The chemokine receptor CXCR5 is pivotal for ectopic mucosa-associated lymphoid tissue neogenesis in chronic Helicobacter pylori-induced inflammation

Abstract

Ectopic lymphoid follicles are a key feature of chronic inflammatory autoimmune and infectious diseases, such as rheumatoid arthritis, Sjögren's syndrome, and Helicobacter pylori-induced gastritis. Homeostatic chemokines are considered to be involved in the formation of such tertiary lymphoid tissue. High expression of CXCL13 and its receptor, CXCR5, has been associated with the formation of ectopic lymphoid follicles in chronic infectious diseases. Here, we defined the role of CXCR5 in the development of mucosal tertiary lymphoid tissue and gastric inflammation in a mouse model of chronic H. pylori infection. CXCR5-deficient mice failed to develop organized gastric lymphoid follicles despite similar bacterial colonization density as infected wild-type mice. CXCR5 deficiency altered Th17 responses but not Th1-type cellular immune responses to H. pylori infection. Furthermore, CXCR5-deficient mice exhibited lower H. pylori-specific serum IgG and IgA levels and an overall decrease in chronic gastric immune responses. In conclusion, the development of mucosal tertiary ectopic follicles during chronic H. pylori infection is strongly dependent on the CXCL13/CXCR5 signaling axis, and lack of de novo lymphoid tissue formation attenuates chronic immune responses.

Fig. 1

Comparable Helicobacter pylori gastric colonization in Wt and CXCR5^−/− mice. a Schematic representation of the experimental H. pylori infection model. b The degree of H. pylori colonization in the gastric mucosa of Wt (black squares; n = 7) and CXCR5^−/− (open circles; n = 10) mice was assessed by colony counting 5 and 7 months after infection. The degree of colonization was comparable 5 and 7 months after infection. Statistical significance was calculated by unpaired Mann–Whitney test. NS nonsignificant. c H. pylori specimen were also detected by immunohistochemical staining with an anti-H. pylori antiserum and hematoxylin counterstaining 7 months after infection (in red; examples indicated by arrows). A lower (upper panels) and higher magnification (lower panels) of each group is shown; scale bars, 100 μm

Fig. 2

Helicobacter pylori-induced formation of ectopic lymphoid follicles in Wt but not in CXCR5^−/− mice. Hematoxylin–eosin stainings of paraffin-embedded gastric mucosal tissue sections of three representative H. pylori-infected Wt mice (a) and CXCR5^−/− mice (b) 5–17 months after infection. Eleven out of 13 CXCR5^−/− mice completely lacked lymphoid aggregates (one representative example, left panel), and two CXCR5^−/− mice showed loose inflammatory aggregates (middle and right panel). Scale bars, 100 μm

Fig. 3

Reduced mucosal proliferation and reactive epithelial changes in Helicobacter pylori-infected CXCR5^−/− mice. a Mucosal proliferation is indicated by Ki67-staining in red (indicated by an arrow). A lower (upper panels) and higher magnification (lower panels) of each group is shown; scale bars, 100 μm. b Reactive epithelial changes including epithelial hyperplasia and epithelial regeneration were semi-quantitated using a score of 0–2. The black bar indicates the mean of Wt (n = 12), and the gray bar the mean of CXCR5^−/− (n = 13) mice. Statistically significant differences between Wt and CXCR5^−/− mice are indicated by asterisks; *P < 0.05; unpaired Student's t test

Fig. 4

Lack of segregated ectopic follicular tissue formation in CXCR5^−/− compared to Wt mice. Paraffin sections with ectopic lymphoid follicles of two representative Wt (a) and sections with loose lymphoid aggregates of two CXCR5^−/− mice (b) 5–17 months after infection were immunohistochemically stained for B (B220 in red) and T (CD3 in blue) cells, for macrophages (F4/80 in red) and B cells (B220 in blue), and for blood vessel formation (CD31 in red, indicated by arrows) and B cells (B220 in blue). Scale bars, 100 μm

Fig. 5

Mucosal tertiary lymphoid tissues in WT mice exhibit lymph node-like structures. Paraffin sections with ectopic lymphoid follicles of representative Wt mice were immunohistochemically stained for the formation of HEVs (PNAd, peripheral lymph node addressin, in red, indicated by arrows; a), regulatory T cells (Foxp3 in red; c), lymphocytic proliferation (Ki67 in red; d), together with B220 staining (in blue) for B cells and plasma cells (CD138 in red, indicated by an arrow; e). Scale bars, 100 μm. Immunofluorescence and immunohistochemical stainings of frozen sections show CD11c⁺ DCs (in blue) scattered within the T cell zone (CD3⁺ T cells in green; b) and FDCs (in red) located within the B cell follicle (f). Scale bar, 100 μm

Fig. 6

Helicobacter pylori-specific Th17 cell proliferation was reduced, but the Th1 responses were unaltered in CXCR5^−/− mice. Seven months after infection, cell suspensions from mesenteric LNs and spleens of Wt (black bars; n = 6–7) and CXCR5^−/− mice (gray bars; n = 6–7) were restimulated in vitro (1 × 10⁶ cells) with or without H. pylori sonicate (100 μg/ml) for 72 h. Undiluted supernatants were analyzed for IFN-γ (a), IL-17A (b), and IL-22 (c) by ELISA. The mean cytokine release from cells of non-infected Wt mice (n = 3) and CXCR5^−/− mice (n = 3) was subtracted from each individual-infected animal. Bars represent the arithmetic means ± SEM. *P < 0.05 and **P < 0.01; unpaired Mann–Whitney test

Fig. 7

Decreased serum levels of anti-Helicobacter pylori-specific IgG and IgA in CXCR5^−/− mice. a Six to seven months after infection, blood sera from Wt (black bar; n = 17) and CXCR5^−/− mice (gray bar; n = 18) were analyzed for the presence of H. pylori-specific IgG (a) and IgA (b) Abs by ELISA. Non-infected Wt (hatched black bar; n = 5) and CXCR5^−/− mice (hatched gray bar; n = 5) served as controls. Sera were diluted 1:500 for IgA and 1:400 for IgG measurements, and the absorbance at 450 nm represents the relative anti-H. pylori IgG or IgA value. Data represent the arithmetic means ± SEM of two independent experiments. **P < 0.01, unpaired Mann–Whitney test