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  • Multiple subsets of Breg cells in mice

    2022-01-14

    Multiple subsets of Breg cells in mice have been identified that could suppress the T cell-mediated response. However, a lack of consensus surface markers for Breg cells made it difficult to judge whether Breg cells are a special B cell subset or whether all Chlorambucil have an immuno-regulatory function under inflammation induction, and it impossible to apply this important immunosuppressive B cell to clinical practice. Therefore, investigating the mechanisms of Breg induction and finding its specific transcription factors have become the most important challenge in the study of Breg. It has been reported that the B cells that act as the regulatory cells in colitis was CD1d+ B cells in MLN (Mizoguchi et al., 2002) or the splenic CD5+CD1d+ B cells (Yanaba et al., 2011). In the studies of our group, we have identified CD11b+ B cells as the regulatory B cells that played important roles in the suppression of inflammatory immune responses in autoimmune diseases, such as IBD and EAH. CD11b, as an integrin subunit, combines with the β2 chain CD18 to form Mac-1, which is an integrin heterodimer with important effects on myeloid cell migration and adherence to stimulated tissue (Pawlus et al., 2013; Abram and Lowell, 2009). Studies also highlighted the protective role of CD11b in B cells following activation during inflammatory responses, including the maintenance of autoreactive B cell tolerance (Watanabe et al., 2000), inhibition of T cell activity (Liu et al., 2015), recruitment of Treg cells (Wang et al., 2019), as well as regulation of TLR4-dependent inflammatory responses (Faridi et al., 2017). Although best known for its expression on B1 cells (Griffin and Rothstein, 2011), our previous study of a chronic liver disease model showed that CD11b was universally expressed on all of the B cell subsets (Liu et al., 2015), and we first suggested CD11b as a surface marker and regulatory molecule for Breg cells. In this study, we found that during the process of colitis CD11b expression was widely increased in B cells of gut-associated lymphoid tissue, the splenic B cells, as well as several reported regulatory B cells, such as marginal zone (MZ), marginal zone precursor (MZP) and CD1dhi CD5+ B cells (Fig. 1), implying that CD11b might be crucial for the regulatory function of B cells. Although IEL mainly consists of CD8+ T cells, there is also a certain proportion of B cells. It has been reported that after the activation, B cells in peyer’s patches differentiate into germinal center B cells and plasmablasts, and then traffic into the intestinal lamina propria. Both the IEL and LPL are effect site of the colitis, so we checked their immuno-regulatory functions together. Furthermore, the down-expression of CD11b led to severer colitis (Fig. 3), and CD11b-knockout B cells lost their regulatory function in the EAH model (Liu et al., 2015). Our data supports the notion that CD19+CD11b+B cells are likely to be a subset of B cells with immuno-regulatory functions in autoimmune diseases. In the models of colitis, B cells have been reported to be activated to produce IL10 to suppress inflammation (Rosser and Mauri, 2015; Mizoguchi et al., 2002). Activated B cells responding to antigen stimuli convert to a highly activated state and led to increased rates of glycolysis to support cell proliferation (Kunisawa, 2017). Our published data in an EAE model suggested that CD11b expression in B cells might be related to B cell activation, and CD11b+ B cells are proliferative (Liu et al., 2015). During the process of colitis, the B cells isolated from PPs expressed increased ki67 (Supplementary Fig. S2A), implying that the B cells in colitic mice were active and proliferative. The major finding in this paper is that HIF-1α is a promoter of Itgam gene transcription. The mucosal inflammatory lesions in mouse IBD models were revealed to be profoundly hypoxic or even anoxic (Colgan and Taylor, 2010; Taylor and Colgan, 2017), which could promote the activity and survival of mucosal immune cells through HIF-1 activation. HIF-1α has been reported to play a protective role in IBD (Fluck et al., 2016; Marks et al., 2017; Wang et al., 2017). It seems that HIF-1α is involved in the suppressive function of CD11b in B cells. We found that B cells from colitic mice had a greater expression of the transcriptional regulator HIF-1α than those naïve B cells (Fig. 2A). These findings are consistent with a study demonstrating that IgA plasma cells in the iLP seem to prefer using glycolysis as an energy supply, compared to naïve B cells in the PPs (Kunisawa, 2017). Knockdown with siRNA or protein inhibition of HIF-1α led to the down-expression of CD11b in B cells (Fig. 2), which implies that HIF-1α might be involved in the transcriptional regulation of CD11b. The transcriptional regulation of CD11b is very important in its expression. The transcription factors Sp1, Sp3, and PU.1 have been shown to bind to CD11b promoter to induce its expression during monocytic lineage differentiation (Shelley and Arnaout, 1991; Pahl et al., 1993; Chen et al., 1993). MS-2, another putative transcription factor, was also reported to prime the expression of CD11b (Farokhzad et al., 1996). Furthermore, ZBP-89, as a zinc finger transcription factor, was demonstrated to repress CD11b gene transcription and subsequently inhibit monocytes differentiating into macrophages in vitro (Park et al., 2003). HIF-1α is a key transcription factor for regulating the expression of target genes by binding to HRE elements in the promoter region (Palazon et al., 2014), and our results showed that HIF-1α bound onto the HRE II or HRE IV regions in the Itgam promoter to promote its transcription. Nevertheless, HIF-1α inhibition or knockdown did not absolutely suppress CD11b transcription, which implies that LPS or inflammation might first trigger an inflammatory response, cause HIF-1α activity and accumulation, and subsequently promote CD11b transcription, but there might be other factors involved in the regulation of CD11b expression.