Pharmacological Activation of TRPC6 Channel Prevents Colitis Progression.

We recently reported that transient receptor potential canonical (TRPC) 6 channel activity contributes to intracellular Zn2+ homeostasis in the heart. Zn2+ has also been implicated in the regulation of intestinal redox and microbial homeostasis. This study aims to investigate the role of TRPC6-mediated Zn2+ influx in the stress resistance of the intestine. The expression profile of TRPC1-C7 mRNAs in the actively inflamed mucosa from inflammatory bowel disease (IBD) patients was analyzed using the GEO database. Systemic TRPC3 knockout (KO) and TRPC6 KO mice were treated with dextran sulfate sodium (DSS) to induce colitis. The Zn2+ concentration and the mRNA expression levels of oxidative/inflammatory markers in colon tissues were quantitatively analyzed, and gut microbiota profiles were compared. TRPC6 mRNA expression level was increased in IBD patients and DSS-treated mouse colon tissues. DSS-treated TRPC6 KO mice, but not TRPC3 KO mice, showed severe weight loss and increased disease activity index compared with DSS-treated WT mice. The mRNA abundances of antioxidant proteins were basically increased in the TRPC6 KO colon, with changes in gut microbiota profiles. Treatment with TRPC6 activator prevented the DSS-induced colitis progression accompanied by increasing Zn2+ concentration. We suggest that TRPC6-mediated Zn2+ influx activity plays a key role in stress resistance against IBD, providing a new strategy for treating colitis.

Functional role of IL-19 in a mouse model of L-arginine-induced pancreatitis and related lung injury.

IL-19 is a member of IL-10 family and is mainly produced by macrophages. Acute pancreatitis (AP) is an inflammatory disease characterized by acinar cell injury and necrosis. In the present study, the role of IL-19 in AP and AP-associated lung injury in mice was explored using L-arginine-induced pancreatitis. Experimental pancreatitis was induced by intraperitoneal injection of L-arginine in wild-type (WT) and IL-19 gene deficient (IL-19 KO) mice. In L-arginine treated mice, the serum amylase level was significantly increased in IL-19 KO mice, and interstitial edema, analyzed using hematoxylin and eosin (H&E)-stained sections, was aggravated mildly in IL-19 KO mice compared to WT mice. Compared to WT mice treated with L-arginine, mRNA expression of tumor necrosis factor (TNF)-α was significantly upregulated in IL-19 KO mice treated with L-arginine. In WT mice, IL-19 mRNA was equally expressed in the pancreas of both control and L-arginine treated mice. The condition of lung alveoli in WT and IL-19 KO mice treated with L-arginine was then evaluated. In mice with L-arginine-induced pancreatitis, alveolar area was remarkedly decreased, and expression of lung myeloperoxidase was significantly increased in IL-19 KO mice compared to WT mice. In the lungs, mRNA expressions of IL-6 and inducible nitric oxide synthase were significantly increased in IL-19 KO mice compared to WT mice. In summary, IL-19 was proposed to alleviate L-arginine-induced pancreatitis by regulating TNF-α production and to protect against AP-related lung injury by inhibiting neutrophil migration.

Interleukin-19 Gene-Deficient Mice Promote Liver Fibrosis via Enhanced TGF-ß Signaling, and the Interleukin-19-CCL2 Axis Is Important in the Direction of Liver Fibrosis.

IL-19 is a cytokine discovered by homologous searching with IL-10 and is produced by non-immune cells, such as keratinocytes, in addition to immune cells, such as macrophages. Liver fibrosis results from the inflammation and activation of hepatic stellate cells via chronic liver injury. However, the participation of IL-19 in liver fibrosis remains to be sufficiently elucidated. Our group studied the immunological function of IL-19 in a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis. IL-19 gene-deficient (KO) mice and body weight-matched wild-type (WT) mice were used. A liver fibrosis mouse model was created via CCl4 administration (two times per week) for 8 weeks. In CCl4-induced liver fibrosis, serum analysis revealed that IL-19 KO mice had higher ALT levels compared to WT mice. IL-19 KO mice had worse fibrosis, as assessed by morphological evaluation of total area stained positive with Azan and Masson trichrome. In addition, the expression of α-SMA was increased in liver tissues of IL-19 KO mice compared to WT mice. Furthermore, mRNA expression levels of TGF-ß and α-SMA were enhanced in IL-19 KO mice compared to WT mice. In vitro assays revealed that IL-19-high expressing RAW264.7 cells inhibited the migration of NIH3T3 cells via the inhibited expression of CCL2 in the presence of CCl4 and IL-4. These findings indicate that IL-19 plays a critical role in liver fibrosis by affecting TGF-ß signaling and the migration of hepatic stellate cells during liver injury. Enhancement of the IL-19 signaling pathway is a potential treatment for liver fibrosis.

L-arginine-induced pancreatitis aggravated by inhibiting Na+/Ca2+ exchanger 1.

Na+/Ca2+ exchangers (NCX) are an exchange transporter of Na+ and Ca2+ ions on the plasma membrane. There are three types of NCX: NCX1, NCX2, and NCX3. We have been working for many years to understand the role of NCX1 and NCX2 in gastrointestinal motility. In this study, we focused on the pancreas, an organ closely related to the gastrointestinal tract, and used a mouse model of acute pancreatitis to investigate a possible role for NCX1 in the pathogenesis of pancreatitis. We characterized a model of acute pancreatitis induced by excessive doses of L-arginine. We administered the NCX1 inhibitor SEA0400 (1 mg/kg) 1 hr prior to L-arginine-induced pancreatitis and evaluated pathological changes. Mice treated with NCX1 inhibitors show exacerbation of the disease with decreased survival and increased amylase activity in response to L-arginine-induced experimental acute pancreatitis, and this exacerbation correlates with increased autophagy mediated by LC3B and p62. These results suggest that NCX1 has a role in regulating pancreatic inflammation and acinar cell homeostasis.

[Recent topics on interorgan communication networks and gut microbiota].

The living body is composed of diverse organ systems, each of which has its own characteristic control mechanisms and complex in vivo responses. Between the brain and organs such as the heart, kidney, liver, pancreas, gastrointestinal tract, and even muscles, there is a sophisticated and complex regulatory system. Coordinated interactions through communication between organs are essential for maintaining health. In this review, we introduce four research trends in inter-organ networks, with a focus on the digestive system: 1) Inter-organ networks on metabolic systems, 2) Inter-organ networks originating from the gastrointestinal tract, 3) Intestinal bacteria, that is one of the biggest topics in recent years, 4) Research results on the involvement of gut microbiota in the inter-organ network between the kidney and the gastrointestinal tract. An integrated understanding and investigation of the regulatory mechanisms of inter-organ communication networks are expected to extend healthy life span and improve quality of life.

Stress decreases contraction of the colon, and the effects of stress are different among the regions of the colon.

Stress affects a variety of organs. Diarrhea and constipation are closely related to stress, which involves the gastrointestinal motility of the colon. We compared the gastrointestinal motility of the proximal, mid, and distal colon in mice with stress. Stress was applied by water immersion restraint. Colon motility was measured using an isotonic transducer in the direction of the circular muscles. Electric field stimulation-induced contractions in stressed mice were reduced compared to control mice in the proximal and distal colon. On the other hand, in the mid colon, contraction in control mice and stressed mice were almost same. This interesting difference between the regions may provide a clue to the functional abnormalities in gastrointestinal motility associated with stress.

Redox-dependent internalization of the purinergic P2Y6 receptor limits colitis progression.

After ligand stimulation, many G protein­coupled receptors (GPCRs) undergo ß-arrestin­dependent desensitization, during which they are internalized and either degraded or recycled to the plasma membrane. Some GPCRs are not subject to this type of desensitization because they lack the residues required to interact with ß-arrestins. We identified a mechanism of redox-dependent alternative internalization (REDAI) that promotes the internalization and degradation of the purinergic P2Y6 receptor (P2Y6R). Synthetic and natural compounds containing electrophilic isothiocyanate groups covalently modified P2Y6R at Cys220, which promoted the ubiquitylation of Lys137 and receptor internalization and degradation in various mouse and human cultured cell lines. Endogenous electrophiles also promoted ligand-dependent P2Y6R internalization and degradation. P2Y6R is highly abundant in inflammatory cells and promotes the pathogenesis of colitis. Deficiency in P2Y6R protected mice against experimentally induced colitis, and mice expressing a form of P2Y6R in which Cys220 was mutated to nonmodifiable serine were more sensitive to the induction of colitis. Several other GPCRs, including A2BAR, contain cysteine and lysine residues at the appropriate positions to mediate REDAI, and isothiocyanate stimulated the internalization of A2BAR and of a form of P2Y2R with insertions of the appropriate residues. Thus, endogenous and exogenous electrophiles may limit colitis progression through cysteine modification of P2Y6R and may also mediate internalization of other GPCRs.

IL-19 Contributes to the Development of Nonalcoholic Steatohepatitis by Altering Lipid Metabolism.

Interleukin (IL)-19, a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. Nonalcoholic steatohepatitis (NASH) is a disease that has progressed from nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. We evaluated the functions of IL-19 in a NAFLD/NASH mouse model using a 60% high fat diet with 0.1% methionine, without choline, and with 2% cholesterol (CDAHFD). Wild-type (WT) and IL-19 gene-deficient (KO) mice were fed a CDAHFD or standard diet for 9 weeks. Liver injury, inflammation, and fibrosis induced by CDAHFD were significantly worse in IL-19 KO mice than in WT mice. IL-6, TNF-α, and TGF-ß were significantly higher in IL-19 KO mice than in WT mice. As a mechanism using an in vitro experiment, palmitate-induced triglyceride and cholesterol contents were decreased by the addition of IL-19 in HepG2 cells. Furthermore, addition of IL-19 decreased the expression of fatty acid synthesis-related enzymes and increased ATP content in HepG2 cells. The action of IL-19 in vitro suppressed lipid metabolism. In conclusion, IL-19 may play an important role in the development of steatosis and fibrosis by directly regulating liver metabolism and may be a potential target for the treatment of liver diseases.

[Therapeutic application utilizing the anti-inflammatory effect of IL-19].

Interleukin-19 (IL-19) is a member of the IL-10 family and is an anti-inflammatory cytokine produced mainly by macrophages, epithelial cells, and vascular smooth muscle cells. In addition, receptors for IL-19, IL-20 receptor 1 and IL-20 receptor 2, are also expressed in the cells mentioned above. The last 10 years from the finding of IL-19, investigations underline the anti-inflammatory role of IL-19 in the human diseases such as psoriasis, asthma, arteriosclerosis, and inflammatory bowel disease. If it is a pro-inflammatory cytokine, therapeutic applications may include the use of neutralizing antibodies, however, because IL-19 exhibits anti-inflammatory effects, recombinant products may be useful in therapeutic applications. However, the therapeutic applications of IL-19 for human disease have not yet been developed. In this review, we present the new findings on the preventive and therapeutic effects of IL-19 on various mouse disease models. Increasing knowledge about mouse disease models will increase the feasibility of future human disease applications.

Ablation of interleukin-19 improves motor function in a mouse model of amyotrophic lateral sclerosis.

Neuroinflammation by activated microglia and astrocytes plays a critical role in progression of amyotrophic lateral sclerosis (ALS). Interleukin-19 (IL-19) is a negative-feedback regulator that limits pro-inflammatory responses of microglia in an autocrine and paracrine manner, but it remains unclear how IL-19 contributes to ALS pathogenesis. We investigated the role of IL-19 in ALS using transgenic mice carrying human superoxide dismutase 1 with the G93A mutation (SOD1G93A Tg mice). We generated IL-19-deficient SOD1G93A Tg (IL-19-/-/SOD1G93A Tg) mice by crossing SOD1G93A Tg mice with IL-19-/- mice, and then evaluated disease progression, motor function, survival rate, and pathological and biochemical alternations in the resultant mice. In addition, we assessed the effect of IL-19 on glial cells using primary microglia and astrocyte cultures from the embryonic brains of SOD1G93A Tg mice and IL-19-/-/SOD1G93A Tg mice. Expression of IL-19 in primary microglia and lumbar spinal cord was higher in SOD1G93A Tg mice than in wild-type mice. Unexpectedly, IL-19-/-/SOD1G93A Tg mice exhibited significant improvement of motor function. Ablation of IL-19 in SOD1G93A Tg mice increased expression of both neurotoxic and neuroprotective factors, including tumor necrosis factor-α (TNF-α), IL-1ß, glial cell line-derived neurotrophic factor (GDNF), and transforming growth factor ß1, in lumbar spinal cord. Primary microglia and astrocytes from IL-19-/-/SOD1G93A Tg mice expressed higher levels of TNF-α, resulting in release of GDNF from astrocytes. Inhibition of IL-19 signaling may alleviate ALS symptoms.

Interleukin-19 Abrogates Experimental Autoimmune Encephalomyelitis by Attenuating Antigen-Presenting Cell Activation.

Interleukin-19 (IL-19) acts as a negative-feedback regulator to limit proinflammatory response of macrophages and microglia in autocrine/paracrine manners in various inflammatory diseases. Multiple sclerosis (MS) is a major neuroinflammatory disease in the central nervous system (CNS), but it remains uncertain how IL-19 contributes to MS pathogenesis. Here, we demonstrate that IL-19 deficiency aggravates experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by promoting IL-17-producing helper T cell (Th17 cell) infiltration into the CNS. In addition, IL-19-deficient splenic macrophages expressed elevated levels of major histocompatibility complex (MHC) class II, co-stimulatory molecules, and Th17 cell differentiation-associated cytokines such as IL-1ß, IL-6, IL-23, TGF-ß1, and TNF-α. These observations indicated that IL-19 plays a critical role in suppression of MS pathogenesis by inhibiting macrophage antigen presentation, Th17 cell expansion, and subsequent inflammatory responses. Furthermore, treatment with IL-19 significantly abrogated EAE. Our data suggest that IL-19 could provide significant therapeutic benefits in patients with MS.

Gastrointestinal motility modulation by stress is associated with reduced smooth muscle contraction through specific transient receptor potential channel.

Excessive stress response causes disability in social life. There are many diseases caused by stress, such as gastrointestinal motility disorders, depression, eating disorders, and cardiovascular diseases. Transient receptor potential (TRP) channels underlie non-selective cation currents and are downstream effectors of G protein-coupled receptors. Ca2+ influx is important for smooth muscle contraction, which is responsible for gastrointestinal motility. Little is known about the possible involvement of TRP channels in the gastrointestinal motility disorders due to stress. The purpose of this study was to measure the changes in gastrointestinal motility caused by stress and to elucidate the mechanism of these changes. The stress model used the water immersion restraint stress. Gastrointestinal motility, especially the ileum, was recorded responses to electric field stimulation (EFS) by isometric transducer. EFS-induced contraction was significantly reduced in the ileum of stressed mouse. Even under the conditions treated with atropine, EFS-induced contraction was significantly reduced in the ileum of stressed mouse. In addition, carbachol-induced, neurokinin A-induced, and substance P-induced contractions were all significantly reduced in the ileum of stressed mouse. Furthermore, the expression of TRPC3 was decreased in the ileum of stressed mouse. These results suggest that the gastrointestinal motility disorders due to stress is associated with specific non-selective cation channel.

Interleukin-19 as an Immunoregulatory Cytokine.

IL-19 is a type of anti-inflammatory cytokine. Since the receptor for IL-19 is common to IL-20 and IL-24, it is important to clarify the role of each of the three cytokines. If three different cytokines bind to the same receptor, these three may have been produced to complement the other two. However, perhaps it is unlikely. Recently, the existence of a novel receptor for IL-19 was suggested. The distinction between the roles of the three cytokines still makes sense. On the other hand, because T cells do not produce IL-19, their role in acquired immunity is limited or indirect. It has been reported that IL-19 causes inflammation in some diseases but does not have an anti-inflammatory effect. In this review, we introduce the current role of IL-19 in each disease. In addition, we will describe the molecular mechanism of IL-19 and its development for the prevention of diseases. IL-19 was previously considered an anti-inflammatory cytokine, but we would like to propose it as an immunoregulatory cytokine.

Deficiency of interleukin-19 exacerbates lipopolysaccharide/D-galactosamine-induced acute liver failure.

Interleukin (IL)-19 is a cytokine clustered in the IL-20 cytokine superfamily with both anti-inflammatory and pro-inflammatory aspects depending on the etiology of inflammatory disease. The function of IL-19 has been evaluated in cutaneous and inflammatory bowel diseases, but has not been studied in liver diseases. Here, we examined the effect of IL-19 on acute liver failure (ALF) using two mouse models of ALF: lipopolysaccharide and D-galactosamine (LPS/GalN)-induced model and concanavalin A (ConA)-induced model. In the LPS/GalN-induced ALF model, which is mainly caused by the innate immune response of liver macrophages, IL-19 knockout (KO) mice showed increased plasma level of liver deviation enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) compared with wild-type (WT) mice. In histopathology of liver sections, IL-19 KO mice exacerbated liver injury with marked hemorrhagic lesions and hepatocellular death in the liver compared with WT mice. In this model, mRNA expressions of pro-inflammatory chemokines, CCL2 and CCL5 were increased in liver tissue from IL-19 KO mice compared with WT mice. Moreover, the mRNA expressions of IL-19 and its receptor subunit were induced in liver tissue by LPS/GalN administration. However, there is no difference in liver injury between WT and IL-19KO in the ConA-induced ALF model induced by CD4+ T cell activation. These data suggest that IL-19 has a protective effect against inflammation-mediated liver injury, which is dependent on the etiology.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-7.

Breakthroughs in Medicinal Chemistry [...].

Interleukin-19 enhances cytokine production induced by lipopolysaccharide and inhibits cytokine production induced by polyI:C in BALB/c mice.

Interleukin (IL)-19 is a cytokine of the IL-10 family. There are many reports on the involvement of IL-19 in several human diseases. There also are many reports elucidating the role of IL-19 using mouse disease models. Most reports use C57BL/6 mice, whereas few reports use BALB/c mice, in terms of the mouse disease model that the researchers used in the present study. To date, research on the role of IL-19 is diversified, yet some basic mechanisms are still unclear. In this study, we administered lipopolysaccharide (LPS), polyI:C, and CpG to BALB/c mice, measured more than 20 cytokines in the blood and compared them with that of the wild-type and IL-19-deficient (IL-19 KO) mice. LPS is associated with bacterial infection, polyI:C is associated with viral infection, and CpG is associated with both bacterial and viral infections. Among the cytokines measured, the results of experiments using LPS revealed that the production of some cytokines was suppressed in IL-19 KO mice. Interestingly, the experiments using polyI:C revealed that production of some cytokines was enhanced in IL-19 KO mice. However, the experiments using CpG have shown that the production of only one cytokine was enhanced in IL-19 KO mice. These results revealed that cytokine production in the blood was regulated by IL-19, and the type of regulation was dependent on the administered stimulant.

Fucoxanthin Ameliorates Atopic Dermatitis Symptoms by Regulating Keratinocytes and Regulatory Innate Lymphoid Cells.

Fucoxanthin (FX) is a xanthophyll that is contained abundantly in marine plants. The biological action of FX includes its antioxidant and anti-lipogenic activities, while the precise action of its mechanisms on skin cells has not yet been clarified. The current study examined the effect of FX in comparison with tacrolimus (TAC) on NC/Nga mice, which are an atopic dermatitis (AD) model. FX topical treatment dramatically ameliorated itching behavior over the TAC treatment, which was insufficient for improvement of AD symptoms. In Nc/Nga mice, FX or TAC applied to the skin inhibited eosinophil infiltration with decreased expression of Il-33. FX also stimulated Il-2, Il-5, Il-13, Il-10, and TGF-ß expression levels, and Sca1+Il-10+TGF-ß+ regulatory innate lymphoid cells (ILCreg) were dominantly observed in FX treated skin epidermal keratinocytes and dermal layers. This combined evidence demonstrated that FX exerts anti-inflammatory effects on keratinocytes and ameliorates AD symptoms by regulating ILCreg to normalize immune responses in an atopic dermatitis model.

[Recent progress in the pathophysiological role of interleukin-19].

Cytokine signal is essential for the biological function including development, maintenance of homeostasis and progression of disease. There are growing evidences that signaling via pro-inflammatory cytokines underlie a variety of immunological diseases such as psoriasis, atopic dermatitis, inflammatory bowel disease, and metabolic syndromes, in which cytokine signals are known as a potential therapeutic target of antibody drugs. In contrast, anti-inflammatory cytokines, which is represented by IL-10, largely contribute to suppression of inflammation and restoration of injured tissues. IL-19 is a member of IL-10 cytokine family, which comprises IL-20 cytokine subfamily with IL-20, IL-22, IL-24, and IL-26. IL-19 is produced by myeloid and epithelial cells with stimulation of bacterial components and cytokines. Although IL-19 has been originally recognized as a potential Th2-related cytokine, in recent researches, it has been reported that this cytokine upregulates Th17 response to reflect and promote progression of Th17-related disease including psoriasis. On the other hand, IL-19 has anti-inflammatory effects on inflammatory diseases such as infectious skin disease, inflammatory bowel disease, and cardiovascular disease. Therefore, IL-19 may exert pleiotropic effects dependent on the pathological mechanism of inflammatory diseases. In this review, we summarize recent studies about IL-19 and introduce the pathophysiological and therapeutic role of IL-19 in inflammatory diseases.

Ibudilast attenuates doxorubicin-induced cytotoxicity by suppressing formation of TRPC3 channel and NADPH oxidase 2 protein complexes.

BACKGROUND AND PURPOSE: Doxorubicin is a highly effective anticancer agent but eventually induces cardiotoxicity associated with increased production of ROS. We previously reported that a pathological protein interaction between TRPC3 channels and NADPH oxidase 2 (Nox2) contributed to doxorubicin-induced cardiac atrophy in mice. Here we have investigated the effects of ibudilast, a drug already approved for clinical use and known to block doxorubicin-induced cytotoxicity, on the TRPC3-Nox2 complex. We specifically sought evidence that this drug attenuated doxorubicin-induced systemic tissue wasting in mice. EXPERIMENTAL APPROACH: We used the RAW264.7 macrophage cell line to screen 1,271 clinically approved chemical compounds, evaluating functional interactions between TRPC3 channels and Nox2, by measuring Nox2 protein stability and ROS production, with and without exposure to doxorubicin. In male C57BL/6 mice, samples of cardiac and gastrocnemius muscle were taken and analysed with morphometric, immunohistochemical, RT-PCR and western blot methods. In the passive smoking model, cells were exposed to DMEM containing cigarette sidestream smoke. KEY RESULTS: Ibudilast, an anti-asthmatic drug, attenuated ROS-mediated muscle toxicity induced by doxorubicin treatment or passive smoking, by inhibiting the functional interactions between TRPC3 channels and Nox2, without reducing TRPC3 channel activity. CONCLUSIONS AND IMPLICATIONS: These results indicate a common mechanism underlying induction of systemic tissue wasting by doxorubicin. They also suggest that ibudilast could be repurposed to prevent muscle toxicity caused by anticancer drugs or passive smoking.