Fiber-Rich Barley Increases Butyric Acid-Producing Bacteria in the Human Gut Microbiota.

Butyric acid produced in the intestine by butyric acid-producing bacteria (BAPB) is known to suppress excessive inflammatory response and may prevent chronic disease development. We evaluated whether fiber-rich barley intake increases BAPB in the gut and concomitantly butyric acid in feces. Eighteen healthy adults received granola containing functional barley (BARLEYmax®) once daily for four weeks. Fecal DNA before intake, after intake, and one month after intake was analyzed using 16S rRNA gene sequencing to assess microbial diversity, microbial composition at the order level, and the proportion of BAPB. Fecal butyric acid concentration was also measured. There were no significant differences in diversities and microbial composition between samples. The proportion of BAPB increased significantly after the intake (from 5.9% to 8.2%). However, one month after stopping the intake, the proportion of BAPB returned to the original value (5.4%). Fecal butyric acid concentration increased significantly from 0.99 mg/g feces before intake to 1.43 mg/g after intake (p = 0.028), which decreased significantly to 0.87 mg/g after stopping intake (p = 0.008). As BAPB produce butyric acid by degrading dietary fiber, functional barley may act as a prebiotic, increasing BAPB and consequently butyric acid in the intestine.

Comparison of the prebiotic properties of native chicory and synthetic inulins using swine fecal cultures.

Inulin-type fructans are known to exert different effects on the fermentation profile depending on the average and range of the degree of polymerization (DP). Here, swine fecal cultures were used to investigate the prebiotic properties of native chicory inulin (NIN), extracted from the chicory root, and synthetic inulin (SIN), which has a narrower DP distribution than NIN. Both NIN and SIN showed prebiotic effects, but NIN exhibited a significant decrease in pH and increase in the production of propionate and butyrate compared to SIN. There were also differences in the production of succinate and lactate, the precursors of propionate and butyrate, and the relative abundance of associated genes. Furthermore, NIN induced the growth of certain species of Bifidobacterium and Lactobacillus more strongly than SIN. These results suggest that NIN and SIN exhibit different prebiotic properties due to differences in DP, and that NIN might be more beneficial to host health.

Effects of BARLEYmax and high-ß-glucan barley line on short-chain fatty acids production and microbiota from the cecum to the distal colon in rats.

We investigated whether supplementation with the barley line BARLEYmax (Tantangara; BM), which contains three fermentable fibers (fructan, ß-glucan, and resistant starch), modifies the microbiota in cecal and distal colonic digesta in addition to short-chain fatty acids (SCFAs) production more favorably than supplementation with a high-ß-glucan barley line (BG012; BG). Male Sprague-Dawley rats were randomly divided into 3 groups that were fed an AIN-93G-based diet that contained 5% fiber provided by cellulose (control), BM or BG. Four weeks after starting the respective diets, the animals were sacrificed and digesta from the cecum, proximal colon and distal colon were collected and the SCFA concentrations were quantified. Microbiota in the cecal and distal colonic digesta were analyzed by 16S rRNA sequencing. The concentrations of acetate and n-butyrate in cecal digesta were significantly higher in the BM and BG groups than in the control group, whereas the concentration of total SCFAs in cecal digesta was significantly higher only in the BM group than in the control group. The concentrations of acetate and total SCFAs in the distal colonic digesta were significantly higher only in the BM group than in the control group. The abundance of Bacteroidetes in cecal digesta was significantly higher in the BM group than in the control group. In contrast, the abundance of Firmicutes in cecal digesta was significantly lower in the BM and BG groups than in the control group. These results indicated that BM increased the concentration of total SCFAs in the distal colonic digesta. These changes might have been caused by fructan and resistant starch in addition to ß-glucan. In conclusion, fermentable fibers in BM reached the distal colon and modified the microbiota, leading to an increase in the concentration of total SCFAs in the distal colonic digesta, more effectively compared with the high-ß-glucan barley line (BG).

Smad2/3 and IRF4 play a cooperative role in IL-9-producing T cell induction.

IL-9 is a pleiotropic cytokine that can regulate autoimmune and allergic responses. Th9 cells can develop from naive T cells or Th2 cells through stimulation by TGF-ß in vitro. In this study, we demonstrated that Smad2 and Smad3 are necessary for IL-9 production from T cells in an OVA-induced asthma model using T cell-specific Smad2- and Smad3-deficient mice. Smad2 and Smad3 were also redundantly essential for TGF-ß signaling to induce histone modifications for Il9 transcription. Although Smad2/3 was recruited to the Il9 promoter by TGF-ß stimulation, they are not sufficient to activate the Il9 promoter. By the screening the transcription factors, we found that IFN regulatory factor 4 (IRF4) was essential for the Smad2/3-mediated Il9 promoter activation. In addition, Smad2/3 physically interacted with IRF4, and Smad2/3 did not bind to the Il9 promoter and could not induce Th9 in IRF4-deficient T cells. Similarly, IRF4 could not stimulate Il9 transcription in the absence of Smad2/3, and TGF-ß enhanced IRF4 recruitment to the Il9 promoter in a Smad2/3-dependent manner. We propose that Smad2/3 and IRF4 cooperatively transactivate the Il9 promoter and play an important role in regulating allergic immune responses by inducing Th9 cells.

SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-{gamma} and IL-17A production.

Regulatory T cells (T(reg) cells) maintain immune homeostasis by limiting inflammatory responses. SOCS1 (suppressor of cytokine signaling 1), a negative regulator of cytokine signaling, is necessary for the suppressor functions of T(reg) cells in vivo, yet detailed mechanisms remain to be clarified. We found that Socs1(-/-) T(reg) cells produced high levels of IFN-γ and rapidly lost Foxp3 when transferred into Rag2(-/-) mice or cultured in vitro, even though the CNS2 (conserved noncoding DNA sequence 2) in the Foxp3 enhancer region was fully demethylated. Socs1(-/-) T(reg) cells showed hyperactivation of STAT1 and STAT3. Because Foxp3 expression was stable and STAT1 activation was at normal levels in Ifnγ(-/-)Socs1(-/-) T(reg) cells, the restriction of IFN-γ-STAT1 signaling by SOCS1 is suggested to be necessary for stable Foxp3 expression. However, Ifnγ(-/-)Socs1(-/-) T(reg) cells had hyperactivated STAT3 and higher IL-17A (IL-17) production compared with Ifnγ(-/-)Socs1(+/+) T(reg) cells and could not suppress colitis induced by naive T cells in Rag2(-/-) mice. In vitro experiments suggested that cytokines produced by Socs1(-/-) T(reg) cells and Ifnγ(-/-)Socs1(-/-) T(reg) cells modulated antigen-presenting cells for preferential Th1 and Th17 induction, respectively. We propose that SOCS1 plays important roles in T(reg) cell integrity and function by maintaining Foxp3 expression and by suppressing IFN-γ and IL-17 production driven by STAT1 and STAT3, respectively.

Pyridone 6, a pan-JAK inhibitor, ameliorates allergic skin inflammation of NC/Nga mice via suppression of Th2 and enhancement of Th17.

Atopic dermatitis (AD) is a common pruritic inflammatory disease triggered by a defective skin barrier and immunodysregulation. AD has been considered a typical example of a Th2 response associated with allergic disease. In the early phases of the disease, symptoms include IgE hyperproduction, eosinophil accumulation, and mast cell activation; in the chronic phase, a Th1-dominant immune response is also observed at the sites of AD skin lesions. The role of IL-17-producing Th (Th17) cells in AD has not been established. In the current study, we found that pyridone 6 (P6), a pan-JAK inhibitor, delayed the onset and reduced the magnitude of skin disease in an AD-like skin-disease model of NC/Nga mice. P6 reduced IFN-γ and IL-13, whereas it enhanced IL-17 and IL-22 expression. In vitro, P6 also inhibited both Th1 and Th2 development, whereas it promoted Th17 differentiation from naive T cells when present within a certain range of concentrations. This was probably because P6 strongly inhibited STAT1, STAT5, and STAT6 phosphorylation, whereas STAT3 phosphorylation was less efficiently suppressed by P6 at the same concentration. Furthermore, IL-22 protects keratinocytes from apoptosis induced by IFN-γ, and administration of IL-17 and IL-22 partially ameliorated skin diseases in NC/Nga mice. These results suggested that the JAK inhibitor P6 is therapeutic for AD by modulating the balance of Th2 and Th17.

Histone 3 lysine 9 (H3K9) methyltransferase recruitment to the interleukin-2 (IL-2) promoter is a mechanism of suppression of IL-2 transcription by the transforming growth factor-ß-Smad pathway.

Suppression of IL-2 ßproduction from T cells is an important process for the immune regulation by TGF-ß. However, the mechanism by which this suppression occurs remains to be established. Here, we demonstrate that Smad2 and Smad3, two major TGF-ß-downstream transcription factors, are redundantly essential for TGF-ß-mediated suppression of IL-2 production in CD4(+) T cells using Smad2- and Smad3-deficient T cells. Both Smad2 and Smad3 were recruited into the proximal region of the IL-2 promoter in response to TGF-ß. We then investigated the histone methylation status of the IL-2 promoter. Although both histone H3 lysine 9 (H3K9) and H3K27 trimethylation have been implicated in gene silencing, only H3K9 trimethylation was increased in the proximal region of the IL-2 promoter in a Smad2/3-dependent manner, whereas H3K27 trimethylation was not. The H3K9 methyltransferases Setdb1 and Suv39h1 bound to Smad3 and suppressed IL-2 promoter activity in collaboration with Smad3. Overexpression of Suv39h1 in 68-41 T cells strongly inhibited IL-2 production in response to T cell receptor stimulation irrespective of the presence or absence of TGF-ß, whereas Setdb1 overexpression only slightly suppressed IL-2 production. Silencing of Suv39h1 by shRNA reverted the suppressive effect of TGF-ß on IL-2 production. Furthermore, TGF-ß induced Suv39h1 recruitment to the proximal region of the IL-2 promoter in wild type primary T cells; however, this was not observed in Smad2(-/-)Smad3(+/-) T cells. Thus, we propose that Smads recruit H3K9 methyltransferases Suv39h1 to the IL-2 promoter, thereby inducing suppressive histone methylation and inhibiting T cell receptor-mediated IL-2 transcription.

miR126 positively regulates mast cell proliferation and cytokine production through suppressing Spred1.

The protein known as Spred1 (Sprouty-related Ena/VASP homology-1 domain-containing protein) has been identified as a negative regulator of growth factor-induced ERK/mitogen-activated protein kinase activation. Spred1 has also been implicated as the target of microRNA-126 (miR126), a miRNA located within the Egfl7 gene, and is involved in the regulation of vessel development through its role in regulating VEGF signaling. In this study, we examined the role of miR126 and Spred1 in the hematopoietic system, as miR126 has been shown to be overexpressed in leukemic cells. miR126 levels were down-regulated during mast cell differentiation from bone marrow cells, whereas Spred1 expression was inversely up-regulated. Overexpression of miR126 suppressed Spred1 expression and enhanced ERK activity in primary bone marrow cells and MC9 mast cells, which were associated with elevated FcεRI-mediated cytokine production. To confirm the effect of Spred1 reduction in vivo, we generated hematopoietic cell-specific Spred1-conditional knockout mice. These mice showed increased numbers of mast cells, and Spred1-deficient bone marrow-derived mast cells were highly activated by cross-linking of Fcε-R stimulation as well as by IL-3 and SCF stimulation. These results suggest that Spred1 negatively regulates mast cell activation, which is modulated by miR126.

Suppression of SOCS3 in macrophages prevents cancer metastasis by modifying macrophage phase and MCP2/CCL8 induction.

Inflammation has been demonstrated to play important roles in tumorigenesis, tumor progression, and metastasis. STAT3 has been shown to be frequently activated in a variety of human cancer cells and STAT3 signaling promotes the growth and survival of tumor cells. However, the role of STAT3 of myeloid cells associated with tumors is currently unknown. Suppressor of cytokine signaling-3 (SOCS3) has been shown to be a negative regulator of STAT3. In this study, we used macrophage specific SOCS3 conditional knockout (cKO) mice to investigate the effect of the hyperactivation of STAT3 in macrophages on tumor development and metastasis. In a subcutaneous transplantation model of B16F10 melanoma cells, although tumor sizes were not significantly different, SOCS3-cKO mice survived longer than wild-type (WT) mice did. SOCS3-cKO mice exhibited fewer lung and liver metastatic tumor nodules than WT mice when B16F10 was challenged intravenously. SOCS3(-/-) macrophages stimulated with tumor lysates in vitro exhibited prolonged STAT3 phosphorylation and produced less amount of TNFα and IL-6, and higher amount of MCP2/CCL8 than WT macrophages. MCP/CCL8 was induced via STAT3 and exhibited anti-tumor metastatic effect in WT mice. These data suggest that hyperactivation of STAT3 in myeloid cells simultaneously exerted an anti-inflammatory as well as anti-tumor effects. Thus, the targeted inhibition of SOCS3 activity in macrophages may be therapeutic for the suppression of tumor metastasis.

Transcription factor Smad-independent T helper 17 cell induction by transforming-growth factor-ß is mediated by suppression of eomesodermin.

Transforming growth factor-ß (TGF-ß) has been shown to be required for Th17 cell differentiation via Smad-independent mechanisms. The molecular mechanism underlying this pathway remains to be clarified, however. We searched for genes regulated by TGF-ß through the Smad-independent pathway by using Smad2 and Smad3 double-deficient T cells and identified the transcription factor Eomesodermin (Eomes), whose expression was suppressed by TGF-ß via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway. Inhibition of JNK strongly suppressed disease in an in vivo EAE model as well as in vitro Th17 cell induction. Overexpression of Eomes substantially suppressed Th17 cell differentiation, whereas ablation of Eomes expression could substitute for TGF-ß in Th17 cell induction in primary T cells. Eomes suppressed Rorc and Il17a promoters by directly binding to the proximal region of these promoters. In conclusion, the suppression of Eomes by TGF-ß via the JNK pathway is an important mechanism for Smad-independent Th17 cell differentiation.

Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways: regulation of T-cell inflammation by SOCS1 and SOCS3.

Various cytokines are involved in the regulation of the immune system and inflammation. Dysregulation of cytokine signaling can cause a variety of diseases, including allergy, autoimmune diseases, inflammation, and cancer. Most cytokines use the so-called janus kinase/signal transducer and activator of transcription pathway, and this pathway is negatively regulated by suppressors of cytokine signaling (SOCS) proteins. SOCS proteins bind to janus kinase and to certain cytokine receptors and signaling molecules, thereby suppressing further signaling events. Studies have shown that SOCS proteins are key physiological regulators of inflammation. Recent studies have also demonstrated that SOCS1 and SOCS3 are important regulators of adaptive immunity.