ABSTRACT
BACKGROUND: SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn's disease (CD), suggesting a role in inflammation. RESULTS: We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa. CONCLUSIONS: This study identifies SP140 as a druggable epigenetic therapeutic target for CD.
Subject(s)
Crohn Disease , Tumor Necrosis Factor Inhibitors , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , Crohn Disease/genetics , Crohn Disease/metabolism , Cytokines/genetics , Cytokines/metabolism , Epigenesis, Genetic , Humans , Macrophages , Transcription Factors/geneticsABSTRACT
BACKGROUND: Vagus nerve stimulation has been suggested to affect immune responses, partly through a neuronal circuit requiring sympathetic innervation of the splenic nerve bundle and norepinephrine (NE) release. Molecular and cellular mechanisms of action remain elusive. Here, we investigated the therapeutic value of this neuromodulation in inflammatory bowel disease (IBD) by applying electrical splenic nerve bundle stimulation (SpNS) in mice with dextran sulfate sodium (DSS)-induced colitis. METHODS: Cuff electrodes were implanted around the splenic nerve bundle in mice, whereupon mice received SpNS or sham stimulation. Stimulation was applied 6 times daily for 12 days during DSS-induced colitis. Colonic and splenic tissues were collected for transcriptional analyses by qPCR and RNA-sequencing (RNA-seq). In addition, murine and human splenocytes were stimulated with lipopolysaccharide (LPS) in the absence or presence of NE. Single-cell RNA-seq data from publicly available data sets were analyzed for expression of ß-adrenergic receptors (ß-ARs). RESULTS: Colitic mice undergoing SpNS displayed reduced colon weight/length ratios and showed improved Disease Activity Index scores with reduced Tumor Necrosis Factor α mRNA expression in the colon compared with sham stimulated mice. Analyses of splenocytes from SpNS mice using RNA-seq demonstrated specific immune metabolism transcriptome profile changes in myeloid cells. Splenocytes showed expression of ß-ARs in myeloid and T cells. Cytokine production was reduced by NE in mouse and human LPS-stimulated splenocytes. CONCLUSIONS: Together, our results demonstrate that SpNS reduces clinical features of colonic inflammation in mice with DSS-induced colitis possibly by inhibiting splenic myeloid cell activation. Our data further support exploration of the clinical use of SpNS for patients with IBD.
Subject(s)
Colitis , Inflammatory Bowel Diseases , Animals , Colitis/chemically induced , Colitis/therapy , Colon/metabolism , Colon/pathology , Dextran Sulfate/toxicity , Disease Models, Animal , Electric Stimulation , Humans , Inflammatory Bowel Diseases/chemically induced , Inflammatory Bowel Diseases/therapy , Lipopolysaccharides/adverse effects , Mice , Mice, Inbred C57BLABSTRACT
Clinical trials suggest that vagus nerve stimulation presents an alternative approach to classical immune suppression in Crohn's disease. T cells capable of producing acetylcholine (ChAT+ T cells) in the spleen are essential mediators of the anti-inflammatory effect of vagus nerve stimulation. Besides the spleen, ChAT+ T cells are found abundantly in Peyer's patches of the small intestine. However, the role of ChAT+ T cells in colitis pathogenesis is unknown. Here, we made use of CD4creChATfl/fl mice (CD4ChAT-/- mice) lacking ChAT expression specifically in CD4+ T cells. Littermates (ChATfl/fl mice) served as controls. In acute dextran sulfate sodium (DSS)-induced colitis (7 days of 2% DSS in drinking water), CD4ChAT-/- mice showed attenuated colitis and lower intestinal inflammatory cytokine levels compared with ChATfl/fl mice. In contrast, in a resolution model of DSS-induced colitis (5 days of 2% DSS followed by 7 days without DSS), CD4ChAT-/- mice demonstrated a worsened colitis recovery and augmented colonic histological inflammation scores and inflammatory cytokine levels as compared with ChATfl/fl mice. In a transfer colitis model using CD4+CD45RBhigh T cells, T cells from CD4ChAT-/- mice induced a similar level of colitis compared with ChATfl/fl T cells. Together, our results indicate that ChAT+ T cells aggravate the acute innate immune response upon mucosal barrier disruption in an acute DSS-induced colitis model, whereas they are supporting the later resolution process of this innate immune-driven colitis. Surprisingly, ChAT expression in T cells seems redundant in the context of T cell-driven colitis.NEW & NOTEWORTHY By using different mouse models of experimental colitis, we provide evidence that in dextran sulfate sodium-induced colitis, ChAT+ T cells capable of producing acetylcholine worsen the acute immune response, whereas they support the later healing phase of this innate immune-driven colitis.
Subject(s)
Acetylcholine/metabolism , CD4-Positive T-Lymphocytes/immunology , Colitis, Ulcerative/immunology , Immunity, Innate , Animals , Choline O-Acetyltransferase/genetics , Choline O-Acetyltransferase/metabolism , Colitis, Ulcerative/etiology , Female , Intestinal Mucosa/metabolism , Mice , Mice, Inbred C57BL , Sodium Dodecyl Sulfate/toxicityABSTRACT
BACKGROUND: Both the parasympathetic and sympathetic nervous system exert control over innate immune responses. In inflammatory bowel disease, sympathetic innervation in intestinal mucosa is reduced. Our aim was to investigate the role of sympathetic innervation to the intestine on regulation of the innate immune responses. METHODS: In lipopolysaccharide (LPS)-stimulated macrophages, we evaluated the effect of adrenergic receptor activation on cytokine production and metabolic profile. In vivo, the effect of sympathetic denervation on mucosal innate immune responses using 6-hydroxydopamine (6-OHDA), or using surgical transection of the superior mesenteric nerve (sympathectomy) was tested in Rag1-/- mice that lack T- and B-lymphocytes. RESULTS: In murine macrophages, adrenergic ß2 receptor activation elicited a dose-dependent reduction of LPS-induced cytokines, reduced LPS-induced glycolysis and increased maximum respiration. Sympathectomy led to a significantly decreased norepinephrine concentration in intestinal tissue. Within 14 days after sympathectomy, mice developed clinical signs of colitis, colon oedema and excess colonic cytokine production. Both 6-OHDA and sympathectomy led to prominent goblet cell depletion and histological damage of colonic mucosa. CONCLUSIONS: We conclude that the sympathetic nervous system plays a regulatory role in constraining innate immune cell reactivity towards microbial challenges, likely via the adrenergic ß2 receptor.
Subject(s)
Colitis/immunology , Immunity, Innate , Immunity, Mucosal , Intestinal Mucosa/immunology , Intestinal Mucosa/innervation , Sympathetic Nervous System/immunology , Adrenergic beta-2 Receptor Agonists/pharmacology , Albuterol/pharmacology , Animals , Cells, Cultured , Colitis/pathology , Colon/drug effects , Colon/pathology , Cytokines/genetics , Cytokines/immunology , Female , Humans , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/immunology , Male , Mice, Inbred C57BL , Mice, Knockout , Oxidopamine/pharmacologyABSTRACT
BACKGROUND & AIMS: Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity. METHODS: We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble ß-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (ex vivo) and the human mast cell line HMC-1. RESULTS: α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble ß-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate ß-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine. CONCLUSIONS: In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble ß-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity.
Subject(s)
Abdominal Pain/microbiology , Fungi/growth & development , Gastrointestinal Microbiome , Hyperalgesia/microbiology , Intestines/microbiology , Irritable Bowel Syndrome/microbiology , Abdominal Pain/physiopathology , Abdominal Pain/prevention & control , Abdominal Pain/psychology , Adult , Animals , Antifungal Agents/pharmacology , Anxiety, Separation/psychology , Behavior, Animal , Case-Control Studies , Cell Degranulation/drug effects , Cell Line , Disease Models, Animal , Dysbiosis , Fecal Microbiota Transplantation , Feces/microbiology , Female , Fungi/drug effects , Gastrointestinal Microbiome/drug effects , Humans , Hyperalgesia/physiopathology , Hyperalgesia/prevention & control , Hyperalgesia/psychology , Intestinal Mucosa/metabolism , Intestines/innervation , Irritable Bowel Syndrome/physiopathology , Irritable Bowel Syndrome/prevention & control , Irritable Bowel Syndrome/psychology , Male , Mast Cells/drug effects , Mast Cells/metabolism , Maternal Deprivation , Middle Aged , Pain Measurement , Pain Perception , Pain Threshold , Protein Kinase Inhibitors/pharmacology , Rats, Long-Evans , Syk Kinase/antagonists & inhibitors , Syk Kinase/metabolism , beta-Glucans/pharmacologyABSTRACT
BACKGROUND: Although never evaluated for efficacy, n-3 (ω-3) long-chain polyunsaturated fatty acids (LCPUFAs) are commercially offered as treatment for irritable bowel syndrome (IBS). OBJECTIVE: This study was designed to investigate, in a mast cell-dependent model for visceral hypersensitivity, whether this pathophysiologic mechanism can be reversed by dietary LCPUFA treatment via peroxisome proliferator-activated receptor γ (PPARG) activation. METHODS: Maternally separated rats were subjected to hypersensitivity-inducing acute stress at adult age. Reversal was attempted by protocols with tuna oil-supplemented diets [4% soy oil (SO) and 3% tuna oil (SO-T3) or 3% SO and 7% tuna oil (SO-T7)] and compared with control SO diets (7% or 10% SO) 4 wk after stress. The PPARG agonist rosiglitazone was evaluated in a 1 wk preventive protocol (30 mg · kg⻹ · d⻹). Erythrocytes were assessed to confirm LCPUFA uptake and tissue expression of lipoprotein lipase and glycerol kinase as indicators of PPARG activation. Colonic mast cell degranulation was evaluated by toluidine blue staining. In vitro, human mast cell line 1 (HMC-1) cells were pretreated with rosiglitazone, eicosapentaenoic acid, or docosahexaenoic acid, stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore or compound 48/80 and evaluated for tumor necrosis factor α (TNF-α) and ß-hexosaminidase release. RESULTS: Stress led to visceral hypersensitivity in all groups. Hypersensitivity was not reversed by SO-T3 or control treatment [prestress vs. 24 h poststress vs. posttreatment area under the curve; 76 ± 4 vs. 128 ± 12 (P < 0.05) vs. 115 ± 14 and 82 ± 5 vs. 127 ± 16 (P < 0.01) vs. 113 ± 19, respectively]. Comparison of SO-T7 with its control showed similar results [74 ± 6 vs. 103 ± 13 (P < 0.05) vs. 115 ± 17 and 66 ± 3 vs. 103 ± 10 (P < 0.05) vs. 117 ± 11, respectively]. Erythrocytes showed significant LCPUFA uptake in the absence of colonic PPARG activation. Rosiglitazone induced increased PPARG target gene expression, but did not prevent hypersensitivity. Mast cell degranulation never differed between groups. Rosiglitazone and LCPUFAs significantly reduced PMA/calcium ionophore-induced TNF-α release but not degranulation of HMC-1 cells. CONCLUSION: Dietary LCPUFAs did not reverse stress-induced visceral hypersensitivity in maternally separated rats. Although further research is needed, claims concerning LCPUFAs as a treatment option in IBS cannot be confirmed at this point and should be regarded with caution.
Subject(s)
Autonomic Nervous System/physiopathology , Colon/innervation , Dietary Supplements , Disease Models, Animal , Fatty Acids, Omega-3/therapeutic use , Fish Oils/therapeutic use , Irritable Bowel Syndrome/diet therapy , Animals , Animals, Newborn , Autonomic Nervous System/drug effects , Autonomic Nervous System/immunology , Biomarkers/blood , Biomarkers/metabolism , Cell Degranulation/drug effects , Cell Line , Colon/drug effects , Colon/immunology , Colon/metabolism , Erythrocytes/drug effects , Erythrocytes/enzymology , Erythrocytes/metabolism , Fatty Acids, Omega-3/administration & dosage , Fatty Acids, Omega-3/metabolism , Female , Fish Oils/administration & dosage , Fish Oils/metabolism , Hypoglycemic Agents/pharmacology , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Intestinal Mucosa/innervation , Intestinal Mucosa/metabolism , Irritable Bowel Syndrome/immunology , Irritable Bowel Syndrome/metabolism , Irritable Bowel Syndrome/physiopathology , Male , Mast Cells/drug effects , Mast Cells/immunology , Mast Cells/physiology , Maternal Deprivation , PPAR gamma/antagonists & inhibitors , PPAR gamma/metabolism , Rats, Long-Evans , TunaABSTRACT
Innervation of the intestinal mucosa by the sympathetic nervous system is well described but the effects of adrenergic receptor stimulation on the intestinal epithelium remain equivocal. We therefore investigated the effect of sympathetic neuronal activation on intestinal cells in mouse models and organoid cultures, to identify the molecular routes involved. Using publicly available single-cell RNA sequencing datasets we show that the α2A isoform is the most abundant adrenergic receptor in small intestinal epithelial cells. Stimulation of this receptor with norepinephrine or a synthetic specific α2A receptor agonist promotes epithelial proliferation and stem cell function, while reducing differentiation in vivo and in intestinal organoids. In an anastomotic healing mouse model, adrenergic receptor α2A stimulation resulted in improved anastomotic healing, while surgical sympathectomy augmented anastomotic leak. Furthermore, stimulation of this receptor led to profound changes in the microbial composition, likely because of altered epithelial antimicrobial peptide secretion. Thus, we established that adrenergic receptor α2A is the molecular delegate of intestinal epithelial sympathetic activity controlling epithelial proliferation, differentiation, and host defense. Therefore, this receptor could serve as a newly identified molecular target to improve mucosal healing in intestinal inflammation and wounding.
Subject(s)
Epithelial Cells , Intestines , Animals , Mice , Cell Proliferation , Intestinal Mucosa , Receptors, Adrenergic , Receptors, Adrenergic, alpha-2/genetics , Wound Healing/physiologyABSTRACT
OBJECTIVES: Repeated exposure to stress leads to mast cell degranulation, microscopic inflammation, and subsequent visceral hypersensitivity in animal models. To what extent this pathophysiological pathway has a role in patients with the irritable bowel syndrome (IBS) has not been properly investigated. The objective of this study was to assess the relationship between visceral hypersensitivity, microscopic inflammation, and the stress response in IBS. METHODS: Microscopic inflammation of the colonic mucosa was evaluated by immunohistochemistry in 66 IBS patients and 20 healthy volunteers (HV). Rectal sensitivity was assessed by a barostat study using an intermittent pressure-controlled distension protocol. Salivary cortisol to a psychological stress was measured to assess the stress response. RESULTS: Compared with HV, mast cells, T cells, and macrophages were decreased in IBS patients. Similarly, λ-free light chain (FLC)-positive mast cells were decreased but not immunoglobulin E (IgE)- and IgG-positive mast cells. There were no differences between hypersensitive and normosensitive IBS patients. No relation was found between any of the immune cells studied and the thresholds of discomfort, urge, first sensation, or IBS symptoms (e.g., abdominal pain, stool-related complaints, bloating). Finally, stress-related symptoms and the hypothalamic-pituitary-adrenal-axis response to stress were not correlated with the number of mast cells or the presence of visceral hypersensitivity. CONCLUSIONS: Although the number of mast cells, macrophages, T cells, and λFLC-positive mast cells is decreased in IBS compared with HV, this is not associated with the presence of visceral hypersensitivity or abnormal stress response. Our data question the role of microscopic inflammation as an underlying mechanism of visceral hypersensitivity, but rather suggest dysregulation of the mucosal immune system in IBS.
Subject(s)
Intestinal Mucosa/immunology , Irritable Bowel Syndrome/immunology , Irritable Bowel Syndrome/physiopathology , Rectum/physiopathology , Adult , Biopsy, Needle , Cell Count , Colon/immunology , Colon/pathology , Colon/physiopathology , Colonoscopy , Female , Humans , Hydrocortisone/blood , Immunohistochemistry , Intestinal Mucosa/pathology , Irritable Bowel Syndrome/pathology , Irritable Bowel Syndrome/psychology , Macrophages/immunology , Macrophages/pathology , Male , Mast Cells/immunology , Mast Cells/pathology , Middle Aged , Pressure , Sensory Thresholds , Stress, Psychological/physiopathology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Young AdultABSTRACT
BACKGROUND AND AIMS: Histone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD]. METHODS: CES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn's disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter. RESULTS: CES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis. CONCLUSIONS: We demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.
Subject(s)
Carboxylic Ester Hydrolases , Colitis , Crohn Disease , Histone Deacetylase Inhibitors , Inflammatory Bowel Diseases , Animals , Carboxylic Ester Hydrolases/metabolism , Colitis/chemically induced , Colitis/drug therapy , Colitis/metabolism , Crohn Disease/drug therapy , Crohn Disease/metabolism , Histone Deacetylase Inhibitors/pharmacology , Humans , Inflammatory Bowel Diseases/metabolism , Intestinal Mucosa/metabolism , Lipopolysaccharides , Mice , Monocytes , Myeloid CellsABSTRACT
BACKGROUND: Mast cell activation is thought to be involved in visceral hypersensitivity, one of the main characteristics of the irritable bowel syndrome (IBS). A study was therefore undertaken to investigate the effect of the mast cell stabiliser ketotifen on rectal sensitivity and symptoms in patients with IBS. METHODS: 60 patients with IBS underwent a barostat study to assess rectal sensitivity before and after 8 weeks of treatment. After the initial barostat, patients were randomised to receive ketotifen or placebo. IBS symptoms and health-related quality of life were scored. In addition, mast cells were quantified and spontaneous release of tryptase and histamine was determined in rectal biopsies and compared with biopsies from 22 age- and gender-matched healthy volunteers. RESULTS: Ketotifen but not placebo increased the threshold for discomfort in patients with IBS with visceral hypersensitivity. This effect was not observed in normosensitive patients with IBS. Ketotifen significantly decreased abdominal pain and other IBS symptoms and improved quality of life. The number of mast cells in rectal biopsies and spontaneous release of tryptase were lower in patients with IBS than in healthy volunteers. Spontaneous release of histamine was mostly undetectable but was slightly increased in patients with IBS compared with healthy volunteers. Histamine and tryptase release were not altered by ketotifen. CONCLUSIONS: This study shows that ketotifen increases the threshold for discomfort in patients with IBS with visceral hypersensitivity, reduces IBS symptoms and improves health-related quality of life. Whether this effect is secondary to the mast cell stabilising properties of ketotifen or H(1) receptor antagonism remains to be further investigated. Trial Registration Number NTR39, ISRCTN22504486.
Subject(s)
Histamine H1 Antagonists/therapeutic use , Irritable Bowel Syndrome/drug therapy , Ketotifen/therapeutic use , Viscera/physiopathology , Adult , Aged , Cell Count , Double-Blind Method , Elasticity/drug effects , Female , Histamine H1 Antagonists/adverse effects , Histamine Release/drug effects , Humans , Irritable Bowel Syndrome/pathology , Irritable Bowel Syndrome/physiopathology , Ketotifen/adverse effects , Male , Mast Cells/pathology , Middle Aged , Pressure , Quality of Life , Rectum/metabolism , Rectum/physiopathology , Sensory Thresholds/drug effects , Sensory Thresholds/physiology , Treatment Outcome , Tryptases/metabolism , Viscera/drug effects , Viscera/innervation , Young AdultABSTRACT
In 2020, three articles were published on a protein that can activate the immune system by binding to macrophage-inducible C-type lectin receptor (Mincle). In the articles, the protein was referred to as 'SAP130, a subunit of the histone deacetylase complex.' However, the Mincle ligand the authors aimed to investigate is splicing factor 3b subunit 3 (SF3B3). This splicing factor is unrelated to SAP130 (Sin3A associated protein 130, a subunit of the histone deacetylase-dependent Sin3A corepressor complex). The conclusions in the three articles were formulated for SF3B3, while the researchers used qPCR primers and antibodies against SAP130. We retraced the origins of the ambiguity about the two proteins and found that Online Mendelian Inheritance in Man (OMIM) added a Nature publication on SF3B3 as a reference for Sin3A associated protein 130 in 2016. Subsequently, companies such as Abcam referred to OMIM and the Nature article in their products for both SF3B3 and SAP130. In turn, the mistake by OMIM followed in the persistent and confusing use of 'SAP130' (spliceosome-associated protein 130) as an alternative symbol for SF3B3. With this report, we aim to eliminate the persistent confusion and separate the literature regarding the two proteins.
Subject(s)
Immunohistochemistry/standards , RNA Splicing Factors/genetics , Antibodies, Monoclonal , Gene Expression , Humans , Ileum/metabolism , Ileum/pathology , Inflammation , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Liver/metabolism , RNA Splicing Factors/metabolism , Terminology as TopicABSTRACT
Antimicrobial responses play an important role in maintaining intestinal heath. Recently we reported that miR-511 may regulate TLR4 responses leading to enhanced intestinal inflammation. However, the exact mechanism remained unclear. In this study we investigated the effect of miR-511 deficiency on anti-microbial responses and DSS-induced intestinal inflammation. miR-511-deficient mice were protected from DSS-induced colitis as shown by significantly lower disease activity index, weight loss and histology scores in the miR-511-deficient group. Furthermore, reduced inflammatory cytokine responses were observed in colons of miR-511 deficient mice. In vitro studies with bone marrow-derived M2 macrophages showed reduced TLR3 and TLR4 responses in miR-511-deficient macrophages compared to WT macrophages. Subsequent RNA sequencing revealed Wdfy1 as the potential miR-511 target. WDFY1 deficiency is related to impaired TLR3/TLR4 immune responses and the expression was downregulated in miR-511-deficient macrophages and colons. Together, this study shows that miR-511 is involved in the regulation of intestinal inflammation through downstream regulation of TLR3 and TLR4 responses via Wdfy1.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Colitis/genetics , MicroRNAs/metabolism , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , Animals , Colitis/chemically induced , Colitis/microbiology , Colitis/pathology , Colon/pathology , Dextran Sulfate , Female , Gene Expression Regulation , Inflammation/genetics , Inflammation/pathology , Lipopolysaccharides , Macrophages/metabolism , Macrophages/microbiology , Male , Mice, Inbred C57BL , MicroRNAs/genetics , Monocytes/metabolismABSTRACT
ß-glucan consumption is known for its beneficial health effects, but the mode of action is unclear. While humans and mice lack the required enzymes to digest ß-glucans, certain intestinal microbes can digest ß-glucans, triggering gut microbial changes. Curdlan, a particulate ß-glucan isolated from Alcaligenes faecalis, is used as a food additive. In this study we determined the effect of curdlan intake in mice on the intestinal microbiota and dextran sodium sulfate (DSS)-induced intestinal inflammation. The effect of curdlan on the human intestinal microbiota was assessed using i-screen, an assay for studying anaerobic microbial interactions. Mice received oral gavage with vehicle or curdlan for 14 days followed by DSS for 7 days. The curdlan-fed group showed reduced weight loss and colonic inflammation compared to the vehicle-fed group. Curdlan intake did not induce general microbiota community changes, although a specific Bifidobacterium, closely related to Bifidobacterium choerinum, was observed to be 10- to 100-fold more prevalent in the curdlan-fed group under control and colitis conditions, respectively. When tested in i-screen, curdlan induced a global change in the microbial composition of the healthy intestinal microbiota from a human. Overall, these results suggest that dietary curdlan induces microbiota changes that could reduce intestinal inflammation.
Subject(s)
Bifidobacterium/drug effects , Colitis/drug therapy , Diet/methods , Gastrointestinal Microbiome/drug effects , beta-Glucans/pharmacology , Animals , Colitis/chemically induced , Colon/metabolism , Dextran Sulfate , Humans , MiceABSTRACT
BACKGROUND AND AIMS: Tyrosine kinase 2 [TYK2] is required for the signalling of key cytokines in the pathogenesis of inflammatory bowel disease [IBD]. We assessed the efficacy of a novel selective TYK2 inhibitor [TYK2i] in experimental colitis, using pharmacological and genetic tools. METHODS: At onset of T cell transfer colitis, RAG1-/- mice received vehicle or TYK2i daily by oral gavage. T cells lacking TYK2 kinase activity [TYK2KE] were used to confirm selectivity of the inhibitor. To this end, RAG1-/- or RAG1-/-TYK2KE animals were transferred with either wild type [WT] or TYK2KE-CD45RBhigh colitogenic T cells. Loss of body weight, endoscopic disease, the disease activity index [DAI], and histopathology scores were recorded. Tissues were analysed ex vivo for lymphocyte populations by flow cytometry. The impact of TYK2 inhibition on human DC-T cell interactions were studied using autologous Revaxis specific T cell assays. RESULTS: TYK2i [70 mg/kg] prevented weight loss and limited endoscopic activity during T cell transfer colitis. TYK2i [70 mg/kg] decreased DAI. Whereas transfer of WT T cells into RAG-/-TYK2KE hosts induced colitis, TYK2KE T cells transferred into RAG1-/-TYK2KErecipients failed to do so. Ex vivo analysis showed a decrease in colon tissue Th1 cells and an increase in Th17 cells upon transfer of TYK2KE-CD45RBhigh cells. In human antigen-triggered T cells, TYK2i displayed reduced Th1 differentiation, similar to murine Th1 cells. CONCLUSIONS: Oral administration of TYK2i, as well as transfer of T cells lacking TYK2 activity, reduced human Th1 differentiation and ameliorated the course of murine T cell transfer colitis. We conclude that TYK2 is a promising drug target for the treatment of IBD.
Subject(s)
Administration, Oral , Adoptive Transfer , Inflammatory Bowel Diseases/enzymology , Inflammatory Bowel Diseases/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , TYK2 Kinase/antagonists & inhibitors , Animals , Cell Differentiation/immunology , Cytokines/immunology , Disease Models, Animal , Female , Flow Cytometry , Homeodomain Proteins , Humans , Mice , Signal Transduction , Th1 Cells/metabolismABSTRACT
BACKGROUND: Recent evidence demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagates in intestinal epithelial cells expressing Angiotensin-Converting Enzyme 2 (ACE2), implying that these cells represent an important entry site for the viral infection. Nicotinic receptors (nAChRs) have been put forward as potential regulators of inflammation and of ACE2 expression. As vagus nerve stimulation (VNS) activates nAChRs, we aimed to investigate whether VNS can be instrumental in affecting intestinal epithelial ACE2 expression. METHODS: By using publicly available datasets we qualified epithelial ACE2 expression in human intestine, and assessed gene co-expression of ACE2 and SARS-CoV-2 priming Transmembrane Serine Protease 2 (TMPRSS2) with nAChRs in intestinal epithelial cells. Next, we investigated mouse and human ACE2 expression in intestinal tissues after chronic VNS via implanted devices. RESULTS: We show co-expression of ACE2 and TMPRSS2 with nAChRs and α7 nAChR in particular in intestinal stem cells, goblet cells, and enterocytes. However, VNS did not affect ACE2 expression in murine or human intestinal tissue, albeit in colitis setting. CONCLUSIONS: ACE2 and TMPRSS2 are specifically expressed in epithelial cells of human intestine, and both are co-expressed with nAChRs. However, no evidence for regulation of ACE2 expression through VNS could be found. Hence, a therapeutic value of VNS with respect to SARS-CoV-2 infection risk through ACE2 receptor modulation in intestinal epithelia could not be established.
ABSTRACT
Irritable bowel syndrome (IBS) is a heterogenic, functional gastrointestinal disorder of the gut-brain axis characterized by altered bowel habit and abdominal pain. Preclinical and clinical results suggested that, in part of these patients, pain may result from fungal induced release of mast cell derived histamine, subsequent activation of sensory afferent expressed histamine-1 receptors and related sensitization of the nociceptive transient reporter potential channel V1 (TRPV1)-ion channel. TRPV1 gating properties are regulated in lipid rafts. Miltefosine, an approved drug for the treatment of visceral Leishmaniasis, has fungicidal effects and is a known lipid raft modulator. We anticipated that miltefosine may act on different mechanistic levels of fungal-induced abdominal pain and may be repurposed to IBS. In the IBS-like rat model of maternal separation we assessed the visceromotor response to colonic distension as indirect readout for abdominal pain. Miltefosine reversed post-stress hypersensitivity to distension (i.e. visceral hypersensitivity) and this was associated with differences in the fungal microbiome (i.e. mycobiome). In vitro investigations confirmed fungicidal effects of miltefosine. In addition, miltefosine reduced the effect of TRPV1 activation in TRPV1-transfected cells and prevented TRPV1-dependent visceral hypersensitivity induced by intracolonic-capsaicin in rat. Miltefosine may be an attractive drug to treat abdominal pain in IBS.
Subject(s)
Abdominal Pain/drug therapy , Antifungal Agents/administration & dosage , Irritable Bowel Syndrome/drug therapy , Phosphorylcholine/analogs & derivatives , Abdominal Pain/metabolism , Abdominal Pain/microbiology , Abdominal Pain/psychology , Animals , Female , Fungi/drug effects , Fungi/physiology , Gastrointestinal Microbiome/drug effects , Humans , Irritable Bowel Syndrome/metabolism , Irritable Bowel Syndrome/microbiology , Irritable Bowel Syndrome/psychology , Male , Maternal Deprivation , Mycobiome/drug effects , Phosphorylcholine/administration & dosage , Rats , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolismABSTRACT
BACKGROUND: Enhanced colorectal sensitivity (i.e. visceral hypersensitivity) is thought to be a pathophysiological mechanism in irritable bowel syndrome (IBS). In healthy men a circadian variation in rectal perception to colonic distention was described. Disturbed day and night rhythms, which occur in shift work and trans meridian flights, are associated with the prevalence of IBS. This raises the question whether disruptions of circadian control are responsible for the observed pathology in IBS. Prior to investigating altered rhythmicity in relation to visceral hypersensitivity in a rat model for IBS, it is relevant to establish whether normal rats display circadian variation similar to healthy men. METHODOLOGY AND FINDINGS: In rodents colorectal distension leads to reproducible contractions of abdominal musculature. We used quantification of this so called visceromotor response (VMR) by electromyography (EMG) to assess visceral sensitivity in rats. We assessed the VMR in normal male Long Evans rats at different time points of the light/dark cycle. Although a control experiment with male maternal separated rats confirmed that intentionally inflicted (i.e. stress induced) changes in VMR can be detected, normal male Long Evans rats showed no variation in VMR along the light/dark cycle in response to colorectal distension. CONCLUSIONS: In the absence of a daily rhythm of colorectal sensitivity in normal control rats it is not possible to investigate possible aberrancies in our rat model for IBS.
ABSTRACT
ß-Glucans have beneficial health effects due to their immune modulatory properties. Oral administration of ß-glucans affects tumour growth, microbial infection, sepsis, and wound healing. We hypothesized that pre-treatment with orally delivered soluble and particulate ß-glucans could ameliorate the development of aggravate dextran sulfate sodium (DSS) induced intestinal inflammation. To study this, mice were orally pre-treated with ß-glucans for 14 days. We tested curdlan (a particulate ß-(1,3)-glucan), glucan phosphate (a soluble ß-(1,3)-glucan), and zymosan (a particle made from Saccharomyces cerevisiae, which contains around 55% ß-glucans). Weight loss, colon weight, and feces score did not differ between ß-glucan and vehicle treated groups. However, histology scores indicated that ß-glucan-treated mice had increased inflammation at a microscopic level suggesting that ß-glucan treatment worsened intestinal inflammation. Furthermore, curdlan and zymosan treatment led to increased colonic levels of inflammatory cytokines and chemokines, compared to vehicle. Glucan phosphate treatment did not significantly affect cytokine and chemokine levels. These data suggest that particulate and soluble ß-glucans differentially affect the intestinal immune responses. However, no significant differences in other clinical colitis scores between soluble and particulate ß-glucans were found in this study. In summary, ß-glucans aggravate the course of dextran sulfate sodium (DSS)-induced intestinal inflammation at the level of the mucosa.
Subject(s)
Colitis/metabolism , Colon/drug effects , Inflammation/metabolism , Intestinal Mucosa/drug effects , beta-Glucans/adverse effects , Administration, Oral , Animals , Chemokines/metabolism , Colitis/chemically induced , Colitis/pathology , Cytokines/metabolism , Dextran Sulfate , Glucans/adverse effects , Inflammation/chemically induced , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Mice, Inbred C57BL , Zymosan/adverse effectsABSTRACT
BACKGROUND: The histamine-1 receptor (H1R) antagonist ketotifen increased the threshold of discomfort in hypersensitive IBS patients. The use of peripherally restricted and more selective H1R antagonists may further improve treatment possibilities. We examined the use of fexofenadine and ebastine to reverse post-stress visceral hypersensitivity in maternally separated rats. METHODS: The visceromotor response to colonic distension was assessed in adult maternally separated and nonhandled rats pre- and 24 hours post water avoidance. Subsequently rats were treated with vehicle alone or different dosages of fexofenadine (1.8 and 18 mg/kg) or ebastine (0.1 and 1.0 mg/kg) and re-evaluated. Colonic tissue was collected to assess relative RMCP-2 and occludin expression levels by Western blot and histamine-1 receptor by RT-qPCR. ß-hexosaminidase release by RBL-2H3 cells was used to establish possible mast cell stabilizing properties of the antagonists. KEY RESULTS: Water avoidance only induced enhanced response to distension in maternally separated rats. This response was reversed by 1.8 and 18 mg/kg fexofenadine. Reversal was also obtained by 1.0 but not 0.1 mg/kg ebastine. RMCP-2 expression levels were comparable in these two ebastine treatment groups but occludin was significantly higher in 1.0 mg/kg treated rats. There were no differences in histamine-1 receptor expression between nonhandled and maternally separated rats. Fexofenadine but not ebastine showed mast cell stabilizing quality. CONCLUSIONS: Our results indicate that the peripherally restricted 2(nd) generation H1-receptor antagonists fexofenadine and ebastine are capable of reversing post stress visceral hypersensitivity in rat. These data justify future IBS patient trials with these well tolerated compounds.