TRAF6 signaling in T cells is crucial for the pathogenicity of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an incurable chronic autoimmune disease affecting the central nervous system (CNS). Although IL-17-producing helper T (Th17) cells are thought to be one of the exacerbating factors in MS, the underlying pathogenic mechanism is incompletely understood. TNF receptor-associated factor 6 (TRAF6) deficient T cells exhibited enhanced Th17 cell differentiation, however, the physiological relevance of TRAF6 in T cells remains unknown. Here, we induced experimental autoimmune encephalomyelitis (EAE) in T cell-specific TRAF6 deficient (TRAF6ΔT) mice to investigate the role of TRAF6 in T cells during the course of MS using an EAE model. Although Th17 cell differentiation was enhanced in TRAF6ΔT mice, mutant mice were resistant to EAE. In contrast, TRAF6 loss did not affect regulatory T-cell differentiation. Consistent with the severity of EAE, a small number of infiltrating T cells and a small area of demyelination were observed in the CNS of TRAF6ΔT mice. Moreover, myelin oligodendrocyte glycoprotein-induced IL-17 production in TRAF6-deficient T cells was significantly suppressed. We further confirmed lower levels of CD69 and granulocyte-macrophage colony-stimulating factor in Th17 cells of TRAF6ΔT mice than in wild-type mice. In contrast, the expression of IL-10 and cytotoxic T-lymphocyte-associated protein 4 in T cells was significantly elevated in the absence of TRAF6 because of enhanced T-cell receptor signaling. Collectively, TRAF6 signaling in T cells contributes to the pathogenesis of EAE by regulating the pathogenicity and autoantigen reactivity of Th17 cells.

Transcriptional coregulator Ess2 controls survival of post-thymic CD4+ T cells through the Myc and IL-7 signaling pathways.

Ess2, also known as Dgcr14, is a transcriptional co-regulator of CD4+ T cells. Ess2 is located in a chromosomal region, the loss of which has been associated with 22q11.2 deletion syndrome (22q11DS), which causes heart defects, skeletal abnormalities, and immunodeficiency. However, the specific association of Ess2 with 22q11DS remains unclear. To elucidate the role of Ess2 in T-cell development, we generated Ess2 floxed (Ess2fl/fl) and CD4+ T cell-specific Ess2 KO (Ess2ΔCD4/ΔCD4) mice using the Cre/loxP system. Interestingly, Ess2ΔCD4/ΔCD4 mice exhibited reduced naïve T-cell numbers in the spleen, while the number of thymocytes (CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+) in the thymus remained unchanged. Furthermore, Ess2ΔCD4/ΔCD4 mice had decreased NKT cells and increased γδT cells in the thymus and spleen. A genome-wide expression analysis using RNA-seq revealed that Ess2 deletion alters the expression of many genes in CD4 single-positive thymocytes, including genes related to the immune system and Myc target genes. In addition, Ess2 enhanced the transcriptional activity of c-Myc. Some genes identified as Ess2 targets in mice show expressional correlation with ESS2 in human immune cells. Moreover, Ess2ΔCD4/ΔCD4 naïve CD4+ T cells did not maintain survival in response to IL-7. Our results suggest that Ess2 plays a critical role in post-thymic T-cell survival through the Myc and IL-7 signaling pathways.

Involvement of clusterin expression in the refractory response of pancreatic cancer cells to a MEK inhibitor.

Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway is essential for tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). To date, however, almost all clinical trials of inhibitor targeting this pathway have failed to improve the outcome of patients with PDAC. We found that implanted MIA Paca2, a human PDAC cell line sensitive to a MAPK inhibitor, PD0325901, became refractory within a week after treatment. By comparing the expression profiles of MIA Paca2 before and after acquisition of the refractoriness to PD0325901, we identified clusterin (CLU) as a candidate gene involved. CLU was shown to be induced immediately after treatment with PD0325901 or expressed primarily in more than half of PDAC cell lines, enhancing cell viability by escaping from apoptosis. A combination of PD0325901 and CLU downregulation was found to synergistically or additively reduce the proliferation of PDAC cells. In surgically resected PDAC tissues, overexpression of CLU in cancer cells was observed immunohistochemically in approximately half of the cases studied. Collectively, our findings highlight the mechanisms responsible for the rapid refractory response to MEK inhibitor in PDAC cells, suggesting a novel therapeutic strategy that could be applicable to patients with PDAC using inhibitor targeting the MAPK signaling pathway and CLU.

Comprehensive lipidomics of lupus-prone mice using LC-MS/MS identifies the reduction of palmitoylethanolamide that suppresses TLR9-mediated inflammation.

Lipid mediators are known to play crucial roles not only in the onset of the inflammatory response but also in the induction of resolution of inflammation. Here, we report that palmitoylethanolamide (PEA), an endogenous N-acylethanolamine, can suppress the inflammation induced by Toll-like receptor (TLR) signaling both in vitro and in vivo. PEA was found to be significantly reduced in the serum and spleen of lupus-prone MRL/lpr mice analyzed by lipidomics. PEA suppressed pro-inflammatory cytokine production in a mouse macrophage cell line stimulated with TLR ligands such as lipopolysaccharide, peptidoglycan, poly (I:C), imiquimod, and CpG-ODN. PEA also inhibited both mRNA and protein levels of IL-6 in bone marrow-derived dendritic cells (BMDCs) and B cells stimulated with CpG-ODN. Augmentation of cell surface CD86 and CD40 on BMDCs and B cells, IgM production, and cell proliferation of B cells in response to CpG-ODN were attenuated by PEA. Moreover, PEA treatment significantly reduced mortality and serum IL-6 levels in mice injected with CpG-ODN plus D-galactosamine. Taken together, PEA ameliorates inflammation induced by TLR signaling, which could be a novel therapeutic target for inflammatory disorders.

Protease inhibitory activity of secretory leukocyte protease inhibitor ameliorates murine experimental colitis by protecting the intestinal epithelial barrier.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestine, and the dysfunction of intestinal epithelial barrier (IEB) may trigger the onset of IBD. Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that has been implicated in the tissue-protective effect in the skin and lung. We found that SLPI was induced in lipopolysaccharides-treated colon carcinoma cell line and in the colon of dextran sulfate sodium (DSS)-treated mice. SLPI-deficient mice were administered DSS to induce colitis and sustained severe inflammation compared with wild-type mice. The colonic mucosa of SLPI-deficient mice showed more severe inflammation with neutrophil infiltration and higher levels of proinflammatory cytokines compared with control mice. Moreover, neutrophil elastase (NE) activity in SLPI-deficient mice was increased and IEB function was severely impaired in the colon, accompanied with the increased number of apoptotic cells. Importantly, we demonstrated that DSS-induced colitis was ameliorated by administration of protease inhibitor SSR69071 and recombinant SLPI. These results suggest that the protease inhibitory activity of SLPI protects from colitis by preventing IEB dysfunction caused by excessive NE activity, which provides insight into the novel function of SLPI in the regulation of gut homeostasis and therapeutic approaches for IBD.

Development of a portable reverse transcription loop-mediated isothermal amplification system to detect the E1 region of Chikungunya virus in a cost-effective manner.

Chikungunya fever is a mosquito-borne disease cause of persistent arthralgia. The current diagnosis of Chikungunya virus (CHIKV) relies on a conventional reverse transcription polymerase chain reaction assay. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and simple tool used for DNA-based diagnosis of a variety of infectious diseases. In this study, we established an RT-LAMP system to recognize CHIKV by targeting the envelope protein 1 (E1) gene that could also detect CHIKV at a concentration of 8 PFU without incorrectly detecting other mosquito-borne viruses. The system also amplified the E1 genome in the serum of CHIKV-infected mice with high sensitivity and specificity. Moreover, we established a dry RT-LAMP system that can be transported without a cold chain, which detected the virus genome in CHIKV-infected patient samples with high accuracy. Thus, the dry RT-LAMP system has great potential to be applied as a novel CHIKV screening kit in endemic areas.

Downregulation of ZNF395 Drives Progression of Pancreatic Ductal Adenocarcinoma through Enhancement of Growth Potential.

BACKGROUND: Progression of pancreatic intraepithelial neoplasia (PanIN) to invasive carcinoma is a critical factor impacting the prognosis of patients with pancreatic tumors. However, the molecular mechanisms involved are not fully understood. We have reported that the process frequently involves loss of chromosome 8p, causing downregulation of DUSP4, thus conferring invasive ability on cancer cells. Here, we focus on ZNF395, whose expression was also found to be decreased by 8p loss and was predicted to be a growth suppressor gene. METHODS: Pancreatic cancer cell lines inducibly expressing ZNF395 were established to assess the functional significance of ZNF395 in pancreatic carcinogenesis. Immunohistochemistry was also performed to analyze the expression levels of ZNF395 in pancreatic cancer tissues. RESULTS: Induction of ZNF395 in pancreatic cancer cells resulted in marked activation of JNK and suppression of their proliferation through a delay in cell cycle progression. Immunohistochemistry revealed that ZNF395 was expressed ubiquitously in both normal pancreatic ducts and PanINs but was significantly reduced in invasive cancers, especially those showing poor differentiation. CONCLUSION: ZNF395 acts as a novel tumor suppressor gene. Its downregulation caused by 8p loss in intraepithelial cells accelerates their proliferation through dysregulation of the cell cycle, leading to progression to invasive cancer.

Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 µM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites.

DUSP4 is involved in the enhanced proliferation and survival of DUSP4-overexpressing cancer cells.

Dual-specificity phosphatase 4 (DUSP4), a MAP kinase phosphatase, has been regarded as a tumor suppressor gene in several cancers. However, high-level expression of DUSP4 is occasionally observed in specific cancers and its functional significance in carcinogenesis is not fully understood. In the present study, we showed that downregulation of DUSP4 suppressed the proliferation of cancer cell lines exhibiting high expression of DUSP4 by inducing apoptosis and cell cycle arrest at G2/M phase. Expression microarray analyses and pathway analyses revealed that downregulation of DUSP4 activated the p53 signaling pathway, and might be involved in cell growth suppression. Aberrant accumulation of p53 and induction of p53 downstream target genes were further investigated. Furthermore, cell growth suppression following downregulation of DUSP4 was markedly attenuated in p53-deleted cells established using the CRISPR/Cas9 system. These findings suggest that constitutive expression of DUSP4 in cancer cells contributes to enhanced proliferation through escape from apoptosis and cell cycle arrest. We propose that DUSP4 could be a novel therapeutic target for cancers overexpressing it.

Fucosyltransferase 8 plays a crucial role in the invasion and metastasis of pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. It is an aggressive malignancy associated with poor prognosis because of recurrence, metastasis, and treatment resistance. Aberrant glycosylation of cancer cells triggers their migration and invasion and is considered one of the most important prognostic cancer biomarkers. The current study aimed to identify glycan alterations and their relationship with the malignant potential of PDAC. METHODS: Using a lectin microarray, we evaluated glycan expression in 62 PDAC samples. Expression of fucosyltransferase 8 (FUT8), the only enzyme catalyzing core fucosylation, was investigated by immunohistochemistry. The role of FUT8 in PDAC invasion and metastasis was confirmed using an in vitro assay and a xenograft peritoneal metastasis mouse model. RESULTS: The microarray data demonstrated that core fucose-binding lectins were significantly higher in carcinoma than in normal pancreatic duct tissues. Similarly, FUT8 protein expression was significantly higher in carcinoma than in normal pancreatic duct tissues. High FUT8 protein expression was significantly associated with lymph-node metastases and relapse-free survival. FUT8 knockdown significantly reduced the invasion in PDAC cell lines and impaired peritoneal metastasis in the xenograft model. CONCLUSIONS: The findings of this study provide evidence that FUT8 plays a pivotal role in PDAC invasion and metastasis and might be a therapeutic target for this disease.

Oral administration of antibiotics results in fecal occult bleeding due to metabolic disorders and defective proliferation of the gut epithelial cell in mice.

Antibiotics sometimes exert adverse effects on the pathogenesis of colitis due to the dysbiosis resulting from the disruption of gut homeostasis. However, the precise mechanisms underlying colitogenic effects of antibiotic-induced colitis are largely unknown. Here, we show a novel murine fecal occult bleeding model induced by the combinatorial treatment of ampicillin and vancomycin, which is accompanied by an enlarged cecum, upregulation of pro-inflammatory cytokines IL-6 and IL-12, a reduction in Ki-67-positive epithelial cell number and an increase in the apoptotic cell number in the colon. Moreover, gas chromatography-tandem mass analysis showed that various kinds of metabolites, including glutamic acid and butyric acid, were significantly decreased in the cecal contents. In addition, abundance of butyric acid producer Clostridiales was dramatically reduced in the enlarged cecum. Interestingly, supplementation of monosodium glutamate or its precursor glutamine suppressed colonic IL-6 and IL-12, protected from cell apoptosis and prevented fecal occult blood indicating that the reduced level of glutamic acid is a possible mechanism of antibiotic-induced fecal occult bleeding. Our data showed a novel mechanism of antibiotic-induced fecal occult bleeding providing a new insight into the clinical application of glutamic acid for the treatment of antibiotic-induced colitis.

Ess2 bridges transcriptional regulators and spliceosomal complexes via distinct interacting domains.

Transcription and pre-mRNA splicing are complex, coupled processes that involve transcriptional co-regulators. Ess2 (also termed Dgcr14) is a nuclear protein that enhances the transcriptional activity of retinoic acid receptor-related orphan receptor gamma/gamma-t (Rorγ/γt). Ess2 is also a component of the spliceosomal C complex (containing U2, U5 and U6 snRNAs). However, the domains in Ess2 that function in splicing and transcription have not been identified. To elucidate the roles of Ess2 in splicing and transcription, we performed RNA immunoprecipitation (RIP) assays to detect Ess2-interacting snRNAs. We found that Ess2 associated with U6 snRNA as well as U1 and U4 snRNAs. Experiments using Ess2 deletion mutants showed that a C-terminus deletion mutant of Ess2 (1-399 a. a.) lost its ability to associate with snRNAs, whereas the N-terminus domain of Ess2 (1-200 a. a.) associated with Rorγ/γt, but not with snRNAs. Interestingly, experiments using anti-ROR common antibody showed that Rors also associated with U4 and U6 snRNAs. Ess2 knockdown in a T cell hybridoma (68-41 cells) abrogated the interaction between spliceosomes and Rors. An Ess2-dependent association was also found between an lncRNA (Rmrp) and Rors. We thus propose that Ess2 associates with both transcriptional factors and spliceosomal complexes and modulates splicing reactions coupled with transcription factors.

Autoimmune sialadenitis is associated with the upregulation of chemokine/chemokine receptor pairs in T cell-specific TRAF6-deficient mice.

Sialadenitis is an inflammatory condition affecting the salivary glands including the parotid, submandibular, and sublingual glands. There are several different types of sialadenitis, each with different sites of predilection. However, the pathogenic mechanism underlying the tissue specificity of sialadenitis is largely unknown. TRAF6 is a cytoplasmic adaptor protein that is necessary for the activation of dendritic cells in response to Toll-like receptor ligands, thereby regulating innate immune responses. We previously demonstrated that T cell-specific TRAF6-deficient mice (TRAF6ΔT mice) spontaneously develop systemic inflammatory disease. Here, we show that salivary secretion is reduced in TRAF6ΔT mice due to sialadenitis that occurs in the parotid and submandibular glands, but not the sublingual glands. Consistent with pathological findings, both CD4+ and CD8+ T cells predominantly infiltrated the submandibular glands; however, sublingual infiltration was rare in TRAF6ΔT mice. The TH1 cytokine IFN-γ, the TH1 cell attractant chemokine CCL2, and its cognate receptor CCR2 were upregulated concomitantly in both the submandibular and sublingual glands. Interestingly, the TH17 cell attractant chemokine CCL20 and its cognate receptor CCR6 were selectively increased in the submandibular glands, but not in the sublingual glands of TRAF6ΔT mice. Thus, the expression of TRAF6 in T cells might be implicated in tissue-specific sialadenitis by regulating the chemokine-chemokine receptor system.

The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondii.

The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."

Secondary Unconjugated Bile Acids Induce Hepatic Stellate Cell Activation.

Hepatic stellate cells (HSCs) are key players in liver fibrosis, cellular senescence, and hepatic carcinogenesis. Bile acids (BAs) are involved in the activation of HSCs, but the detailed mechanism of this process remains unclear. We conducted a comprehensive DNA microarray study of the human HSC line LX-2 treated with deoxycholic acid (DCA), a secondary unconjugated BA. Additionally, LX-2 cells were exposed to nine BAs and studied using immunofluorescence staining, enzyme-linked immunosorbent assay, and flow cytometry to examine the mechanisms of HSC activation. We focused on the tumor necrosis factor (TNF) pathway and revealed upregulation of genes related to nuclear factor kappa B (NF-κB) signaling and senescence-associated secretory phenotype factors. α-Smooth muscle actin (α-SMA) was highly expressed in cells treated with secondary unconjugated BAs, including DCA, and a morphological change associated with radial extension of subendothelial protrusion was observed. Interleukin-6 level in culture supernatant was significantly higher in cells treated with secondary unconjugated BAs. Flow cytometry showed that the proportion of cells highly expressing α-SMA was significantly increased in HSCs cultured with secondary unconjugated BAs. We demonstrated that secondary unconjugated BAs induced the activation of human HSCs.

Flt3 Ligand Is Essential for Survival and Protective Immune Responses during Toxoplasmosis.

Dendritic cells (DCs) are critical for resistance to Toxoplasma gondii, and infection with this pathogen leads to increased numbers of DCs at local sites of parasite replication and in secondary lymphoid organs, but the factors that regulate this expansion are poorly understood. The cytokine Flt3 ligand (Flt3L) is critical for the generation and maintenance of DCs, and Flt3L(-/-) mice were found to be highly susceptible to acute toxoplasmosis. This phenotype correlated with decreased production of IL-12 and IFN-γ, as well as impaired NK cell responses. Surprisingly, despite low basal numbers of DCs, Flt3L(-/-) mice infected with T. gondii displayed an expansion of CD8α(+) and CD11b(lo)CD8α(-) DCs. Infection also induced an expansion of parasite-specific CD4(+) and CD8(+) T cells in Flt3L(-/-) mice; however, these cells were reduced in number and displayed impaired ability to produce IFN-γ relative to wild-type controls. Exogenous IL-12 treatment partially restored NK and T cell responses in Flt3L(-/-) mice, as well as acute resistance; however, these mice eventually succumbed to toxoplasmic encephalitis, despite the presence of large numbers of DCs and T cells in the brain. These results highlight the importance of Flt3L for resistance to toxoplasmosis and demonstrate the existence of Flt3L-independent pathways that can mediate infection-induced expansion of DCs and T cell priming.

Amelioration of collagen-induced arthritis by a novel S1P1 antagonist with immunomodulatory activities.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking through the type 1 sphingosine 1-phosphate receptor (S1P(1)) and participates in many pathological conditions, including autoimmune diseases. We developed a novel S1P(1)-selective antagonist, TASP0277308, which is structurally unrelated to S1P. This antagonist competitively inhibited S1P-induced cellular responses, such as chemotaxis and receptor internalization. Furthermore, differing from previously reported S1P(1) antagonists, TASP0277308 demonstrated in vivo activities to induce lymphopenia, a block in T cell egress from the thymus, displacement of marginal zone B cells, and upregulation of CD69 expression on both T and B cells, all of which recapitulate phenotypes of S1P(1)-deficient lymphocytes. In a mouse collagen-induced arthritis model, TASP0277308 significantly suppressed the development of arthritis, even after the onset of disease. These findings provide the first chemical evidence to our knowledge that S1P(1) antagonism is responsible for immunosuppression in the treatment of autoimmune diseases and also resolve the discrepancies between genetic and chemical studies on the functions of S1P(1) in lymphocytes.

Tonic TCR and IL-1ß signaling mediate phenotypic alterations of naive CD4+ T cells.

Inert naive CD4+ T (TN) cells differentiate into functional T helper (Th) or regulatory T (Treg) cell subsets upon encountering antigens, mediating properly directed immune responses. Although all TN cells can differentiate into any of the Th and Treg cell subsets, heterogeneity exists among TN cells. By constructing reporter mice to detect ongoing T cell receptor (TCR) signaling, we identify that interleukin (IL)-1ß signaling affects TN cell characteristics, independent of tonic TCR signaling, which also alters TN cell phenotypes. IL-1ß reversibly attenuates the differentiation potential of TN cells toward Treg cells. IL-1ß signaling is elevated in the splenic TN cells, consequently attenuating their differentiation potential toward Treg cells. Aberrant elevation of IL-1ß signaling augments colitogenic activities of TN cells. TN cells in patients with colitis exhibited elevated IL-1ß signaling. We demonstrate that phenotypic alteration in TN cells by IL-1ß is an important mechanism in the regulation of immune responses.

SLP-76 is required for high-affinity IgE receptor- and IL-3 receptor-mediated activation of basophils.

Basophils have been reported to play a critical role in allergic inflammation by secreting IL-4 in response to IL-3 or high-affinity IgE receptor (FcεRI)-cross-linking. However, the signaling pathways downstream of FcεRI and the IL-3 receptor in basophils have yet to be determined. In the present study, we used mice deficient in SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76kDa) to demonstrate critical functions of this adaptor molecule in transducing FcεRI- and IL-3 receptor-mediated signals that induce basophil activation. Although SLP-76 was dispensable for in vivo differentiation, as well as IL-3-induced in vitro proliferation of basophils, IL-4 production induced by both stimuli was completely ablated by SLP-76 deficiency. Biochemical analyses revealed that IL-3-induced phosphorylation of phospholipase C (PLC) γ2 and Akt, but not STAT5, was severely reduced in SLP-76-deficient basophils, whereas FcεRI cross-linking phosphorylation of PLCγ2, but not Akt, was abrogated by SLP-76 deficiency, suggesting important differences in the requirement of SLP-76 for Akt activation between FcεRI- and IL-3 receptor-mediated signaling pathways in basophils. Because IL-3-induced IL-4 production was sensitive to calcineurin inhibitors and an intracellular calcium chelator, in addition to PI3K inhibitors, SLP-76 appears to regulate FcεRI- and IL-3 receptor-induced IL-4 production via mediating PLCγ2 activation in basophils. Taken together, these findings indicate that SLP-76 is an essential signaling component for basophil activation downstream of both FcεRI and the IL-3 receptor.

Saireito, a Japanese herbal medicine, alleviates leaky gut associated with antibiotic-induced dysbiosis in mice.

Antibiotics disrupt normal gut microbiota and cause dysbiosis, leading to a reduction in intestinal epithelial barrier function. Disruption of the intestinal epithelial barrier, which is known as "leaky gut", results in increased intestinal permeability and contributes to the development or exacerbation of gastrointestinal diseases such as inflammatory bowel disease and irritable bowel syndrome. We have previously reported on a murine model of intestinal epithelial barrier dysfunction associated with dysbiosis induced by the administration of ampicillin and vancomycin. Saireito, a traditional Japanese herbal medicine, is often used to treat autoimmune disorders including ulcerative colitis; the possible mechanism of action and its efficacy, however, remains unclear. In this study, we examined the efficacy of Saireito in our animal model for leaky gut associated with dysbiosis. C57BL/6 mice were fed a Saireito diet for the entirety of the protocol (day1-28). To induce colitis, ampicillin and vancomycin were administered in drinking water for the last seven consecutive days (day22-28). As previously demonstrated, treatment with antibiotics caused fecal occult bleeding, cecum enlargement with black discoloration, colon inflammation with epithelial cell apoptosis, and upregulation of pro-inflammatory cytokines. Oral administration of Saireito significantly improved antibiotics-induced fecal occult bleeding and cecum enlargement by suppressing inflammation in the colon. Furthermore, Saireito treatment ensured the integrity of the intestinal epithelial barrier by suppressing apoptosis and inducing cell adhesion proteins including ZO-1, occludin, and E-cadherin in intestinal epithelial cells, which in turn decreased intestinal epithelial permeability. Moreover, the reduced microbial diversity seen in the gut of mice treated with antibiotics was remarkably improved with the administration of Saireito. In addition, Saireito altered the composition of gut microbiota in these mice. These results suggest that Saireito alleviates leaky gut caused by antibiotic-induced dysbiosis. Our findings provide a potentially new therapeutic strategy for antibiotic-related gastrointestinal disorders.