LFs. There was a significant correlation between the number of lymphoid follicles and e CD20-positive B cells (R² = 0.411, p = 0.006), f BAFF-positive B cells (R² = 0.498, p < 0.0001) and g LFs with organized germinal centers (R² = 0.497, p < 0.0001) in patients with CF. This is a quantitative assessment of the data demonstrated in the representative images in Fig. 1. Mann-Whitney U test was used to perform the statistical analysis (a-d). Box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. Squares in (a) indicate outliers. Correlation coefficients were calculated using the Pearson rank method. A total of 27 CF subjects were analyzed. *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001, CF versus normal controls

Fig. 3

LFs, B cells, BAFF⁺ and IgG⁺ B cells were increased in uninfected Cftr ^−/− mice. a Representative confocal images of triple-color immunofluorescence staining of representative pulmonary LFs from uninfected Cftr ^−/− mice (top two rows) and wild type (WT) controls (bottom row) from cohort 1. B cells are identified by staining for CD45R and red fluorophore. BAFF positive cells have a grey color. IgG positive B cells were identified by staining with green fluorophore. 4′6-diamidino-2-phenylindole (blue) was used to counterstain the nuclei. The final panel in each row is a merged file of CD45R, BAFF and IgG staining. In the Cftr ^−/− mice images (top row), the magnification is 60X. In the wild type control images (bottom row), the magnification is 100X. The images shown are representative of LFs in Cftr ^−/− mice (n = 6) and WT controls (n = 7). Cftr ^−/− and WT lung b Lymphoid follicles, c B cells, d BAFF⁺ B cells and e IgG⁺ B cells were quantified. Mann-Whitney U test was used to perform the statistical analysis (b-e). Box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. *p ≤ 0.05, **p ≤ 0.001, Cftr ^−/− mice versus WT

Fig. 4

LFs, B cells, BAFF⁺ and CXCR4⁺ B cells were increased in uninfected Cftr ^−/− mice. A second cohort of uninfected Cftr ^−/− mice bred, housed and maintained at a different facility were also analyzed (cohort 2). a Representative confocal images of triple-color immunofluorescence staining of representative pulmonary LFs from Cftr ^−/− mice (top row) and wild type (WT) controls (bottom row). B cells are identified by staining for CD20 and red fluorophore. BAFF positive B cells were identified by staining with green fluorophore. CXCR4 positive B cells have a grey color. 4′6-diamidino-2-phenylindole (blue) was used to counterstain the nuclei. The final panel in each row is a merged file of CD20, BAFF and CXCR4 staining. In both the WT and Cftr ^−/− images the magnification is 60X. The images shown are representative of LFs in Cftr ^−/− mice (n = 8) and WT controls (n = 4). Cftr ^−/− and WT lung b Lymphoid follicles and c B cells were quantified. Mann-Whitney U test was used to perform the statistical analysis (b-c). Box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. **p ≤ 0.005, Cftr ^−/− mice versus WT

Fig. 5

Cftr ^−/− B cells have increased MHC class II and increased IL-6 response to LPS MHC class II expression was measured on B cells from lung mononuclear cells a isolated from uninfected Cftr^−/− and wild type controls with directly conjugated antibodies by flow cytometry. b A representative histogram of MHC class II expression on lung B cells from uninfected Cftr ^−/− mice (gray histogram) and wild type controls (white histogram). Isotype control staining is represented by dotted line. MHC class II expression was also measured on B cell splenocytes isolated from uninfected Cftr ^−/− mice and wild type controls (c). d IL-6 production from B cells isolated from the spleens of Cftr ^−/− (n = 7) and WT mice (n = 4) stimulated with media or lipopolysaccharide (LPS) for 48 h was determined. The concentration of IL-6 in supernatants was determined by ELISA. Box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. *p ≤ 0.05, Cftr ^−/− mice versus WT.

Fig. 6

TLR4⁺ B cells are increased in patients with CF. a Representative confocal images of triple-color immunofluorescence staining of representative pulmonary lymphoid follicles (LFs) from subjects with CF (bottom panels) and normal controls (top panels). B cells are identified by staining with red fluorophore for CD20. TLR4 is identified by staining with green fluorophore. 4′6-diamidino-2-phenylindole (blue) was used to counterstain the nuclei. The final panel in each row is a merged file of CD20, TLR4 and DAPI staining. The percentage of TLR4⁺ B cells was quantified in lung tissue from CF subjects (n = 7) and normal controls (n = 5). There was significant increase in TLR4⁺ B cells in subjects with CF compared to controls (b). Images were taken at 10X using a Nikon Eclipse 80i microscope. Mann-Whitney U test was used to perform the statistical analysis. Box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. *p = 0.003, CF versus normal controls