Transcription factor c‐Rel mediates communication between commensal bacteria and mucosal lymphocytes

The NF‐κB transcription factor c‐Rel plays a crucial role in promoting and regulating immune responses and inflammation. However, the function of c‐Rel in modulating the mucosal immune system is poorly understood. T follicular helper (Tfh) cells and IgA production in gut‐associated lymphoid tissues (GALT) such as Peyer's patches (PPs) are important for maintaining the intestinal homeostasis. Here, c‐Rel was identified as an essential factor regulating intestinal IgA generation and function of Tfh cells. Genetic deletion of c‐Rel resulted in the aberrant formation of germinal centers (GCs) in PPs, significantly reduced IgA generation and defective Tfh cell differentiation. Supporting these findings, the Ag‐specific IgA response to Citrobacter rodentium was strongly impaired in c‐Rel‐deficient mice. Interestingly, an excessive expansion of segmented filamentous bacteria (SFB) was observed in the small intestine of animals lacking c‐Rel. Yet, the production of IL‐17A, IgA, and IL‐21, which are induced by SFB, was impaired due to the lack of transcriptional control by c‐Rel. Collectively, the transcriptional activity of c‐Rel regulates Tfh cell function and IgA production in the gut, thus preserving the intestinal homeostasis.

translocation of active NF-κB dimers into the nucleus. 2,3 Functional analysis of NF-κB proteins in vivo revealed that both, T cell-dependent B cells responses and the germinal center (GC) B cell development are dependent on the activity of NF-κB, although the contribution of individual subunits is still unclear. 5 Interestingly, the NF-κB transcription factor c-Rel was shown to be indispensable for IgG, but not for IgA responses, 6 suggesting that c-Rel is not required for the class switch to IgA. However, it has to be noted that the serum IgA levels, but not intestinal IgA concentrations were measured in this study.
IgA-producing B lymphocytes and plasma cells are mainly located in the gut-associated lymphoid tissues including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), cryptopaches, and the cecal patch. 7 IgA derived from the small intestine and colon is secreted as a dimer into the intestinal lumen. 8 Secretory IgA is not only important for preventing invasion of pathogens into the intestinal tissue, but it is also involved in maintaining gut homeostasis. Previous studies revealed that the majority of bacterial species in the gut lumen are bound to IgA and that commensal bacteria utilize secretory IgA for gut colonization thus directly contributing to the host-microbial symbiosis. 9 Recent observations have indicated the specific role of follicular helper T cells (Tfh), responsible for IgA selection and synthesis in PPs. 10 Defective secretion of intestinal IgA, smaller PPs, and reduced frequencies of Tfh cells have been described for germ-free (GF) mice devoid of microbiota. 11 Thus, intestinal commensals promote IgA production and GC formation in PPs, with Tfh cells essentially supporting this process.
NF-κB activity is associated with B cell development, proliferation, and IgG production. 12 However, whether the NF-κB canonical pathway containing hetero-and homodimers of c-Rel also play an important role in IgA generation has not been investigated in detail yet. We thus examined whether c-Rel is required for regulating the interactions between Tfh cells and Ig-secreting B and plasma cells in the intestine at steadystate conditions.

Mice
C57BL/6 WT mice were purchased from the Jackson Laboratory. Both WT and rel -/mice on C57BL/6 background were maintained under specific pathogen-free conditions at the animal facility of BMFZ Marburg, Germany. 8-12-week-old female mice were used for experiments.
All experiments were performed in accordance with the protocols approved by the local authorities (Study Nr. Ex-22-2020, RP Giessen, Germany).

Flow cytometry
The single-cell suspensions derived from PPs, mesenteric lymph nodes

Infection with Citrobacter rodentium
All mice used in the experiments were 8-12 weeks old females. Animals were orally gavaged with 1 × 10 10 colony forming units (CFU/ml) of nalidixic acid-resistant C. rodentium in a total volume of 200 μl of PBS.
For analysis of bacterial burden, colonic feces from infected WT and c-Rel-deficient mice were collected into tubes containing LB medium.
Serial dilutions were made from the collected samples and the number of colonies was determined at day 14 post-infection.

Fluorescence in situ hybridization
Small intestinal tissues were fixed overnight using the methacarn fixative solution prior to embedding into paraffin. For FISH analysis, 3-

Enzyme-linked immunosorbent assay
For detection of fecal IgA small intestinal feces were homogenized in PBS and centrifuged at 12,000 rpm for 5 min. IgA in fecal supernatants was measured using a primary rabbit-anti-mouse IgA Ab (Rockland), and a secondary rabbit-anti-mouse IgA-HRP

Statistical analysis
Statistical analysis was performed using Student's t-test. All results are expressed as mean ± SEM, unless otherwise indicated. A P < 0.05 was

Mucosal T and B lymphocytes from c-Rel-deficient mice exhibit several functional defects
The analysis of lymphocytes derived from rel -/mice revealed that both T and B cells require c-Rel for various aspects of their activity. 13 Although mucosal and systemic immune systems contain the same T cell subsets, only intestinal T lymphocytes are constitutively activated by Ags and molecules originating from commensal microbiota and food. 14 Previously, we and others showed that the development of thymic Foxp3 + CD4 + regulatory T cells (Tregs) is regulated by the activity of NF-κB subunit c-Rel. [15][16][17][18] We accordingly observed lower frequencies of Foxp3 + Tregs in mucosa-associated lymphoid tissue (MALT) such as PPs and mLNs in the absence of c-Rel ( Supplementary   Fig. S1A,B). Interestingly, despite low Treg percentages in mice lacking c-Rel, these animals did not develop overt autoimmune or inflammatory responses mediated by effector CD4 + T or B cells. When we searched for activated and memory T cells using the activation marker CD44 (CD44 + CD62L -CD4 + T lymphocytes), we observed significantly decreased frequencies of effector/memory CD4 + T cells in mLNs and PPs of c-Rel-deficient mice as compared to WT counterparts, which might explain the lack of autoimmunity and inflammation in aged rel -/mice ( Supplementary Fig. S1C,D). Ab production in naive and immunized mice was particularly demonstrated for IgG1 and IgG2a but not for IgG3 and IgM in the absence of c-Rel. 20 GCs within PPs generate high-affinity IgA against food and bacterial antigens. We here demonstrate that both, the formation of GCs as well as that of highly activated GC PNA + B220 + B cells were significantly impaired in c-Rel-deficient mice (Fig. 1E, F) . The frequency of IL-21 + cells within PP CD4 + T lymphocytes is presented in the right panel (n = 4 mice per group). The Student's t-test was applied for statistical analysis (**P = 0.001-0.01; ***P < 0.001; data are shown as mean ± SEM) c-Rel ( Fig. 2A, B). Moreover, mice lacking this transcription factor had also substantially decreased percentages of IgA + B220plasma cells in the intestinal lamina propria (Supplementary Fig. S3A,B). Compared to WT mice, IgA concentrations in feces were decreased in animals lacking c-Rel (Fig. 2C). To further examine the impact of c-Rel on generating Ag-specific IgA responses in vivo, we orally infected c-Rel-deficient and WT mice with the murine intestinal pathogen C. rodentium. Of note, c-Rel-deficient mice showed an increased susceptibility to C. rodentium infection than WT mice (Fig. 2D). Consistent with this, the infection with C. rodentium induced significantly higher amounts of pathogenspecific IgA antibodies in the intestine of WT mice as compared to animals lacking c-Rel (Fig. 2E). Taken together, the function of intestinal T and B lymphocytes that sense Ags of commensals and pathogens is strongly dependent on the activity of the transcription factor c-Rel.  (Fig. 3B and C). We and others have recently described a defective secretion of IL-23 in the gut of c-Rel-deficient mice, which might explain the reduced IL-17A production. 25,26 Similarly, we found a defective expression of IL-21 (Fig. 3D), a Tfh and Th17 cell-derived cytokine, which is also induced following the colonization of GF mice with SFB. 23 Previously, a direct binding of c-Rel to the Il21 promoter sequence was described. 26 Notably, although the lack of intestinal IgA production in c-Rel-deficient mice was associated with a dominant expansion of SFB, IL-17A, and IL-21 cytokines, which are normally induced by this bacterium, were reduced in the absence of c-Rel.

Aberrant growth of segmented filamentous bacteria in the intestine of c-Rel deficient mice
Together, these findings indicate that c-Rel is essential for the appropriate communication of the mucosal immune system with commensal bacteria, resulting in a stable composition of gut microbiota, formation, and secretion of protective IgA and cytokines IL-21 and IL-17A.

CONCLUDING REMARKS
In conclusion, our data suggest that the transcription factor c-Rel