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B cells have been implicated in the pathogenesis of multiple sclerosis, but the mechanisms that guide B cell activation in the periphery and subsequent migration to the CNS remain incompletely understood. We previously showed that systemic inflammation induces an accumulation of B cells in the spleen in a CCR6/CCL20-dependent manner. In this study, we evaluated the role of CCR6/CCL20 in the context of myelin oligodendrocyte glycoprotein (MOG) protein-induced (B cell-dependent) experimental autoimmune encephalomyelitis (EAE). We found that CCR6 is upregulated on murine B cells that migrate into the CNS during neuroinflammation. In addition, human B cells that migrate across CNS endothelium in vitro were found to be CCR6 + , and we detected CCL20 production by activated CNS-derived human endothelial cells as well as a systemic increase in CCL20 protein during EAE. Although mice that lack CCR6 expression specifically on B cells exhibited an altered germinal center reaction in response to MOG protein immunization, CCR6-deficient B cells did not exhibit any competitive disadvantage in their migration to the CNS during EAE, and the clinical and pathological presentation of EAE induced by MOG protein was unaffected. These data, to our knowledge, provide new information on the role of B cell-intrinsic CCR6 expression in a B cell-dependent model of neuroinflammation., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Microbial and host cell interactions stimulate rabbit B cells to diversify the primary Ab repertoire in GALT. B cells at the base of appendix follicles begin proliferating and diversifying their V-(D)-J genes around 1 wk of age, ∼5 d after B cells first begin entering appendix follicles. To gain insight into the microbial and host cell interactions that stimulate B cells to diversify the primary Ab repertoire, we analyzed B cell trafficking within follicles during the first week of life. We visualized B cells, as well as chemokines that mediate B cell homing in lymphoid tissues, by in situ hybridization, and we examined B cell chemokine receptor expression by flow cytometry. We found that B cells were activated and began downregulating their BCRs well before a detectable B cell proliferative region appeared at the follicle base. The proliferative region was similar to germinal center dark zones, in that it exhibited elevated CXCL12 mRNA expression, and B cells that upregulated CXCR4 mRNA in response to signals acquired from selected intestinal commensals localized in this region. Our results suggest that after entering appendix follicles, B cells home sequentially to the follicle-associated epithelium, the follicular dendritic cell network, the B cell/T cell boundary, and, ultimately, the base of the follicle, where they enter a proliferative program and diversify the primary Ab repertoire., (Copyright © 2014 by The American Association of Immunologists, Inc.)

In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Innate immune responses provoke the accumulation of leukocytes at sites of inflammation. In addition to monocytes and granulocytes, B cells also participate in antimicrobial innate immune responses; however, the mechanisms for accumulation of B cells to sites of inflammation are not well understood. To study B cell accumulation following systemic inflammation, we used a model synthetic ligand that stimulates a specific pattern recognition molecule, nucleotide-binding oligomerization domain-containing protein 1 (Nod1). Upon exposure to Nod1 agonists, both B cells and neutrophils rapidly accumulate within the spleen, and dendritic cells migrate into the periarterial lymphoid sheath. Nod1 stimulation led to a marked increase in several chemokines within the spleen, including CXCL13, CCL2, and CCL20. Whereas the lymphotoxin pathway was critical for the induction of the B cell chemoattractant CXCL13 in response to Nod1 agonists, B cell accumulation within the spleen following Nod1-induced systemic inflammation was independent of the lymphotoxin pathway. In contrast, a CCR6/CCL20 chemokine loop instructed rapid increase of B cells in the spleen in response to systemic administration of Nod1 agonists in a TNF-α-dependent manner. Moreover, CCR6 was required to regulate Nod1-mediated B cell responses. These results reveal a novel mechanism of B cells during inflammation and shed light on how B cells participate in innate immune responses to microbial stimulation.

The significance of Th17 cells and IL-17A signaling in host defense and disease development has been demonstrated in various infection and autoimmune models. Additionally, the generation of Th17 cells is highly influenced by microbes. However, the specific bacterial components capable of shaping Th17 responses have not been well defined. The goals of this study were to understand how a bacterial toxin, cholera toxin (CT), modulates Th17-dominated response in isolated human CD4(+) T cells, and what are the mechanisms associated with this modulation. CD4(+) cells isolated from human peripheral blood were treated with CT. The levels of cytokine production and specific Th cell responses were determined by ELISA, Luminex assay, and flow cytometry. Along with the decreased production of other proinflammatory cytokines (IFN-γ, TNF-α, and IL-2), we found that CT could directly enhance the IL-17A production through a cAMP-dependent pathway. This enhancement is specific for IL-17A but not for IL-17F, IL-22, and CCL20. Interestingly, CT could increase IL-17A production only from Th17-committed cells, such as CCR6(+)CD4(+) T cells and in vitro-differentiated Th17 cells. Furthermore, we also demonstrated that this direct effect occurs at a transcriptional level because CT stimulates the reporter activity in Jurkat and primary CD4(+) T cells transfected with the IL-17A promoter-reporter construct. This study shows that CT has the capacity to directly shape Th17 responses in the absence of APCs. Our findings highlight the potentials of bacterial toxins in the regulation of human Th17 responses.

The skin as the outmost epithelial tissue is under frequent physical, chemical, and biological assaults. To counter the assaults and maintain the local tissue homeostasis, the skin is stationed with various innate or innate-like lymphocytes such as γδT cells. Increasing evidence suggests that an intrathymically programmed process is involved in coordinated expression of multiple homing molecules on specific γδT cell subsets to direct their localization in different regions of the skin for the protective functions. However, detailed molecular events underlying the programmed skin distribution of specific γδT cell subsets are not fully understood. We report in this study that the temporally and spatially regulated downregulation of chemokine receptor CCR6 on fetal thymic Vγ3(+) epidermal γδT precursors is involved in their thymic egress and proper localization in the epidermis. Failure of downregulation of CCR6 in the mature Vγ3(+) epidermal γδT precursor cells due to the constitutive expression of transgenic CCR6 resulted in their abnormal accumulation in the fetal thymus and reduced numbers of the epidermal γδT cells. In addition, the transgenic expression of CCR6 on the Vγ3(+) γδT cells also improperly increased their distribution in dermis of the skin. Those findings advanced our understanding of the molecular basis regulating the tissue specific distribution of various innate-like γδT cell lymphocytes in the skin.

T cell subset-specific migration to inflammatory sites is tightly regulated and involves interaction of the T cells with the endothelium. Th17 cells often appear at different inflammatory sites than Th1 cells, or both subsets appear at the same sites but at different times. Differences in T cell subset adhesion to endothelium may contribute to subset-specific migratory behavior, but this possibility has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo and we characterized their migratory phenotype by flow cytometry and quantitative RT-PCR. More Th17 than Th1 cells interacted with E-selectin. Fewer Th17 than Th1 cells bound to TNF-α-activated E-selectin-deficient endothelial cells, and intravital microscopy studies demonstrated that Th17 cells engage in more rolling interactions with TNF-α-treated microvessels than Th1 cells in wild-type mice but not in E-selectin-deficient mice. Th17 adhesion to ICAM-1 was dependent on integrin activation by CCL20, the ligand for CCR6, which is highly expressed by Th17 cells. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. These data provide evidence that E-selectin- and ICAM-1-dependent adhesion of Th17 and Th1 cells with endothelium are quantitatively different.

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.

IL-17A is a cytokine secreted by the newly described Th17 cells implicated in rheumatoid arthritis (RA). Less is known about its receptors in synoviocytes. IL-17RA and IL-17RC were found to be overexpressed in RA peripheral whole blood and their expression was detected locally in RA synovium. In vitro, IL-17A synergized with TNF-alpha to induce IL-6, IL-8, CCL-20, and matrix metalloproteinase-3. Using microarrays, a specific up-regulation of Glu-Leu-Arg+ CXC chemokines was observed in IL-17A-treated synoviocytes. Using both posttranslational inhibitions by silencing interfering RNA and extracellular blockade by specific inhibitors, we showed that both IL-17RA and IL-17RC are implicated in IL-17A-induced IL-6 secretion, whereas in the presence of TNF-alpha, the inhibition of both receptors was needed to down-regulate IL-17A-induced IL-6 and CCL-20 secretion. Thus, IL-17A-induced IL-6, IL-8, and CCL20 secretion was dependent on both IL-17RA and IL-17RC, which are overexpressed in RA patients. IL-17A-induced pathogenic effects may be modulated by IL-17RA and/or IL-17RC antagonism.