Novel 5-HT7 receptor antagonists modulate intestinal immune responses and reduce severity of colitis.

Inflammatory bowel disease (IBD) encompasses a number of debilitating chronic gastrointestinal (GI) inflammatory disorders, including Crohn's disease and ulcerative colitis. In both conditions, mucosal inflammation is a key clinical presentation and is associated with altered serotonin (5-hydroxytryptamine; 5-HT) signaling. This altered 5-HT signaling is also found across various animal models of colitis. Of the 14 known receptor subtypes, 5-HT receptor type 7 (5-HT7) is one of the most recently discovered. We previously reported that blocking 5-HT signaling, with either a selective 5-HT7 receptor antagonist (SB-269970) or genetic ablation alleviated intestinal inflammation in murine experimental models of colitis. Here, we developed novel antagonists, namely MC-170073 and MC-230078, which target 5-HT7 receptors with high selectivity. We also investigated the in vivo efficacy of these antagonists in experimental colitis by utilizing dextran sulfate sodium (DSS) and the transfer of CD4+CD45RBhigh T cells to induce intestinal inflammation. Inhibition of 5-HT7 receptor signaling with the antagonists, MC-170073 and MC-230078, ameliorated intestinal inflammation in both acute and chronic colitis models, which was accompanied by lower histopathological damage and diminished levels of pro-inflammatory cytokines in comparison to vehicle-treated controls. Together, the data reveal that the pharmacological inhibition of 5-HT7 receptors by these selective antagonists ameliorates the severity of colitis across various experimental models and may, in the future, serve as a potential treatment option for patients with IBD. In addition, these findings support that 5-HT7 is a viable therapeutic target for IBD.

Emerging Roles of Gut Serotonin in Regulation of Immune Response, Microbiota Composition and Intestinal Inflammation.

Although the exact etiology of inflammatory bowel diseases (IBD) is unknown, studies have shown that dysregulated immune responses, genetic factors, gut microbiota, and environmental factors contribute to their pathogenesis. Intriguingly, serotonin (5-hydroxytryptamine or 5-HT) seems to be a molecule with increasingly strong implications in the pathogenesis of intestinal inflammation, affecting host physiology, including autophagy and immune responses, as well as microbial composition and function. 5-HT may also play a role in mediating how environmental effects impact outcomes in IBD. In this review, we aim to explore the production and important functions of 5-HT, including its impact on the gut. In addition, we highlight the bidirectional impacts of 5-HT on the immune system, the gut microbiota, and the process of autophagy and how these effects contribute to the manifestation of intestinal inflammation. We also explore recent findings connecting 5-HT signalling and the influence of environmental factors, particularly diet, in the pathogenesis of IBD. Ultimately, we explore the pleiotropic effects of this ancient molecule on biology and health in the context of intestinal inflammation.

Inhibition of NADPH Oxidase (NOX) 2 Mitigates Colitis in Mice with Impaired Macrophage AMPK Function.

Macrophage adenosine monophosphate-activated protein kinase (AMPK) limits the development of experimental colitis. AMPK activation inhibits NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine secretion in macrophages during inflammation, while increased NOX2 expression is reported in experimental models of colitis and inflammatory bowel disease (IBD) patients. Although there are reductions in AMPK activity in IBD, it remains unclear whether targeted inhibition of NOX2 in the presence of defective AMPK can reduce the severity of colitis. Here, we investigate whether the inhibition of NOX2 ameliorates colitis in mice independent of AMPK activation. Our study identified that VAS2870 (a pan-Nox inhibitor) alleviated dextran sodium sulfate (DSS)-induced colitis in macrophage-specific AMPKß1-deficient (AMPKß1LysM) mice. Additionally, VAS2870 blocked LPS-induced TLR-4 and NOX2 expression, ROS production, nuclear translocation of NF-κB, and pro-inflammatory cytokine secretion in bone marrow-derived macrophages (BMDMs) from AMPKß1LysM mice, whereas sodium salicylate (SS; AMPK ß1 activator) did not. Both VAS2870 and SS inhibited LPS-induced NOX2 expression, ROS production, and pro-inflammatory cytokine secretions in bone marrow-derived macrophages (BMDMs) from wildtype (AMPKß1fl/fl) mice but only VAS2870 inhibited these effects of LPSs in AMPKß1LysM BMDMs. Furthermore, in macrophage cells (RAW 264.7), both SS and VAS2870 inhibited ROS production and the secretion of pro-inflammatory cytokines and reversed the impaired autophagy induced by LPSs. These data suggest that inhibiting NOX2 can reduce inflammation independent of AMPK in colitis.

Parenteral BCG vaccine induces lung-resident memory macrophages and trained immunity via the gut-lung axis.

Aside from centrally induced trained immunity in the bone marrow (BM) and peripheral blood by parenteral vaccination or infection, evidence indicates that mucosal-resident innate immune memory can develop via a local inflammatory pathway following mucosal exposure. However, whether mucosal-resident innate memory results from integrating distally generated immunological signals following parenteral vaccination/infection is unclear. Here we show that subcutaneous Bacillus Calmette-Guérin (BCG) vaccination can induce memory alveolar macrophages (AMs) and trained immunity in the lung. Although parenteral BCG vaccination trains BM progenitors and circulating monocytes, induction of memory AMs is independent of circulating monocytes. Rather, parenteral BCG vaccination, via mycobacterial dissemination, causes a time-dependent alteration in the intestinal microbiome, barrier function and microbial metabolites, and subsequent changes in circulating and lung metabolites, leading to the induction of memory macrophages and trained immunity in the lung. These data identify an intestinal microbiota-mediated pathway for innate immune memory development at distal mucosal tissues and have implications for the development of next-generation vaccine strategies against respiratory pathogens.

Chronic exposure to synthetic food colorant Allura Red AC promotes susceptibility to experimental colitis via intestinal serotonin in mice.

Chemicals in food are widely used leading to significant human exposure. Allura Red AC (AR) is a highly common synthetic colorant; however, little is known about its impact on colitis. Here, we show chronic exposure of AR at a dose found in commonly consumed dietary products exacerbates experimental models of colitis in mice. While intermittent exposure is more akin to a typical human exposure, intermittent exposure to AR in mice for 12 weeks, does not influence susceptibility to colitis. However, exposure to AR during early life primes mice to heightened susceptibility to colitis. In addition, chronic exposure to AR induces mild colitis, which is associated with elevated colonic serotonin (5-hydroxytryptamine; 5-HT) levels and impairment of the epithelial barrier function via myosin light chain kinase (MLCK). Importantly, chronic exposure to AR does not influence colitis susceptibility in mice lacking tryptophan hydroxylase 1 (TPH1), the rate limiting enzyme for 5-HT biosynthesis. Cecal transfer of the perturbed gut microbiota by AR exposure worsens colitis severity in the recipient germ-free (GF) mice. Furthermore, chronic AR exposure elevates colonic 5-HT levels in naïve GF mice. Though it remains unknown whether AR has similar effects in humans, our study reveals that chronic long-term exposure to a common synthetic colorant promotes experimental colitis via colonic 5-HT in gut microbiota-dependent and -independent pathway in mice.

Peripheral serotonin: cultivating companionship with gut microbiota in intestinal homeostasis.

Serotonin, also known as 5-hydroxytryptamine (5-HT), is an evolutionarily ancient and phylogenetically conserved monoamine that regulates multifaceted physiological functions in mammals. 5-HT was, at one time, most extensively studied as a neurotransmitter within the central nervous system but is now known to regulate nonneuronal functions including immune responses in an autocrine-paracrine-endocrine manner. Compelling evidence from intervention studies using germ-free mice or antibiotic-associated microbiota perturbation suggests that novel interactions between 5-HT and the gut microbiota are essential in maintaining intestinal homeostasis. Importantly, recent studies reveal that bidirectional host-microbial interactions mediated by the host serotonergic system can promote distinct changes within the gut microbiota. These changes may potentially lead to a state known as "dysbiosis" that has been strongly associated with various gut pathologies including inflammatory bowel disease (IBD). In this review, we update the current understanding of host-microbiota interaction by focusing on the impact of peripheral 5-HT signaling within this dynamic. We also briefly highlight key environmental risk factors for IBD, such as the Western diet, and draw attention to the interaction of synthetic food colorants with 5-HT signaling that may facilitate future research.

Increased gut serotonin production in response to bisphenol A structural analogs may contribute to their obesogenic effects.

Obesogens are synthetic, environmental chemicals that can disrupt endocrine control of metabolism and contribute to the risk of obesity and metabolic disease. Bisphenol A (BPA) is one of the most studied obesogens. There is considerable evidence that BPA exposure is associated with weight gain, increased adiposity, poor blood glucose control, and nonalcoholic fatty liver disease in animal models and human populations. Increased usage of structural analogs of BPA has occurred in response to legislation banning their use in some commercial products. However, BPA analogs may also cause some of the same metabolic impairments because of common mechanisms of action. One key effector that is altered by BPA and its analogs is serotonin, however, it is unknown if BPA-induced changes in peripheral serotonin pathways underlie metabolic perturbations seen with BPA exposure. Upon ingestion, BPA and its analogs act as endocrine-disrupting chemicals in the gastrointestinal tract to influence serotonin production by the gut, where over 95% of serotonin is produced. The purpose of this review is to evaluate how BPA and its analogs alter gut serotonin regulation and then discuss how disruption of serotonergic networks influences host metabolism. We also provide evidence that BPA and its analogs enhance serotonin production in gut enterochromaffin cells. Taken together, we propose that BPA and many BPA analogs represent endocrine-disrupting chemicals that can influence host metabolism through the endogenous production of gut-derived factors, such as serotonin.

Gut-derived serotonin and its emerging roles in immune function, inflammation, metabolism and the gut-brain axis.

PURPOSE OF REVIEW: To shed light on the recently uncovered diverse role of serotonin (5-hydroxytryptamine; 5-HT) in the regulation of immune functions, inflammation, metabolism, and gut-brain axis. RECENT FINDINGS: Peripheral 5-HT which accounts for approximately 95% of the total is largely synthesized in the gut by enterochromaffin cells. Enterochromaffin cells release 5-HT in response to various stimuli including microbial products. Released 5-HT influences secretomotor, sensory and immune functions as well as inflammatory processes in the gut. 5-HT released from enterochromaffin cells enters circulation and is taken up and concentrated in platelets. 5-HT released from the activated platelets interacts with different organs to alter their metabolic activity. 5-HT also serves as a link in the gut-brain axis. SUMMARY: Emerging evidence regarding the role of peripheral 5-HT in the regulation of various physiological and pathophysiological conditions opens up new targets for researchers to explore and for clinicians to treat and manage different diseases associated with the altered 5-HT signalling.

BMAL1 Regulates the Daily Timing of Colitis.

Many physiological functions exhibit circadian rhythms: oscillations in biological processes that occur in a 24-hour period. These daily rhythms are maintained through a highly conserved molecular pacemaker known as the circadian clock. Circadian disruption has been proposed to cause increased risk of Inflammatory Bowel Disease (IBD) but the underlying mechanisms remain unclear. Patients with IBD experience chronic inflammation and impaired regeneration of intestinal epithelial cells. Previous animal-based studies have revealed that colitis models of IBD are more severe in mice without a circadian clock but the timing of colitis, and whether its inflammatory and regenerative processes have daily rhythms, remains poorly characterized. We tested circadian disruption using Bmal1-/- mutant mice that have a non-functional circadian clock and thus no circadian rhythms. Dextran Sulfate Sodium (DSS) was used to induce colitis. The disease activity of colitis was found to exhibit time-dependent variation in Bmal1+/+ control mice but is constant and elevated in Bmal1-/- mutants, who exhibit poor recovery. Histological analyses indicate worsened colitis severity in Bmal1-/- mutant colon, and colon infiltration of immune system cells shows a daily rhythm that is lost in the Bmal1-/- mutant. Similarly, epithelial proliferation in the colon has a daily rhythm in Bmal1+/+ controls but not in Bmal1-/- mutants. Our results support a critical role of a functional circadian clock in the colon which drives 24-hour rhythms in inflammation and healing, and whose disruption impairs colitis recovery. This indicates that weakening circadian rhythms not only worsens colitis, but delays healing and should be taken into account in the management of IBD. Recognition of this is important in the management of IBD patients required to do shift work.

Autoantibodies in Common Connective Tissue Diseases: A Primer for Laboratory Professionals.

BACKGROUND: Autoimmune connective tissue disorders are a significant health concern throughout the world with an estimated prevalence of 3% to 5%. They are associated with a variety of autoantibodies that play roles in their diagnosis, risk stratification, prognostication, and/or management. While some autoantibodies have been well-characterized for use in clinical laboratories, many more are in the research stage. Rapid transition from research to clinical practice, lack of clinical guidelines, and harmonization across a rapidly growing number of commercially available tests create numerous challenges to clinicians and laboratories. CONTENT: This article briefly discusses common connective tissue disorders and their association with well-known autoantibodies, describes current methods used in clinical laboratories, and outlines their advantages and limitations in the context of these diseases. SUMMARY: Understanding the role of specific autoantibodies and various methodologies for autoantibody testing are important for laboratory professionals who may be introducing/repatriating new tests, updating existing tests, or advising clinicians/patients about testing options/results. Collaboration between laboratory professional staff and clinicians, around the advantages and limitations of each methodology, is also important in their appropriate clinical utilization.

Disruption of autophagy by increased 5-HT alters gut microbiota and enhances susceptibility to experimental colitis and Crohn's disease.

Autophagy, an essential intracellular recycling process, is linked to the pathogenesis of various diseases including Crohn's disease (CD). Factors that lead to the development of impaired autophagy during intestinal inflammation remain largely unexplored. Here, we report the impact of the interaction between serotonin [5-hydroxytryptamine;(5-HT)] and autophagy in colitis in mouse and human studies. In mice, increased gut 5-HT inhibited autophagy and led to enhanced colitis susceptibility. Reciprocally, mice with reduced 5-HT exhibited up-regulated autophagy via the mammalian target of rapamycin pathway, which resulted in significantly decreased colitis. Deletion of autophagy gene, Atg7, in an epithelial-specific manner, in concert with reduced 5-HT, promoted the development of a colitogenic microbiota and abolished the protective effects conferred by reduced 5-HT. Notably, in control and patient peripheral blood mononuclear cells, we uncovered that 5-HT treatment inhibited autophagy. Our findings suggest 5-HT as a previously unidentified therapeutic target in intestinal inflammatory disorders such as CD that exhibits dysregulated autophagy.

The Circadian Clock Gene, Bmal1, Regulates Intestinal Stem Cell Signaling and Represses Tumor Initiation.

BACKGROUND & AIMS: Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known. We tested the nonredundant clock gene Bmal1 in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer. METHODS: Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod (day/night cycle) disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal nontransformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal. RESULTS: Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors show high Yes-associated protein 1 (Hippo signaling) activity but show low Wnt (Wingless and Int-1) activity. Intestinal organoid assays show that loss of Bmal1 increases self-renewal in a Yes-associated protein 1-dependent manner. CONCLUSIONS: Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation. Transcript profiling: GEO accession number: GSE157357.

Tryptophan-derived serotonin-kynurenine balance in immune activation and intestinal inflammation.

Endogenous tryptophan metabolism pathways lead to the production of serotonin (5-hydroxytryptamine; 5-HT), kynurenine, and several downstream metabolites which are involved in a multitude of immunological functions in both health and disease states. Ingested tryptophan is largely shunted to the kynurenine pathway (95%) while only minor portions (1%-2%) are sequestered for 5-HT production. Though often associated with the functioning of the central nervous system, significant production of 5-HT, kynurenine and their downstream metabolites takes place within the gut. Accumulating evidence suggests that these metabolites have essential roles in regulating immune cell function, intestinal inflammation, as well as in altering the production and suppression of inflammatory cytokines. In addition, both 5-HT and kynurenine have a considerable influence on gut microbiota suggesting that these metabolites impact host physiology both directly and indirectly via compositional changes. It is also now evident that complex interactions exist between the two pathways to maintain gut homeostasis. Alterations in 5-HT and kynurenine are implicated in the pathogenesis of many gastrointestinal dysfunctions, including inflammatory bowel disease. Thus, these pathways present numerous potential therapeutic targets, manipulation of which may aid those suffering from gastrointestinal disorders. This review aims to update both the role of 5-HT and kynurenine in immune regulation and intestinal inflammation, and analyze the current knowledge of the relationship and interactions between 5-HT and kynurenine pathways.

Trichuris muris Model: Role in Understanding Intestinal Immune Response, Inflammation and Host Defense.

Several parasites have evolved to survive in the human intestinal tract and over 1 billion people around the world, specifically in developing countries, are infected with enteric helminths. Trichuris trichiura is one of the world's most common intestinal parasites that causes human parasitic infections. Trichuris muris, as an immunologically well-defined mouse model of T. trichiura, is extensively used to study different aspects of the innate and adaptive components of the immune system. Studies on T. muris model offer insights into understanding host immunity, since this parasite generates two distinct immune responses in resistant and susceptible strains of mouse. Apart from the immune cells, T. muris infection also influences various components of the intestinal tract, especially the gut microbiota, mucus layer, epithelial cells and smooth muscle cells. Here, we reviewed the different immune responses generated by innate and adaptive immune components during acute and chronic T. muris infections. Furthermore, we discussed the importance of studying T. muris model in understanding host-parasite interaction in the context of alteration in the host's microbiota, intestinal barrier, inflammation, and host defense, and in parasite infection-mediated modulation of other immune and inflammatory diseases.

Saffron Pre-Treatment Promotes Reduction in Tissue Inflammatory Profiles and Alters Microbiome Composition in Experimental Colitis Mice.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract with an incompletely understood pathogenesis. Long-standing colitis is associated with increased risk of colon cancer. Despite the availability of various anti-inflammatory and immunomodulatory drugs, many patients fail to respond to pharmacologic therapy and some experience drug-induced adverse events. Dietary supplements, particularly saffron (Crocus sativus), have recently gained an appreciable attention in alleviating some symptoms of digestive diseases. In our study, we investigated whether saffron may have a prophylactic effect in a murine colitis model. Saffron pre-treatment improved the gross and histopathological characteristics of the colonic mucosa in murine experimental colitis. Treatment with saffron showed a significant amelioration of colitis when compared to the vehicle-treated mice group. Saffron treatment significantly decreased secretion of serotonin and pro-inflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, in the colon tissues by suppressing the nuclear translocation of NF-κB. The gut microbiome analysis revealed distinct clusters in the saffron-treated and untreated mice in dextran sulfate sodium (DSS)-induced colitis by visualization of the Bray-Curtis diversity by principal coordinates analysis (PCoA). Furthermore, we observed that, at the operational taxonomic unit (OTU) level, Cyanobacteria were depleted, while short-chain fatty acids (SCFAs), such as isobutyric acid, acetic acid, and propionic acid, were increased in saffron-treated mice. Our data suggest that pre-treatment with saffron inhibits DSS-induced pro-inflammatory cytokine secretion, modulates gut microbiota composition, prevents the depletion of SCFAs, and reduces the susceptibility to colitis.

Fecal Calprotectin, CRP and Leucocytes in IBD Patients: Comparison of Biomarkers With Biopsy Results.

BACKGROUND: This study aimed to compare fecal calprotectin (FC) levels with other commonly used parameters as part of patient care during evaluation for inflammatory bowel disease (IBD). METHODS: We recruited adult IBD patients with ulcerative colitis (UC) and Crohn's disease (CD) and compared the results of the patient's biopsy results (i.e., inflamed versus noninflamed) for six sites (i.e., ileum, ascending colon, transverse colon, descending colon, sigmoid colon, rectum) with concentrations of C-reactive protein (CRP), total leucocytes and fecal calprotectin (FC). RESULTS: We found that FC was significantly elevated in a concentration-dependent manner that correlated with the number of active inflammation sites reported in biopsy. Although CRP and leucocyte measurements trended upwards in line with inflammation reported from biopsy, the results were highly variable and highlighted poor reliability of these biomarkers for indicating IBD inflammation. CONCLUSIONS: These results strongly suggest that FC correlates best with biopsy reports and is a superior marker than CRP and leucocytes.

Salicylates Ameliorate Intestinal Inflammation by Activating Macrophage AMPK.

BACKGROUND: Inflammatory bowel diseases are the most common chronic intestinal inflammatory conditions, and their incidence has shown a dramatic increase in recent decades. Limited efficacy and questionable safety profiles with existing therapies suggest the need for better targeting of therapeutic strategies. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular metabolism and has been implicated in intestinal inflammation. Macrophages execute an important role in the generation of intestinal inflammation. Impaired AMPK in macrophages has been shown to be associated with higher production of proinflammatory cytokines; however, the role of macrophage AMPK in intestinal inflammation and the mechanism by which it regulates inflammation remain to be determined. In this study, we investigated the role of AMPK with a specific focus on macrophages in the pathogenesis of intestinal inflammation. METHODS: A dextran sodium sulfate-induced colitis model was used to assess the disease activity index, histological scores, macroscopic scores, and myeloperoxidase level. Proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß were measured by enzyme-linked immunosorbent assay. Transient transfection of AMPKß1 and LC3-II siRNA in RAW 264.7 cells was performed to elucidate the regulation of autophagy by AMPK. The expression of p-AMPK, AMPK, and autophagy markers (eg, LC3-II, p62, Beclin-1, and Atg-12) was analyzed by Western blot. RESULTS: Genetic deletion of AMPKß1 in macrophages upregulated the production of proinflammatory cytokines, aggravated the severity of dextran sodium sulfate-induced colitis in mice, which was associated with an increased nuclear translocation of nuclear factor-κB, and impaired autophagy both in vitro and in vivo. Notably, the commonly used anti-inflammatory 5-aminosalicylic acid (ie, mesalazine) and sodium salicylate ameliorated dextran sodium sulfate-induced colitis through the activation of macrophage AMPK targeting the ß1 subunit. CONCLUSIONS: Together, these data suggest that the development of therapeutic agents targeting AMPKß1 may be effective in the treatment of intestinal inflammatory conditions including inflammatory bowel disease.

Mucins in Intestinal Mucosal Defense and Inflammation: Learning From Clinical and Experimental Studies.

Throughout the gastrointestinal (GI) tract, a distinct mucus layer composed of highly glycosylated proteins called mucins plays an essential role in providing lubrication for the passage of food, participating in cell signaling pathways and protecting the host epithelium from commensal microorganisms and invading pathogens, as well as toxins and other environmental irritants. These mucins can be broadly classified into either secreted gel-forming mucins, those that provide the structural backbone for the mucus barrier, or transmembrane mucins, those that form the glycocalyx layer covering the underlying epithelial cells. Goblet cells dispersed among the intestinal epithelial cells are chiefly responsible for the synthesis and secretion of mucins within the gut and are heavily influenced by interactions with the immune system. Evidence from both clinical and animal studies have indicated that several GI conditions, including inflammatory bowel disease (IBD), colorectal cancer, and numerous enteric infections are accompanied by considerable changes in mucin quality and quantity. These changes include, but are not limited to, impaired goblet cell function, synthesis dysregulation, and altered post-translational modifications. The current review aims to highlight the structural and functional features as well as the production and immunological regulation of mucins and the impact these key elements have within the context of barrier function and host defense in intestinal inflammation.