The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6.

The physiological functions of members of the tumor-necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family in T cell immunity are not well understood. We found that in the presence of interleukin 6 (IL-6), naive TRAF5-deficient CD4(+) T cells showed an enhanced ability to differentiate into the TH17 subset of helper T cells. Accordingly, TH17 cell-associated experimental autoimmune encephalomyelitis (EAE) was greatly exaggerated in Traf5(-/-) mice. Although it is normally linked with TNFR signaling pathways, TRAF5 constitutively associated with a cytoplasmic region in the signal-transducing receptor gp130 that overlaps with the binding site for the transcription activator STAT3 and suppressed the recruitment and activation of STAT3 in response to IL-6. Our results identify TRAF5 as a negative regulator of the IL-6 receptor signaling pathway that limits the induction of proinflammatory CD4(+) T cells that require IL-6 for their development.

TNF Receptor-Associated Factor 5 Limits IL-27 Receptor Signaling in CD4+ T Lymphocytes.

TNF receptor-associated factor 5 (TRAF5) restrains early signaling activity of the IL-6 receptor in naive CD4+ T cells by interacting with the shared gp130 chain, although TRAF5 was initially discovered as a cytoplasmic adaptor protein to activate signaling mediated by TNF receptor family molecules. This leads to the question of whether TRAF5 limits signaling via the receptor for IL-27, which is composed of gp130 and WSX-1. The aim of this study is to clarify the role of TRAF5 in IL-27 receptor signaling and to understand the differential role of TRAF5 on cytokine receptor signaling. We found that Traf5 -/- CD4+ T cells displayed significantly higher levels of phosphorylated STAT1 and STAT-regulated genes Socs3 and Tbx21, as early as 1 h after IL-27 exposure when compared with Traf5 +/+ CD4+ T cells. Upon IL-27 and TCR signals, the Traf5 deficiency significantly increased the induction of IL-10 and promoted the proliferation of CD4+ T cells. Traf5 -/- mice injected with IL-27 displayed significantly enhanced delayed-type hypersensitivity responses, demonstrating that TRAF5 works as a negative regulator for IL-27 receptor signaling. In contrast, IL-2 and proliferation mediated by glucocorticoid-induced TNF receptor-related protein (GITR) and TCR signals were significantly decreased in Traf5 -/- CD4+ T cells, confirming that TRAF5 works as a positive regulator for cosignaling via GITR. Collectively, our results demonstrate that TRAF5 reciprocally controls signals mediated by the IL-27 receptor and GITR in CD4+ T cells and suggest that the regulatory activity of TRAF5 in gp130 is distinct from that in TNF receptor family molecules in a T cell.

IL-33-ILC2 axis promotes anti-tumor CD8+ T cell responses via OX40 signaling.

Group 2 innate lymphoid cells (ILC2s) are resident cells and participate in innate and adaptive immunity. In the tumor microenvironment (TME), ILC2s contribute to both tumorigenesis and inhibition of tumor growth, but the true role of ILC2s in TME construction remains unclear. We show that IL-33 treatment induces an anti-tumor effect in vivo in a mouse model of melanoma in which ILC2s and CD8+ T cells infiltrate into tumor tissue. This anti-tumor effect is dependent on CD8+ T cells, however, IL-33 does not act directly on CD8+ T cells because the cells lack ST2, the receptor for IL-33. ILC2s and CD8+ T cells in tumors of IL-33-treated mice express OX40 ligand (OX40L) and OX40, respectively, and in vivo blockade of OX40L-OX40 interaction canceled the anti-tumor effect of IL-33. Co-culture of CD8+ T cells expressing OX40 with IL-33-stimulated ILC2 expressing OX40L promoted cell activation and proliferation of CD8+ T cells, which was significantly suppressed by administration of anti-OX40L blocking antibody. Thus, the IL-33-ILC2 axis promotes CD8+ T cell responses via OX40/OX40L interaction and exerts an anti-tumor effect.

OX40 Ligand-Mannose-Binding Lectin Fusion Protein Induces Potent OX40 Cosignaling in CD4+ T Cells.

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is induced on activated T cells. Membrane-bound OX40 ligand (OX40L) expressed by activated antigen-presenting cells induces OX40 signaling, which promotes T cell immunity. OX40 agonism would be a potential target for immunotherapy, however, it remains unclear how the activity of OX40 can be successfully controlled by a designer OX40L protein. We prepared a soluble OX40L protein possessing a PA-peptide tag and a collagenous trimerization domain from mannose-binding lectin (MBL), and tested whether PA-MBL-OX40L fusion protein worked as an agonist for OX40. We found that the majority of recombinant PA-MBL-OX40L protein purified from culture supernatants displayed a trimer structure and bound to cell surface OX40 or OX40-Fc fusion protein in a dose-dependent manner. Upon stimulation of CD4+ T cells with TCR/CD3 without CD28, PA-MBL-OX40L displayed significantly increased proliferative and cytokine responses when compared with a benchmark agonistic monoclonal antibody for OX40. Both soluble and immobilized forms of PA-MBL-OX40L induced potent OX40 signaling in CD4+ T cells. Mice administered with PA-MBL-OX40L displayed significantly augmented T cell-mediated delayed-type hypersensitivity responses. Our results suggest that activity of OX40L could be engineered to elicit better T cell responses by rational design of its assembly and architecture.

Dysregulation of Rnf 213 gene contributes to T cell response via antigen uptake, processing, and presentation.

Growing evidence suggest the association between Moyamoya disease (MMD) and immune systems, such as antigen presenting cells in particular. Rnf213 gene, a susceptibility gene for MMD, is highly expressed in immune tissues, however, its function remains unclear. In addition, the physiological role of RNF213 gene polymorphism c.14576G > A (rs112735431), susceptibility variant for MMD, is also poorly understood. By studying Rnf213-knockout (Rnf213-KO) mice with deletion of largest exon32 and Rnf213-knockin (Rnf213-KI) mice with insertion of single-nucleotide polymorphism corresponding to c.14576G > A mutation in MMD patients, we aimed to investigate the role of RNF213 in dendritic cell development, and antigen processing and presentation. First, we found a high level of Rnf213 gene expression in conventional DCs and monocytes. Second, flow cytometric and confocal microscopic analysis revealed ovalbumin protein-pulsed Rnf213-KO and Rnf213-KI DCs showed impaired antigen uptake, proteolysis and reduced numbers of endosomes and lysosomes, and thereby failed to activate and proliferate antigen-specific T cells efficiently. In addition, Rnf213-KI DCs showed a similar phenotype to that of Rnf213-KO BMDCs. In conclusion, our findings suggest the critical role of RNF213 in antigen uptake, processing and presentation.

IQ motif-containing GTPase-activating protein 1 is essential for the optimal maintenance of lung ILC2s.

Group 2 innate lymphoid cells (ILC2s) play critical roles in type 2 immunity and are crucial for pathogenesis of various types of inflammatory disease. IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein that is involved in multiple cellular functions such as cell survival and trafficking. While the roles for IQGAP1 in T and B lymphocytes have been uncovered, the physiological significance of IQGAP1 in innate lymphocytes remains to be elucidated. In the current study, we demonstrate that using bone marrow chimeras, the deficiency of IQGAP1 caused an impaired survival of lung ILC2s in a cell-intrinsic manner and that Iqgap1-/- mice displayed decreased accumulation of ILC2s after administration of papain and thereby reduced the pathology of the disease. Moreover, Iqgap1-/- ILC2s showed a significantly enhanced apoptosis as compared to wild-type ILC2s under both steady-state and inflammatory conditions. Together these results identify for the first time that IQGAP1 is essential for homeostasis of ILC2s in the lung.

Biallelic variants/mutations of IL1RAP in patients with steroid-sensitive nephrotic syndrome.

Nephrotic syndrome (NS) is a renal disease characterized by severe proteinuria and hypoproteinemia. Although several single-gene mutations have been associated with steroid-resistant NS, causative genes for steroid-sensitive NS (SSNS) have not been clarified. While seeking to identify causative genes associated with SSNS by whole-exome sequencing, we found compound heterozygous variants/mutations (c.524T>C; p.I175T and c.662G>A; p.R221H) of the interleukin-1 receptor accessory protein (IL1RAP) gene in two siblings with SSNS. The siblings' parents are healthy, and each parent carries a different heterozygous IL1RAP variant/mutation. Since IL1RAP is a critical subunit of the functional interleukin-1 receptor (IL-1R), we investigated the effect of these variants on IL-1R subunit function. When stimulated with IL-1ß, peripheral blood mononuclear cells from the siblings with SSNS produced markedly lower levels of cytokines compared with cells from healthy family members. Moreover, IL-1R with a variant IL1RAP subunit, reconstituted on a hematopoietic cell line, had impaired binding ability and low reactivity to IL-1ß. Thus, the amino acid substitutions in IL1RAP found in these NS patients are dysfunctional variants/mutations. Furthermore, in the kidney of Il1rap-/- mice, the number of myeloid-derived suppressor cells, which require IL-1ß for their differentiation, was markedly reduced although these mice did not show significantly increased proteinuria in acute nephrotic injury with lipopolysaccharide treatment. Together, these results identify two IL1RAP variants/mutations in humans for the first time and suggest that IL1RAP might be a causative gene for familial NS.

IQGAP1 restrains T-cell cosignaling mediated by OX40.

A costimulatory signal from the tumor necrosis factor receptor (TNFR) family molecule OX40 (CD134), which is induced on activated T cells, is important for T-cell immunity. Aberrant OX40 cosignaling has been implicated in autoimmune and inflammatory disorders. However, the molecular mechanism by which the OX40 cosignaling regulates the T-cell response remains obscure. We found that OX40 associated with a scaffold protein, IQ motif-containing GTPase-activating protein 1 (IQGAP1) after ligation by its ligand OX40L. Naïve CD4+ T cells from Iqgap1-/- mice displayed enhanced proliferation and cytokine secretion upon receiving OX40 cosignaling. A C-terminal IQGAP1 region was responsible for its association with OX40, and TNFR-associated factor 2 (TRAF2) bridged these two proteins. The enhanced cytokine response in Iqgap1-/- T cells was restored by the expression of the C-terminal IQGAP1. Thus, the IQGAP1 binding limits the OX40 cosignaling. Disease severity of experimental autoimmune encephalomyelitis (EAE) was significantly exacerbated in Iqgap1-/- mice as compared to wild-type mice. Additionally, recipient mice with Iqgap1-/- donor CD4+ T cells exhibited significantly higher EAE scores than those with their wild-type counterparts, and OX40 blockade led to a significant reduction in the EAE severity. Thus, our study defines an important component of the OX40 cosignaling that restricts inflammation driven by antigen-activated T cells.

GITR controls intestinal inflammation by suppressing IL-15-dependent NK cell activity.

Glucocorticoid-induced TNFR family related gene (GITR) is a member of the TNFR superfamily that is expressed on cells of the immune system. Although the protective and pathogenic roles of GITR in T cell immunity are well characterized, the role of GITR in innate immunity in the intestinal tissues has not been well clarified. In this study, using a dextran sulfate sodium (DSS)-induced colitis model in mice, we found that GITR-deficiency rendered mice more susceptible to acute intestinal inflammation and that a significantly higher number of activated natural killer (NK) cells was accumulated in the colonic lamina propria of Gitr-/- mice as compared to wild-type mice. Additionally, Rag2-/- Gitr-/- mice, which lack T cells but have NK cells, also displayed more severe colonic inflammation than Rag2-/- mice. In contrast, an anti-GITR agonistic antibody significantly alleviated colitis in Rag2-/- mice. Engagement of GITR inhibited IL-15-mediated activating signaling events in NK cells, which include cell activation and proliferation, and production of cytokines and cytotoxic granules. Taken together, our results provide the first evidence that GITR negatively controls intestinal inflammation through NK cell functions.

Simple amino silyl ether organocatalyst for asymmetric hetero Diels-Alder reaction of isatins with enones.

New two catalysts component system comprising of a primary ß-amino silyl ethers as an organocatalyst and N-protected amino acids as a co-catalyst put together worked as an efficient organocatalyst system in the hetero Diels-Alder reaction of isatins with enones affording the chiral spirooxindole-tetrahydropyranones in good chemical yields and stereoselectivities (up to 94%, up to dr 78:22., up to 85% ee).

ADL Outcome of Stroke by Stroke Type and Time from Onset to Admission to a Comprehensive Inpatient Rehabilitation Ward.

OBJECTIVE: To examine the effect of onset to admission interval (OAI) and stroke type on activities of daily living (ADL) outcome. MATERIALS AND METHODS: Stroke patients (n=3112) admitted to and discharged from comprehensive inpatient rehabilitation wards at Nanakuri Memorial Hospital were classified into 8 OAI segments and by stroke type [intracerebral hemorrhage (ICH) and cerebral infarction (CI)]. Motor subscore of the Functional Independence Measure (FIM-M) on admission, FIM-M at discharge, FIM-M gain, length of stay (LOS), and FIM-M efficiency in the ICH and CI group matched by OAI segment were compared using the Wilcoxon test. Multiple comparisons using the Steel-Dwass test of FIM-M on admission, FIM-M at discharge, FIM-M gain, LOS, and FIM-M efficiency by OAI segments were performed. RESULTS: FIM-M on admission was lower in the ICH group than the CI group in matched OAI segments. However, FIM-M improvement was greater in the ICH group than the CI group, resulting in no difference in FIM-M between groups at discharge. In both groups, the longer the OAI, the lower the FIM-M on admission and at discharge. The distribution pattern of significant differences among OAI segments differed between the groups. LOS tended to be longer and FIM-M efficiency tended to be higher in the ICH group than in the CI group. CONCLUSIONS: The brain mass effect at the time of admission was larger and took longer to decrease in the ICH group than in the CI group. These results may improve prediction of outcomes in comprehensive inpatient rehabilitation wards.

Bmi1 Regulates IκBα Degradation via Association with the SCF Complex.

Bmi1 is a polycomb group protein and regulator that stabilizes the ubiquitination complex PRC1 in the nucleus with no evidently direct link to the NF-κB pathway. In this study, we report a novel function of Bmi1: its regulation of IκBα ubiquitination in the cytoplasm. A deficiency of Bmi1 inhibited NF-κB-mediated gene expression in vitro and a NF-κB-mediated mouse model of arthritis in vivo. Mechanistic analysis showed that Bmi1 associated with the SCF ubiquitination complex via its N terminus and with phosphorylation by an IKKα/ß-dependent pathway, leading to the ubiquitination of IκBα. These effects on NF-κB-related inflammation suggest Bmi1 in the SCF complex is a potential therapeutic target for various diseases and disorders, including autoimmune diseases.

TRAF2 and TRAF5 associated with the signal transducing receptor gp130 limit IL-6-driven transphosphorylation of JAK1 through the inhibition of proximal JAK-JAK interaction.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 constitutively bind to glycoprotein 130 kDa (gp130) and inhibit IL-6-driven activation of signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which limits the differentiation of pro-inflammatory IL-17-producing helper T cells that require IL-6-receptor (IL-6R) signals for their development. However, it is not known how the interaction between TRAF and gp130 negatively regulates STAT3 activity in the IL-6R complex. We hypothesized that TRAF proteins associated with gp130 might limit the activation of Janus kinase that is needed for the activation of STAT3. To test this, we transfected HEK293T cells to express gp130 and TRAF2 or TRAF5 together with two chimeric JAK1 proteins combined with either the N-terminal or the C-terminal protein fragment of firefly luciferase. Using this luciferase fragment complementation system, we found that the recovery of luciferase enzyme activity was coincident with proximal JAK1-JAK1 interaction and phosphorylation of JAK1 in the IL-6R complex and that the expression of TRAF protein significantly inhibited the recovery of luciferase activity. The binding of TRAF to gp130 via the C-terminal TRAF domain was essential for the inhibition. In accordance with this, upon stimulation of endogenous gp130 with a complex of IL-6 and IL-6R, Traf5-/- CD4+ T cells displayed significantly higher amounts of phosphorylated JAK1 than did their wild-type counterparts. Therefore, our results demonstrate that gp130-associated TRAF2 and TRAF5 inhibit the interaction between two JAK proteins in the IL-6R complex that is essential for initiating the JAK-STAT signaling pathway.

TNF receptor associated factor 5 controls oncostatin M-mediated lung inflammation.

Oncostatin M (OSM) is involved in pathogenesis of several human inflammatory diseases including lung inflammation and fibrosis. Although accumulating evidence indicates that OSM mediates lung inflammation, the precise mechanism for OSM on lung inflammation still remains unclear. In this study, we found that OSM receptor was abundantly expressed on endothelial and stromal/fibroblast cells in the lung of mice. In vitro stimulation with OSM upregulated vascular cell adhesion molecule-1 (VCAM-1), which promotes eosinophil infiltration in the lung tissues, on freshly-isolated lung stromal/fibroblast cells from wild-type mice. However, these cells from TNF receptor associated factor 5 (TRAF5)-deficient mice failed to show the increase in VCAM-1 expression after OSM stimulation. Furthermore, Traf5-/- mice showed markedly attenuated lung inflammation in terms of eosinophil infiltration upon intranasal administration with OSM as compared to wild-type mice. These results indicate that TRAF5 is crucially involved in OSM-mediated lung inflammation probably by inducing lung stromal/fibroblast cell activation.

Rbm10 regulates inflammation development via alternative splicing of Dnmt3b.

RNA-binding motif 10 (Rbm10) is an RNA-binding protein that regulates alternative splicing, but its role in inflammation is not well defined. Here, we show that Rbm10 controls appropriate splicing of DNA (cytosine-5)-methyltransferase 3b (Dnmt3b), a DNA methyltransferase, to regulate the activity of NF-κB-responsive promoters and consequently inflammation development. Rbm10 deficiency suppressed NF-κB-mediated responses in vivo and in vitro. Mechanistic analysis showed that Rbm10 deficiency decreased promoter recruitment of NF-κB, with increased DNA methylation of the promoter regions in NF-κB-responsive genes. Consistently, Rbm10 deficiency increased the expression level of Dnmt3b2, which has enzyme activity, while it decreased the splicing isoform Dnmt3b3, which does not. These two isoforms associated with NF-κB efficiently, and overexpression of enzymatically active Dnmt3b2 suppressed the expression of NF-κB targets, indicating that Rbm10-mediated Dnmt3b2 regulation is important for the induction of NF-κB-mediated transcription. Therefore, Rbm10-dependent Dnmt3b regulation is a possible therapeutic target for various inflammatory diseases.

TNFR-Associated Factors 2 and 5 Differentially Regulate the Instructive IL-6 Receptor Signaling Required for Th17 Development.

IL-17-producing CD4(+) T cells (Th17 cells) regulate host defense and immune pathogenesis, and IL-6 plays an important role for the differentiation of Th17 cells. We have previously identified that TNFR-associated factor (TRAF)5 binds to the signal-transducing receptor gp130 through the C-terminal TRAF domain and inhibits Th17 development mediated by IL-6. Although gp130 has TRAF-binding motifs that can be recognized by other TRAF family proteins, it is unclear how TRAFs regulate IL-6-driven Th17 differentiation in general. Using retrovirus-mediated gene complementation and gene silencing approaches, we found that not only TRAF5 but also TRAF2 restrained the IL-6R signaling, whereas TRAF1, TRAF3, TRAF4, and TRAF6 did not. Traf2 silencing further promoted the ability of naive CD4(+) T cells from Traf5(-/-) mice to differentiate into Th17 cells. Notably, TRAF5 but not TRAF2 expressed in naive CD4(+) T cells was rapidly downregulated after TCR triggering, which indicates that TRAF5 specifically inhibits instructive IL-6 signals in the initial stage of Th17 development. Collectively, our results demonstrate a dedicated role for TRAF2 and TRAF5 in the process of IL-6-mediated Th17 development and a differential role for TCR signaling in regulation of TRAF2 and TRAF5. Therefore, both TRAF2 and TRAF5 work as important regulators of the IL-6R signaling needed for Th17 development.

Breakpoint Cluster Region-Mediated Inflammation Is Dependent on Casein Kinase II.

The breakpoint cluster region (BCR) is known as a kinase and cause of leukemia upon fusing to Abl kinase. In this study, we demonstrate that BCR associated with the α subunit of casein kinase II (CK2α), rather than BCR itself, is required for inflammation development. We found that BCR knockdown inhibited NF-κB activation in vitro and in vivo. Computer simulation, however, suggested that the putative BCR kinase domain has an unstable structure with minimal enzymatic activity. Liquid chromatography-tandem mass spectrometry analysis showed that CK2α associated with BCR. We found the BCR functions are mediated by CK2α. Indeed, CK2α associated with adaptor molecules of TNF-αR and phosphorylated BCR at Y177 to establish a p65 binding site after TNF-α stimulation. Notably, p65 S529 phosphorylation by CK2α creates a p300 binding site and increased p65-mediated transcription followed by inflammation development in vivo. These results suggest that BCR-mediated inflammation is dependent on CK2α, and the BCR-CK2α complex could be a novel therapeutic target for various inflammatory diseases.

Mesenteric lymph nodes contribute to proinflammatory Th17-cell generation during inflammation of the small intestine in mice.

T cells of the small intestine, including Th17 cells, are critically involved in host protection from microbial infection, and also contribute to the pathogenesis of small bowel inflammatory disorders. Accumulating evidence suggests that mesenteric lymph nodes (MLNs) play important roles in gut-tropic T-cell generation, although it is still unclear if MLNs are involved in the pathogenesis of small intestine inflammation. To address this issue, we analyzed the roles of both MLNs and Peyer's patches (PPs) by evaluating MLN- or PP-deficient mice in an experimental model of small intestine inflammation, induced by CD3-specific mAb injection. Interestingly, MLNs, but not PPs, were essential for the pathogenesis of intestinal inflammation, in particular the accumulation and infiltration of CD4(+) T-cell populations, including Th17 cells, from the blood. In addition, CD4(+) T-cell accumulation was dependent on the function of the α4 ß7 integrin. Furthermore, MLN removal led to a significantly reduced number of peripheral α4 ß7 (+) CD4(+) effector memory T cells under normal conditions, suggesting that MLNs may play a role in maintaining the number of gut-tropic CD4(+) effector memory T cells circulating in the blood. Taken together, the present study highlights the important role of MLNs in contributing to the pathogenesis of small intestine inflammation.

Interleukin-17 promotes autoimmunity by triggering a positive-feedback loop via interleukin-6 induction.

Dysregulated cytokine expression and signaling are major contributors to a number of autoimmune diseases. Interleukin-17A (IL-17A) and IL-6 are important in many disorders characterized by immune self-recognition, and IL-6 is known to induce the differentiation of T helper 17 (Th17) cells. Here we described an IL-17A-triggered positive-feedback loop of IL-6 signaling, which involved the activation of the transcription factors nuclear factor (NF)-kappaB and signal transducer and activator of transcription 3 (STAT3) in fibroblasts. Importantly, enhancement of this loop caused by disruption of suppressor of cytokine signaling 3 (SOCS3)-dependent negative regulation of the IL-6 signal transducer gp130 contributed to the development of arthritis. Because this mechanism also enhanced experimental autoimmune encephalomyelitis (EAE) in wild-type mice, it may be a general etiologic process underlying other Th17 cell-mediated autoimmune diseases.

Influence of increased amount of exercise on improvements in walking ability of convalescent patients with post-stroke hemiplegia.

[Purpose] This study aimed to determine the effects of increased amount of physical therapy exercise on improvements in the walking ability of patients with stroke. [Subjects and Methods] The subjects were selected from patients with stroke who were hospitalized in the convalescent rehabilitation ward, and included 91 patients who received physical therapy for 2.5-3 exercise sessions per day during 2005-2006 (PT3unit group), and 86 patients who received physical therapy for 4.5-6 exercise sessions per day during 2010-2015 (PT6unit group). The functional independence measure (FIM) score evaluates the walking ability of patients during hospital admission, 2 and 4 weeks after admission, and at discharge. The FIM score was stratified according to the degree of lower limb motor paralysis and subsequently compared between groups. [Results] Among the patients with complete paralysis and severe paralysis, the FIM-Walking scores at 4 weeks after admission and at discharge were significantly higher in the PT6unit group than in the PT3unit group. No significant differences were found between the PT6unit and PT3unit groups for patients with mild and moderate paralysis. [Conclusion] Higher amounts of physical therapy exercise contributed to improvements in the walking ability of patients with complete and severe lower limb paralysis.