Hiroaki Takatori

Lymphoid tissue inducer–like cells are an innate source of IL-17 and IL-22

The interleukin (IL) 17 family of cytokines has emerged to be critical for host defense as well as the pathogenesis of autoimmune and autoinflammatory disorders, and serves to link adaptive and innate responses. Recent studies have identified a new subset of T cells that selectively produce IL-17 (Th17 cells; Bettelli, E., T. Korn, and V.K. Kuchroo. 2007. Curr. Opin. Immunol. 19:652–657; Kolls, J.K., and A. Linden. 2004. Immunity. 21:467–476), but the regulation of IL-17 production by innate immune cells is less well understood. We report that in vitro stimulation with IL-23 induced IL-17 production by recombination activating gene (Rag) 2−/− splenocytes but not Rag2−/− common γ chain−/− splenocytes. We found that a major source of IL-17 was CD4+CD3−NK1.1−CD11b−Gr1−CD11c−B220− cells, a phenotype that corresponds to lymphoid tissue inducer–like cells (LTi-like cells), which constitutively expressed the IL-23 receptor, aryl hydrocarbon receptor, and CCR6. In vivo challenge with the yeast cell wall product zymosan rapidly induced IL-17 production in these cells. Genetic deletion of signal transducer and activator of transcription 3 reduced but did not abrogate IL-17 production in LTi-like cells. Thus, it appears that splenic LTi-like cells are a rapid source of IL-17 and IL-22, which might contribute to dynamic organization of secondary lymphoid organ structure or host defense.

IL-4–Stat6 Signaling Induces Tristetraprolin Expression and Inhibits TNF-α Production in Mast Cells

Increasing evidence has revealed that mast cell–derived tumor necrosis factor α (TNF-α) plays a critical role in a number of inflammatory responses by recruiting inflammatory leukocytes. In this paper, we investigated the regulatory role of interleukin 4 (IL-4) in TNF-α production in mast cells. IL-4 inhibited immunoglobulin E–induced TNF-α production and neutrophil recruitment in the peritoneal cavity in wild-type mice but not in signal transducers and activators of transcription 6 (Stat6)–deficient mice. IL-4 also inhibited TNF-α production in cultured mast cells by a Stat6-dependent mechanism. IL-4–Stat6 signaling induced TNF-α mRNA destabilization in an AU-rich element (ARE)–dependent manner, but did not affect TNF-α promoter activity. Furthermore, IL-4 induced the expression of tristetraprolin (TTP), an RNA-binding protein that promotes decay of ARE-containing mRNA, in mast cells by a Stat6-dependent mechanism, and the depletion of TTP expression by RNA interference prevented IL-4–induced down-regulation of TNF-α production in mast cells. These results suggest that IL-4–Stat6 signaling induces TTP expression and, thus, destabilizes TNF-α mRNA in an ARE-dependent manner.

Stat5a Inhibits IL-12-Induced Th1 Cell Differentiation through the Induction of Suppressor of Cytokine Signaling 3 Expression

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a−/−) CD4+ T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-γ production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a−/− CD4+ T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a−/− CD4+ T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a−/− CD4+ T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4+ T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a−/− CD4+ T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3.

Sox5 and c-Maf cooperatively induce Th17 cell differentiation via RORγt induction as downstream targets of Stat3

A novel isoform of Sox5, Sox5t, and c-Maf activate RORγt to induce Th17 cells. Sox5−/− mice exhibit impaired Th17 differentiation and are thus resistant to EAE and delayed-type hypersensitivity.

B and T lymphocyte attenuator suppresses IL-21 production from follicular Th cells and subsequent humoral immune responses.

We recently showed that mice lacking B and T lymphocyte attenuator (BTLA), a third inhibitory coreceptor expressed on B cells and T cells, exhibit an increased Ag-specific IgG response and gradually develop hyper-γ–globulinemia and autoantibody production. Recent studies revealed that follicular Th (Tfh) cells, which are non-Th1, non-Th2 effector T cells that express CXCR5 and provide help for B cells to produce Ig, also express BTLA. However, the role of BTLA in Tfh cell function remains unknown. In this study, we examined the regulatory role of BTLA in the development and function of Tfh cells. We found that CXCR5+ Tfh cells expressed higher levels of BTLA than did CXCR5− conventional CD4+ T cells. We also found that adoptive transfer of BTLA−/− CD4+ T cells, stimulated under Tfh cell-inducing conditions (Tfh-like cells), to wild-type (WT) mice induced more Ag-specific IgG2a and IgG2b production compared with that of WT Tfh-like cells. By contrast, another adoptive-transfer experiment using BTLA−/− mice as recipients showed that the expression of BTLA on B cells was not involved in the regulation of Tfh-like cell-mediated Ag-specific IgG responses. Moreover, the development of IL-21–producing CXCR5+ Tfh-like cells was significantly increased in BTLA−/− CD4+ T cells compared with WT CD4+ T cells. Furthermore, Tfh-like cell-mediated IgG responses were abolished when IL-21R−/− mice were used as recipients. These results suggest that BTLA signaling suppresses IL-21 production from Tfh cells and subsequent Tfh cell-mediated IgG responses.

Indispensable Role of Stat5a in Stat6-Independent Th2 Cell Differentiation and Allergic Airway Inflammation

It is well-recognized that Stat6 plays a critical role in Th2 cell differentiation and the induction of allergic inflammation. We have previously shown that Stat5a is also required for Th2 cell differentiation and allergic airway inflammation. However, it is the relative importance and redundancy of Stat6 and Stat5a in Th2 cell differentiation and allergic airway inflammation are unknown. In this study we addressed these issues by comparing Stat5a-deficient (Stat5a−/−) mice, Stat6−/− mice, and Stat5a- and Stat6 double-deficient (Stat5a−/− Stat6−/−) mice on the same genetic background. Th2 cell differentiation was severely decreased in Stat6−/−CD4+ T cells, but Stat6-independent Th2 cell differentiation was still significantly observed in Stat6−/−CD4+ T cells. However, even in the Th2-polarizing condition (IL-4 plus anti-IFN-γ mAb), no Th2 cells developed in Stat5a−/−Stat6−/− CD4+ T cells. Moreover, Ag-induced eosinophil and lymphocyte recruitment in the airways was severely decreased in Stat5a−/−Stat6−/− mice compared with that in Stat6−/− mice. These results indicate that Stat5a plays an indispensable role in Stat6-independent Th2 cell differentiation and subsequent Th2 cell-mediated allergic airway inflammation.

Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (P<0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21Cip1, p27Kip1, p15INK4B, and p16INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (P<0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (P<0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.

Overexpression of cell cycle regulator CDCA3 promotes oral cancer progression by enhancing cell proliferation with prevention of G1 phase arrest

BackgroundCell division cycle associated 3 (CDCA3), part of the Skp1-cullin-F-box (SCF) ubiquitin ligase, refers to a trigger of mitotic entry and mediates destruction of the mitosis inhibitory kinase. Little is known about the relevance of CDCA3 to human malignancy including oral squamous cell carcinoma (OSCC). We aimed to characterize the expression state and function of CDCA3 in OSCC.MethodsWe evaluated CDCA3 mRNA and protein expression in both OSCC-derived cell lines and primary OSCCs and performed functional analyses of CDCA3 in OSCC-derived cells using the shRNA system.ResultsThe CDCA3 expression at both the mRNA and protein levels was frequently up-regulated in all cell lines examined and primary tumors (mRNA, 51/69, 74 %; protein, 79/95, 83 %) compared to normal controls (p < 0.001). In contrast, no significant level of CDCA3 protein expression was seen in oral premalignant lesions (OPLs) (n = 20) compared with the expression in OSCCs. Among the clinical variables analyzed, the CDCA3 expression status was closely related to tumor size (p < 0.05). In addition, suppression of CDCA3 expression with shRNA significantly (p < 0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase. Further, there was up-regulation of the cyclin-dependent kinase inhibitors (p21Cip1, p27Kip1, p15INK4B, and p16INK4A) in the knockdown cells.ConclusionThe current results showed that overexpression of CDCA3 occurs frequently during oral carcinogenesis and this overexpression might be associated closely with progression of OSCCs by preventing the arrest of cell-cycle progression at the G1 phase via decreased expression of the cyclin-dependent kinase inhibitors.

Tumor Suppressor p53 Inhibits Systemic Autoimmune Diseases by Inducing Regulatory T Cells

The tumor suppressor p53 plays a central role in tumor suppression by inducing apoptosis, cell cycle arrest, senescence, and DNA repair. In addition to the antitumor functions of p53, accumulating evidence using systemic p53-deficient mice suggests that p53 suppresses autoimmunity. However, it remains unknown how p53 suppresses autoimmunity. In this study, we generated T cell–specific p53-deficient mice (CD4-Cre p53fl/fl mice, or p53 conditional knockout [cKO] mice) and found that aged p53-cKO mice spontaneously developed inflammatory lesions in various organs, including lung, liver, stomach, thyroid gland, submandibular gland, and kidney. Additionally, anti-nuclear Abs and autoantibodies against gastric parietal cells were detected in p53-cKO mice but not in control p53fl/fl mice (p53 wild-type mice). Importantly, the number of Foxp3+CD4+ regulatory T cells (Tregs) in the spleen and lung as well as in vitro differentiation of induced Tregs was significantly reduced in p53-cKO mice as compared with that in p53 wild-type mice. Regarding the mechanisms underlying p53-mediated Treg induction, p53 enhanced the transcription of Foxp3 by binding to the promoter and the conserved noncoding DNA sequence-2 of the Foxp3 gene. Taken together, these results suggest that p53 expressed in T cells functions as a suppressor for autoimmunity by inducing Treg differentiation.

Annexin A10 in Human Oral Cancer: Biomarker for Tumoral Growth via G1/S Transition by Targeting MAPK Signaling Pathways

Background Annexins are calcium and phospholipid binding proteins that form an evolutionary conserved multigene family. Considerable evidence indicates that annexin A10 (ANXA10) is involved in tumoral progression, although little is known about its role in human oral carcinogenesis. In this study, we investigated the involvement of ANXA10 in oral squamous cell carcinoma (OSCC). Methodology/Principal Findings ANXA10 mRNA and protein expressions were assessed by quantitative reverse transcriptase polymerase chain reaction and immunoblotting, and we conducted a proliferation assay and cell-cycle analysis in ANXA10 knockdown cells in vitro. We evaluated the correlation between the ANXA10 expression status in 100 primary OSCCs and the clinicopathological features by immunohistochemistry. ANXA10 mRNA and protein expression levels were up-regulated in all cellular lines examined (n = 7, p<0.05). ANXA10 knockdown cells showed that cellular proliferation decreased by inactivation of extracellular regulated kinase (ERK) (p<0.05), and cell-cycle arrest at the G1 phase resulted from up-regulation of cyclin-dependent kinase inhibitors. ANXA10 protein expression in primary OSCCs was also significantly greater than in normal counterparts (p<0.05), and higher expression was correlated with tumoral size (p = 0.027). Conclusions/Significance Our results proposed for the first time that ANXA10 is an indicator of cellular proliferation in OSCCs. Our results suggested that ANXA10 expression might indicate cellular proliferation and ANXA10 might be a potential therapeutic target for the development of new treatments for OSCCs.