Hirohisa Kawahata

The Influence of Excessive IL-6 Production In Vivo on the Development and Function of Foxp3+ Regulatory T Cells

IL-6 is a proinflammatory cytokine and its overproduction is implicated in a variety of inflammatory disorders. Recent in vitro analyses suggest that IL-6 is a key cytokine that determines the balance between Foxp3+ regulatory T cells (Tregs) and Th17 cells. However, it remains unclear whether excessive IL-6 production in vivo alters the development and function of Foxp3+ Tregs. In this study, we analyzed IL-6 transgenic (Tg) mice in which serum IL-6 levels are constitutively elevated. Interestingly, in IL-6 Tg mice, whereas peripheral lymphoid organs were enlarged, and T cells exhibited activated phenotype, Tregs were not reduced but rather increased compared with wild-type mice. In addition, Tregs from Tg mice normally suppressed proliferation of naive T cells in vitro. Furthermore, Tregs cotransferred with naive CD4 T cells into SCID–IL-6 Tg mice inhibited colitis as successfully as those transferred into control SCID mice. These results indicate that overproduction of IL-6 does not inhibit development or function of Foxp3+ Tregs in vivo. However, when naive CD4 T cells alone were transferred, Foxp3+ Tregs retrieved from SCID–IL-6 Tg mice were reduced compared with SCID mice. Moreover, the Helios− subpopulation of Foxp3+ Tregs, recently defined as extrathymic Tregs, was significantly reduced in IL-6 Tg mice compared with wild-type mice. Collectively, these results suggest that IL-6 overproduced in vivo inhibits inducible Treg generation from naive T cells, but does not affect the development and function of natural Tregs.

Blockade of interleukin-6 signaling suppresses experimental autoimmune uveoretinitis by the inhibition of inflammatory Th17 responses.

The aim of this study was to investigate the effect of anti-mouse IL-6 receptor monoclonal antibody (MR16-1) treatment on CD4 T cell differentiation and compared it to the effect of anti-TNF mAb treatment with using a murine model of experimental autoimmune uveoretinitis (EAU). C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) to induce ocular inflammation treatment with control IgG or MR16-1 or anti-TNF mAb. Helper T cells differentiation was analyzed during the development of EAU. Immunization with IRBP increased the frequency of Th17 cells rather than Th1 cells in the early stage of EAU. Treatment with MR16-1 on the same day as immunization (day 0) or one day after (day 1) suppressed ocular inflammation in EAU mice. Treatment with MR16-1 on day 0 inhibited the induction of Th17 cells in vivo, and inhibited not only IRBP-responsive Th17 cells but also their Th1 counterparts and induced IRBP-responsive regulatory T (Treg) cells in vitro. The administration of anti-TNF mAb had no significant protective effect in EAU mice. The protective effect of anti-IL-6R mAb treatment, but not anti-TNF mAb treatment on EAU correlated with the inhibition of Th17 differentiation. This finding suggests that IL-6 blockade may have a therapeutic effect on human ocular inflammation which is mediated via mechanisms distinct from those of TNF blockade. IL-6 blockade may thus represent an alternative therapy for patients with ocular inflammation who are refractory to anti-TNF mAb therapy.

Comparative analysis of the effects of anti‐IL‐6 receptor mAb and anti‐TNF mAb treatment on CD4+ T‐cell responses in murine colitis

Background: The efficacy of anti‐tumor necrosis factor monoclonal antibody (anti‐TNF mAb) for Crohns disease (CD) is well established, and anti‐interleukin‐6 receptor (anti‐IL‐6R) mAb has also been reported to be effective in CD. It is, however, unclear if the efficacy and mechanisms of both agents are different in CD therapy. Methods: Using an adoptive transfer colitis model, we compared the efficacy of anti‐IL‐6R mAb, anti‐TNF mAb, and TNF receptor‐Fc fusion protein (TNFR‐Fc), and their modes of action on CD4+ T cells. We also investigated the role of Th1 and Th17 cells in colitis using the same model. Results: The histological scores for the anti‐IL‐6R mAb and anti‐TNF mAb groups but not for TNFR‐Fc group were much lower than that for the control group, and the score was the lowest for the anti‐IL‐6R mAb group. The frequency of proliferating CD4+ T cells was reduced in anti‐IL‐6R mAb and anti‐TNF mAb groups, but not in the TNFR‐Fc group, whereas the frequency of apoptotic CD4+ T cells was similar in all groups. Anti‐IL‐6R mAb suppressed the induction of Th17 cells and increased the frequency of lamina propria regulatory T cells, whereas anti‐TNF mAb exerted no influence on CD4+ T‐cell differentiation. A deficiency in interferon‐γ and/or IL‐17 in CD4+ T cells reduced the severity of colitis. Conclusions: Our findings suggest that suppression of the proliferation of pathogenic CD4+ T cells is the major mode of action of biological agents for colitis therapy. Anti‐IL‐6R mAb might have benefits in CD patients with Th17 dominance and impaired Treg frequency. (Inflamm Bowel Dis 2011)

Effect of angiotensin II receptor blocker, olmesartan, on turnover of bone metabolism in bedridden elderly hypertensive women with disuse syndrome.

Although recent studies suggest that several antihypertensive drugs could reduce the risk of bone fracture, it is still unclear how these drugs act on bone remodeling, especially in elderly women with severe osteoporosis with disuse syndrome. In the present study, we investigated the effects of a calcium channel blocker (CCB) and an angiotensin II receptor blocker (ARB) on bone metabolism in elderly bedridden women with hypertension and disuse syndrome.

Leucine-rich alpha 2 glycoprotein promotes Th17 differentiation and collagen-induced arthritis in mice through enhancement of TGF-β-Smad2 signaling in naïve helper T cells

BackgroundLeucine-rich alpha 2 glycoprotein (LRG) has been identified as a serum protein elevated in patients with active rheumatoid arthritis (RA). Although the function of LRG is ill-defined, LRG binds with transforming growth factor (TGF)-β and enhances Smad2 phosphorylation. Considering that the imbalance between T helper 17 (Th17) cells and regulatory T cells (Treg) plays important roles in the pathogenesis of RA, LRG may affect arthritic pathology by enhancing the TGF-β-Smad2 pathway that is pivotal for both Treg and Th17 differentiation. The purpose of this study was to explore the contribution of LRG to the pathogenesis of arthritis, with a focus on the role of LRG in T cell differentiation.MethodsThe differentiation of CD4 T cells and the development of collagen-induced arthritis (CIA) were examined in wild-type mice and LRG knockout (KO) mice. To examine the influence of LRG on T cell differentiation, naïve CD4 T cells were isolated from LRG KO mice and cultured under Treg- or Th17-polarization condition in the absence or presence of recombinant LRG.ResultsIn the CIA model, LRG deficiency led to ameliorated arthritis and reduced Th17 differentiation with no influence on Treg differentiation. By addition of recombinant LRG, the expression of IL-6 receptor (IL-6R) was enhanced through TGF-β-Smad2 signaling. In LRG KO mice, the IL-6R expression and IL-6-STAT3 signaling was attenuated in naïve CD4 T cells, compared to wild-type mice.ConclusionsOur findings suggest that LRG upregulates IL-6R expression in naïve CD4 T cells by the enhancement of TGF-β-smad2 pathway and promote Th17 differentiation and arthritis development.

Continuous infusion of angiotensin II modulates hypertrophic differentiation and apoptosis of chondrocytes in cartilage formation in a fracture model mouse.

Although components of the renin-angiotensin system (RAS) are reported to be expressed in cultured chondrocytes and cartilage, little is known about the precise function of Angiotensin II (Ang II) in chondrocytes. In this study, we employed a rib fracture model mouse to investigate the effect of Ang II on chondrocytes. Ang II type 1 receptor (AT1R) was expressed in chondrocytes in the growth plate of mouse tibia. Continuous infusion of Ang II to rib-fractured mice resulted in a significant increase in the volume of cartilage, suggesting Ang II-induced hypertrophic differentiation of chondrocytes. It was also confirmed by a significant increase in the mRNA expression of Sox9 and runt-related transcription factor 2 (Runx2), which are genes related to chondrocyte differentiation, and type X collagen, matrix metalloproteinase (MMP)-13 and Indian hedgehog (Ihh), which are hypertrophic chondrocyte-specific molecular markers. Chondrocyte hypertrophy with upregulation of these genes was attenuated by administration of olmesartan, an AT1R blocker, but not by hydralazine. Moreover, Ang II infusion significantly suppressed apoptosis of chondrocytes, accompanied by significant induction of mRNA expression of bcl-2 and bcl-xL. Olmesartan, but not hydralazine, significantly attenuated the reduction of apoptotic cells and the increase in anti-apoptotic genes induced by Ang II infusion. Overall, the present study demonstrated that Ang II promoted hypertrophic differentiation of chondrocytes and reduced apoptosis of hypertrophic chondrocytes independently of high blood pressure. The present data indicate the role of Ang II in cartilage, and might provide a new concept for treatment of cartilage diseases.

Therapeutic Effect of Intra‐Articular Injection of Ribbon‐Type Decoy Oligonucleotides for Hypoxia Inducible Factor‐1 on Joint Contracture in an Immobilized Knee Animal Model

Limited range of motion (ROM) as a result of joint contracture in treatment associated with joint immobilization or motor paralysis is a critical issue. However, its molecular mechanism has not been fully clarified and a therapeutic approach is not yet established.

Anti-inflammatory Effect of Electro-acupuncture via Reduction in Colonic Peristalsis in a Mouse Model of Inflammatory Bowel Disease

Conventional therapies for idiopathic inflammatory bowel diseases (IBD), which are chronic nonspecific in- flammatory disorders, are still insufficient. Recently, there has been a focus on acupuncture, one of the major complemen- tary and alternative interventions, and some clinical trials have suggested efficacy of acupuncture for IBD. However, the therapeutic mechanism and effect of acupuncture are still unknown and are controversial because of not only lack of clini- cal trials but also poor experimental investigation. To prevent patients receiving ineffectual therapy, evaluation of its effi- cacy and elucidation of its mechanisms are important. In this study, we employed mice with dextran sulfate sodium (DSS)-induced colitis and investigated the effect of electro-acupuncture on disease activity and colonic inflammation. We performed electro-acupuncture every other day. Mice with colitis showed an increase in disease activity after DSS treat- ment, whereas in mice treated with electro-acupuncture, scores of disease activity, stool consistency and bloody bowel discharge were significantly reduced, consistent with improvement in pathological findings. In addition, recruitment of macrophages and expression of ICAM were inhibited, accompanied by a significant reduction of DSS-induced expression of interleukin (IL)-1� and IL-6. Of importance, acceleration of giant migrating contraction (peristalsis) induced by vagostigmin attenuated these effects of electro-acupuncture. Overall, these data demonstrated that electro-acupuncture suppressed disease activity and colonic inflammation in DSS-induced colitis through a reduction in propagated colonic peristalsis mediated by sympathetic overactivity, suggesting the potential of acupuncture as a relief therapy for IBD.

Beneficial effect of laughter therapy on physiological and psychological function in elders

In the present study we investigated the effect of laughter therapy on physiological and psychological function in older people.

Electric Field Exposure Improves Subjective Symptoms Related to Sleeplessness in College Students: A Pilot Study of Electric Field Therapy for Sleep Disorder

Background: Sleep disorder is a common health problem in modern days. Estab-lishment of safe, non-invasive, convenient and effective treatment is anticipated in the field of complementary and alternative medicine. Objective: We designed a protocol for a randomized controlled trial to investigate the effect of Electric Field (EF) exposure on sleep disorder. Methods: Nineteen college students with sleep disorder, defined as a score of 8 or higher on the Pitzburg Sleep Quality Index, were divided into two groups; EF intervention and sham treatment. EF exposure (50-Hz, 18 kV) was performed for 30 minutes a day for five con-secutive days. Subjective parameters were obtained by an OSA sleep inventory MA version consisting of five factors, and objective parameters were measured using a sleep-scan. Results: Significant improvement in scores of three factors (sleepiness on rising, refreshing and sleep length) was observed after 5 days of EF exposure intervention, as compared to both before intervention and after 5 days of sham treatment. Moreover, improvement rati-os for these three factors were significantly higher in the EF group than in the sham treat-ment group. Analysis of the sleep-scan demonstrated a high improvement ratio for duration of nocturnal awakening in the EF group. Conclusion: The beneficial effect of electric field therapy on sleep disorder in college stu-dents is considered to be beyond a placebo effect. This study raises the therapeutic possibil-ity of electric field exposure.