Ichino Chinen

Aryl hydrocarbon receptor negatively regulates dendritic cell immunogenicity via a kynurenine-dependent mechanism

Although an immunoregulatory role of aryl hydrocarbon receptor (Ahr) has been demonstrated in T cells and macrophages, little is known about its function in dendritic cells (DC). Here, we show that lipopolysaccharide (LPS) and CpG stimulate Ahr expression in bone marrow-derived dendritic cells (BMDC). Furthermore, we found that Ahr is required to induce indoleamine 2,3-dioxygenase (IDO) expression, an immunosuppressive enzyme that catabolizes tryptophan into kynurenine (Kyn) and other metabolites in DC. In the presence of LPS or CpG, Ahr-deficient (Ahr−/−) mature BMDC induced immune responses characterized by reduced Kyn and IL-10 production compared with results observed with tolerogenic mature WT BMDC. In a coculture system with LPS- or CpG-stimulated BMDC and naive T cells, Ahr−/− BMDC inhibited naive T-cell differentiation into regulatory T (Treg) cells, which likely facilitated Th17 cell development and promoted naive T-cell proliferation. Addition of synthetic L-Kyn to the coculture system skewed the differentiation of naive T cells to Treg cells rather than Th17 cells. Taken together, our results demonstrate a previously unknown negatively regulatory role for Ahr in DC-mediated immunogenesis in the presence of LPS or CpG, which, in turn, alters the Kyn-dependent generation of Treg cells and Th17 cells from naive T cells.

Aryl hydrocarbon receptor deficiency in T cells suppresses the development of collagen-induced arthritis

The contributions of aryl hydrocarbon receptor (Ahr) to the pathogenesis of rheumatoid arthritis have not been elucidated. Here, we show that Ahr deficiency ameliorated collagen-induced arthritis, a mouse model of RA. Collagen-immunized Ahr KO mice showed decreased serum levels of such proinflammatory cytokines as IL-1β and IL-6. The Th17 and Th1 cell populations in lymph nodes from these mice decreased and increased, respectively, whereas the percentage of regulatory T cells was unchanged. Interestingly, a lack of Ahr specifically in T cells significantly suppressed collagen-induced arthritis development, whereas Ahr deficiency in macrophages had no effect. These finding indicate that the development of experimental autoimmune arthritis depends on the presence of Ahr in T cells, and that Th1/Th17 balance may be particularly important for this process.

HB-EGF and PDGF Mediate Reciprocal Interactions of Carcinoma Cells with Cancer-Associated Fibroblasts to Support Progression of Uterine Cervical Cancers

Tumor stroma drives the growth and progression of cancers. A heparin-binding epidermal growth factor-like growth factor, HB-EGF, is an EGF receptor ligand that stimulates cell growth in an autocrine or paracrine fashion. While elevated expression of HB-EGF in cancer cells and its contribution to tumor progression are well documented, the effects of HB-EGF expression in the tumor stroma have not been clarified. Here, we show that HB-EGF is expressed in stromal fibroblasts where it promotes cancer cell proliferation. In uterine cervical cancers, HB-EGF was detected immunohistochemically in the stroma proximal to the cancer epithelium. Proliferation of cervical cancer cells in vitro was enhanced by coculture with fibroblasts isolated from tumor tissues of patients with cervical cancer. Inhibition of HB-EGF function or treatment with platelet-derived growth factor (PDGF) inhibitors abrogated cancer cell growth enhanced by cervical cancer-associated fibroblast (CCF) coculture. Furthermore, tumor formation in a mouse xenograft model was enhanced by cotransplantation of CCF or mouse embryonic fibroblasts, but not with embryonic fibroblasts from HB-EGF-deficient mice. Conversely, conditioned medium from cancer cells induced HB-EGF expression in CCF. Mechanistic investigations established that PDGF was the primary factor responsible. Together, our findings indicate that HB-EGF and PDGF reciprocally mediate the interaction of cancer cells with cancer-associated fibroblasts, promoting cancer cell proliferation in a paracrine manner that has implications for novel combinatorial cancer therapies.

Aryl hydrocarbon receptor negatively regulates LPS-induced IL-6 production through suppression of histamine production in macrophages

Macrophages play a pivotal role in innate immune responses to pathogens via toll-like receptors. We previously demonstrated that aryl hydrocarbon receptor (Ahr) in combination with signal transducer and activator of transcription 1 (Stat1) negatively regulates pro-inflammatory cytokine production by inhibiting nuclear factor-κB activation in macrophages after LPS stimulation. Here, we show that Ahr also negatively regulates production of the pro-inflammatory cytokine IL-6 by suppressing histamine production in macrophages stimulated by LPS. We found that Ahr-Sp1 complex, independent of Stat1, represses histidine decarboxylase expression by inhibiting LPS-induced Sp1 phosphorylation on Ser residues in macrophages; this leads to suppression of histamine production. Moreover, we found that loratadine and chlorpromazine, histamine 1 receptor (H1R) antagonists, more effectively impair the production of LPS-induced IL-6 than that of other inflammatory cytokines in Ahr(-/-) macrophages. Collectively, these results demonstrate that Ahr negatively regulates IL-6 production via H1R signaling through the suppression of histamine production in macrophages following LPS stimulation.

The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production to maintain intestinal homeostasis

Aryl hydrocarbon receptor (Ahr), a transcription factor, plays a critical role in autoimmune inflammation of the intestine. In addition, microRNAs (miRNAs), small non-coding oligonucleotides, mediate pathogenesis of inflammatory bowel diseases (IBD). However, the precise mechanism and interactions of these molecules in IBD pathogenesis have not yet been investigated. We analyzed the role of Ahr and Ahr-regulated miRNAs in colonic inflammation. Our results show that deficiency of Ahr in intestinal epithelial cells in mice exacerbated inflammation in dextran sodium sulfate-induced colitis. Deletion of Ahr in T cells attenuated colitis, which was manifested by suppressed Th17 cell infiltration into the lamina propria. Candidate miRNA analysis showed that induction of colitis elevated expression of the miR-212/132 cluster in the colon of wild-type mice, whereas in Ahr (-/-) mice, expression was clearly lower. Furthermore, miR-212/132(-/-) mice were highly resistant to colitis and had reduced levels of Th17 cells and elevated levels of IL-10-producing CD4(+) cells. In vitro analyses revealed that induction of type 1 regulatory T (Tr1) cells was significantly elevated in miR-212/132(-/-) T cells with increased c-Maf expression. Our findings emphasize the vital role of Ahr in intestinal homeostasis and suggest that inhibition of miR-212/132 represents a viable therapeutic strategy for treating colitis.

Anti-human HB-EGF monoclonal antibodies inhibiting ectodomain shedding of HB-EGF and diphtheria toxin binding.

HB-EGF is a member of the EGF family of growth factors that bind and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. ProHB-EGF functions as the receptor for the diphtheria toxin (DT). HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Monoclonal antibodies (mAbs) specific for HB-EGF could be an important tool in HB-EGF research. However, few such mAbs have been established to date. In this study, we newly generated seven clones of hybridoma-derived mAbs by immunizing HB-EGF null mice with recombinant human HB-EGF protein. All mAbs specifically bound to human HB-EGF but not to mouse HB-EGF. Epitope mapping analysis showed that most of the mAbs recognized the EGF-like domain. Although none of the newly isolated mAbs directly inhibited the mitogenic activity of HB-EGF for EGFR-expressing cells, some strongly inhibited DT-binding. Interestingly, some of the mAbs efficiently inhibited ectodomain shedding of proHB-EGF, and consequently prevented the cell growth of the EGFR-expressing cells in a co-culture system with proHB-EGF-expressing cells. Hence, these new anti-HB-EGF mAbs may advance clinical as well as basic research on HB-EGF.

Aryl hydrocarbon receptor in combination with Stat1 regulates LPS-induced inflammatory responses

1. 1. Kimura, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20090560 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20090560%26rft_id%253Dinfo%253Apmid%252F19703987%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo