RELM-β Expression Research Articles

Supplemental Psyllium Fiber Increases Antimicrobial Proteins via the Tuft Cell-ILC2 Circuit and Type II Immune Response in the Mouse Small Intestine

Dietary fibers regulate intestinal barrier function; however, the precise mechanisms remain unclear. This study investigated the effects of psyllium fibers on antimicrobial protein expression, focusing on the type II immunity and tuft cell-group 2 innate lymphoid cell (ILC2) circuit in the small intestine of the mouse. Supplemental psyllium fiber upregulated antimicrobial proteins, such as small proline-rich protein 2A (SPRR2A) and resistin-like beta (RELMβ), in mouse small intestine, evidently affecting cecal microbiota composition. The psyllium fibers also increased the RNA and protein expression of molecules related to ILC2 and tuft cells, such as IL-13, IL-25, DCLK1, Gfi-1b, SH2 domain-containing protein 3C, and Spi-B. In addition, ILC2 inhibitor (disulfiram) and bitter taste receptor blocker administration reduced psyllium-induced SPRR2A and RELMβ expression. Collectively, psyllium supplementation upregulates antimicrobial proteins such as SPRR2A and RELMß via the type II immune response and tuft cell-ILC2 circuit in the mouse small intestine.

Abstract 5669: Rare germline variants on SI gene and the possibility of colorectal cancer development

Abstract Research on cancer and germline variants has been ongoing. The germline variant in CPGs has been linked to cancer development. BRCA 1/2 is a typical case and oncometabolite production due to germline variants of the FH gene can affect the cancer progression. Among the genes related to IEMs, SI (Sucrase-isomaltase) is an enzyme that converts sucrose into glucose and fructose to allow normal metabolism in the small intestine. In addition, SI is highly specifically expressed in the gastrointestinal tract (nTPM = 435.9). So, we focused on and hypothesized the biological traits of the SI gene and alteration caused by germline variants could affect colorectal cancer progression. We used the public cancer databases PCAWG and TCGA and the healthy cohorts 1000G. We confirmed carriers with SI germline variants are the top gene with a high prevalence of IEM-related genes. We also established the hypothesis the SI gene may match “Knudson’s Two-hit hypothesis”. Depending on whether germline variants in IEM-related genes or not, we performed regression and utilized LOH (Loss of heterozygosity) information for each cancer sample from the public database. In COAD and STAD which are in the gastrointestinal tract, SI was confirmed to have a high tendency for LOH according to germline prevalence. Next, we performed DEG analysis and found SI germline carriers had upregulated genes related to the IFIT (Interferon-induced proteins with tetratricopeptide repeats) family. STRING network analysis also showed significance in pathways associated with “Interferon alpha/beta signaling (FDR = 0.00058)”, “Antiviral defense (FDR = 0.0016)”, “Innate immunity (FDR = 0.0317)”, and “Tetratricopeptide repeats (FDR = 0.0413)”. The following GSEA revealed that “KEGG_CYTOSOLIC DNA-SENSING PATHWAY” and “HALLMARK_INTERFERON_ALPHA_RESPONSE” were enriched in the SI germline carriers. In summary, it can be suggested immune regulation caused by SI germline variants may lead to cancer progression. We executed a follow-up analysis using the genes with low expression levels to the SI germline carriers. Among the down-regulated genes in the DEG, REG4 (Regenerating Family Member 4) and RELMβ (Resistin-Like Molecule Beta) genes were confirmed to express and function specifically only in the gastrointestinal tract. RELMβ is essential for preserving energy balance and glucose metabolism. As a validation at the protein level, we conducted IHC (Immunohistochemistry) on tissues from Korean colorectal cancer patients and confirmed lower REG4 and RELMβ expression were associated with poor prognosis. Our results suggest that SI germline carriers may cause colorectal cancer by affecting the immune system due to decreased SI enzyme function. Additionally, the decreased REG4 and RELMβ expression levels are associated with the SI germline variant and may affect cancer progression. Further analyses and experimental validations are still required. Citation Format: Seokhyeon Kim, Jeong Mo Bae, Youngil Koh, Sung-Soo Yoon. Rare germline variants on SI gene and the possibility of colorectal cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5669.

Effect of Lifelong Exposure to Dietary Plant and Marine Sources of n-3 Polyunsaturated Fatty Acids on Morphologic and Gene Expression Biomarkers of Intestinal Health in Early Life.

Altered intestinal health is also associated with the incidence and severity of many chronic inflammatory conditions, which could be attenuated via dietary n-3 PUFA interventions. However, little is known about the effect of lifelong exposure to n-3 PUFA from plant and marine sources (beginning in utero via the maternal diet) on early life biomarkers of intestinal health. Harems of C57Bl/6 mice were randomly assigned to one of three isocaloric AIN-93G modified diets differing in their fat sources consisting of the following: (i) 10% safflower oil (SO, enriched in n-6 PUFA), (ii) 3% flaxseed oil + 7% safflower oil (FX, plant-based n-3 PUFA-enriched diet), or (iii) 3% menhaden fish oil + 7% safflower oil (MO, marine-based n-3 PUFA-enriched diet). Mothers remained on these diets throughout pregnancy and offspring (n = 14/diet) continued on the same parental diet until termination at 3 weeks of age. In ileum, villi:crypt length ratios were increased in both the FX and MO dietary groups compared to SO (p < 0.05). Ileum mRNA expression of critical intestinal health biomarkers was increased by both n-3 PUFA-enriched diets including Relmβ and REG3γ compared to SO (p < 0.05), whereas only the FX diet increased mRNA expression of TFF3 and Muc2 (p < 0.05) and only the MO diet increased mRNA expression of ZO-1 (p < 0.05). In the proximal colon, both the FX and MO diets increased crypt lengths compared to SO (p < 0.05), whereas only the MO diet increased goblet cell numbers compared to SO (p < 0.05). Further, the MO diet increased proximal colon mRNA expression of Relmβ and REG3γ (p < 0.05) and both MO and FX increased mRNA expression of Muc2 compared to SO (p < 0.05). Collectively, these results demonstrate that lifelong exposure to dietary n-3 PUFA, beginning in utero, from both plant and marine sources, can support intestinal health development in early life. The differential effects between plant and marine sources warrants further investigation for optimizing health.

The Mechanisms of Resistin-Like Molecule-β-Mediated Airway Inflammation in Chronic Obstructive Pulmonary Disease via Autophagy.

The role of irreversible airway inflammatory damage in chronic obstructive pulmonary disease (COPD) progression is evident. Autophagy is an essential process in the cellular material metabolic cycle, and a family of resistant vegetative molecules may be involved in the COPD autophagic process. In this study, we investigated the mechanism of resistin-like molecule β (RELMβ) in COPD smoking-induced autophagy. Firstly, the expression differences of RELMβ and autophagy markers between COPD and control groups were analyzed in the Gene Expression Omnibus (GEO) datasets and clinical specimens. Secondly, in vitro and in vivo experiments were conducted using immunoblotting, immunofluorescence, immunohistochemistry, and other methods to investigate the mechanism by which RELMβ promotes airway inflammation through autophagy in a cigarette smoke extract-induced 16HBE cell inflammation model and a cigarette smoke-induced COPD-like mouse model. In addition, immunoprecipitation was used to analyze the binding of RELMβ to the membrane protein TLR4. The expression of RELMβ and autophagy genes p62 and LC3B in lung tissue of COPD patients was significantly increased. RELMβ can mediate the activation of autophagy in 16HBE cells, and through autophagy, it increases the expression of inflammatory cytokines in a cigarette smoke extract-induced 16HBE cell inflammation model. RELMβ promotes cigarette smoke-induced COPD-like mouse airway inflammation through autophagy, and RELMβ can mediate signal transduction through the cell membrane receptor TLR4. The RELMβ binds to TLR4 to encourage signal transduction and that RELMβ can promote inflammation in smoky COPD lungs through autophagy.

Resistin-like Molecule α and Pulmonary Vascular Remodeling: A Multi-Strain Murine Model of Antigen and Urban Ambient Particulate Matter Co-Exposure.

Pulmonary hypertension (PH) has a high mortality and few treatment options. Adaptive immune mediators of PH in mice challenged with antigen/particulate matter (antigen/PM) has been the focus of our prior work. We identified key roles of type-2- and type-17 responses in C57BL/6 mice. Here, we focused on type-2-response-related cytokines, specifically resistin-like molecule (RELM)α, a critical mediator of hypoxia-induced PH. Because of strain differences in the immune responses to type 2 stimuli, we compared C57BL/6J and BALB/c mice. A model of intraperitoneal antigen sensitization with subsequent, intranasal challenges with antigen/PM (ovalbumin and urban ambient PM2.5) or saline was used in C57BL/6 and BALB/c wild-type or RELMα-/- mice. Vascular remodeling was assessed with histology; right ventricular (RV) pressure, RV weights and cytokines were quantified. Upon challenge with antigen/PM, both C57BL/6 and BALB/c mice developed pulmonary vascular remodeling; these changes were much more prominent in the C57BL/6 strain. Compared to wild-type mice, RELMα-/- had significantly reduced pulmonary vascular remodeling in BALB/c, but not in C57BL/6 mice. RV weights, RV IL-33 and RV IL-33-receptor were significantly increased in BALB/c wild-type mice, but not in BALB/c-RELMα-/- or in C57BL/6-wild-type or C57BL/6-RELMα-/- mice in response to antigen/PM2.5. RV systolic pressures (RVSP) were higher in BALB/c compared to C57BL/6J mice, and RELMα-/- mice were not different from their respective wild-type controls. The RELMα-/- animals demonstrated significantly decreased expression of RELMβ and RELMγ, which makes these mice comparable to a situation where human RELMβ levels would be significantly modified, as only humans have this single RELM molecule. In BALB/c mice, RELMα was a key contributor to pulmonary vascular remodeling, increase in RV weight and RV cytokine responses induced by exposure to antigen/PM2.5, highlighting the significance of the genetic background for the biological role of RELMα.

A172 THE LACK OF CHROMOGRANIN A PROTECTS THE COLONIC EPITHELIAL BARRIER FUNCTIONS FROM COLITIS IN MALE MICE AND EXACERBATES COLITIS IN FEMALE MICE

Abstract Background Ulcerative colitis (UC) is associated with compromised mucosal barrier function and colonic epithelial repair in a sex-dependent manner. Chromogranin A (CHGA), a pro-hormone, correlates positively with UC disease severity. In male mice, deletion of CHGA has been shown to decrease the inflammatory process; however, the effect of CHGA on mucosal barrier function and colonic epithelial repair between males and females is unknown. Purpose We investigated whether the lack of CHGA modulates gut barrier function, mucosa integrity, and colonic epithelial repair between males and females in a mice model of colitis. Method Male and female wild-type (WT) and CHGA (CHGA-/-) deficient mice (13-17 weeks old) were given 5% dextran sulfate sodium (DSS) to induce colitis or water for 5-days (n=5-8 mice per group). The disease activity index (DAI) was assessed. Colons were collected, and tumor necrosis factor (TNF)-α and IL-25 concentrations were measured by ELISA. Expression of structural and functional markers specific to epithelial cells, namely, colonocytes (Na-K-Cl cotransporter [Nkcc]1), goblet cells function (resistin-like molecule [Relm]β), and mucin [MUC]2) and stem cells (reserve Hopx, fast-cycling Lgr5, and fetal-like Ly6a cells) were evaluated by qRT-PCR. Result(s) Colitic male CHGA-/- did not show significant changes in DAI compared to WT mice. Conversely, female CHGA-/- mice demonstrated a trend toward higher susceptibility to colitis compared to female WT mice with increased weight loss and bleeding. This was associated with elevated levels of colonic TNF-α and IL-25 (p&amp;lt;0.05) in CHGA-/- females compared to CHGA-/- males. TNF-α levels were not different between female groups at baseline and during colitis. While colitic CHGA-/- female had elevated Relmβ expression (p&amp;lt;0.01) compared to WT mice. No significative change was noted in Relmβ expression between female WT mice at baseline and during colitis. Similarly, Nkcc1 and Muc2 expression was not different between female groups. By contrast, male CHGA-/- were less susceptible to colitis than male WT mice with elevated Nkcc1and a lower Relmβ and Muc2 expression (p&amp;lt;0.01). In colitis, expression of stem cell markers, Hopx and Lgr5, was markedly reduced in all groups, while male WT, CHGA-/-, and female WT had elevated Ly6a expression. However, the magnitude of Hopx and Ly6a expression was associated with sex. Thus, colitic male CHGA-/- mice had a higher Hopx expression than male WT and female CHGA-/ - mice, with a lower reduction of 1.9 compared to 4.9 and 6.6, respectively (p&amp;lt;0.05, 0.01, and 0.0001). While colitic male CHGA-/- mice had elevated Ly6a expression (p&amp;lt;0.05) in contrast to female CHGA-/- mice (p=0.5). The magnitude of the decrease in Lgr5 expression was not different between all groups. Conclusion(s) In the absence of CHGA, male mice preserved their colonic mucosa integrity and repair potential, while female mice suffered significant loss of mucosa integrity and repair potential during colitis. Disclosure of Interest None Declared

The IL-25-dependent tuft cell circuit driven by intestinal helminths requires macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is a key innate immune mediator with chemokine- and cytokine-like properties in the inflammatory pathway. While its actions on macrophages are well-studied, its effects on other cell types are less understood. Here we report that MIF is required for expansion of intestinal tuft cells during infection with the helminth Nippostrongylus brasiliensis. MIF-deficient mice show defective innate responses following infection, lacking intestinal epithelial tuft cell hyperplasia or upregulation of goblet cell RELMβ, and fail to expand eosinophil, type 2 innate lymphoid cell (ILC2) and macrophage (M2) populations. Similar effects were observed in MIF-sufficient wild-type mice given the MIF inhibitor 4-IPP. MIF had no direct effect on epithelial cells in organoid cultures, and MIF-deficient intestinal stem cells could generate tuft cells in vitro in the presence of type 2 cytokines. In vivo the lack of MIF could be fully compensated by administration of IL-25, restoring tuft cell differentiation and goblet cell expression of RELM-β, demonstrating its requirement upstream of the ILC2-tuft cell circuit. Both ILC2s and macrophages expressed the MIF receptor CXCR4, indicating that MIF may act as an essential co-factor on both cell types to activate responses to IL-25 in helminth infection. [Display omitted]

Upregulation of antimicrobial peptide expression in slc26a3-/- mice with colonic dysbiosis and barrier defect

ABSTRACT Genetic defects in SLC26A3 (DRA), an intestinal Cl−/HCO3 − exchanger, result in congenital chloride diarrhea (CLD), marked by lifelong acidic diarrhea and a high risk of inflammatory bowel disease. Slc26a3 −/− mice serve as a model to understand the pathophysiology of CLD and search for treatment options. This study investigates the microbiota changes in slc26a3 −/− colon, the genotype-related causes for the observed microbiota alterations, its inflammatory potential, as well as the corresponding host responses. The luminal and the mucosa-adherent cecal and colonic microbiota of cohoused slc26a3 −/− and wt littermates were analyzed by 16S rRNA gene sequencing. Fecal microbiota transfer from cohoused slc26a3 −/− and wt littermates to germ-free wt mice was performed to analyze the stability and the inflammatory potential of the communities. The cecal and colonic luminal and mucosa-adherent microbiota of slc26a3 −/− mice was abnormal from an early age, with a loss of diversity, of short-chain fatty acid producers, and an increase of pathobionts. The transfer of slc26a3 −/− microbiota did not result in intestinal inflammation and the microbial diversity in the recipient mice normalized over time. A strong increase in the expression of Il22, Reg3β/γ, Relmβ, and other proteins with antimicrobial functions was observed in slc26a3 −/− colon from juvenile age, while the mucosal and systemic inflammatory signature was surprisingly mild. The dysbiotic microbiota, low mucosal pH, and mucus barrier defect in slc26a3 −/− colon are accompanied by a stark upregulation of the expression of a panel of antimicrobial proteins. This may explain the low inflammatory burden in the gut of these mice.

The Inflammatory Response Induced by RELMβ Upregulates IL-8 and IL-1β Expression in Bronchial Epithelial Cells in COPD.

PurposeChronic obstructive pulmonary disease (COPD) is associated with a complex inflammatory regulatory network. Resistin-like molecule β (RELMβ) is highly expressed in the lungs of COPD patients. We aimed to investigate the proinflammatory effect of RELMβ on airway epithelial cells in COPD.MethodsFirst, a GEO dataset was used to analyze the expression of the RELMβ gene in the COPD and control groups as well as the protein levels of RELMβ in the sera of outpatients with COPD and normal control subjects in our hospital. We also stimulated 16HBE bronchial epithelial cells with recombinant RELMβ protein and analyzed the expression of IL-8 and IL-1β. We upregulated and downregulated the gene expression of RELMβ in 16HBE cells and analyzed the expression of the inflammatory cytokines IL-8 and IL-1β. In addition, we also examined the mechanism by which the p38 MAPK signaling pathway contributed to the regulation of IL-8 and IL-1β expression by RELMβ.ResultsRELMβ expression was increased in COPD tissues in different data sets and in the serum of COPD patients in our hospital. IL-8 and IL-1β expression was also increased in COPD tissues with high RELMβ gene expression in different data sets. The RELMβ gene was mainly related to inflammatory factors and inflammatory signaling pathways in the PPI regulatory network. Experiments at the cellular level showed that RELMβ promoted the expression of the inflammatory cytokines IL-8 and IL-1β, and this regulation was mediated by the p38 MAPK signaling pathway.ConclusionRELMβ can promote the expression of the inflammatory cytokines IL-8 and IL-1β in bronchial epithelial cells of patients with COPD and exert inflammatory effects. RELMβ may be a potential target for the treatment of COPD.

Navy Bean Supplementation in Established High-Fat Diet-Induced Obesity Attenuates the Severity of the Obese Inflammatory Phenotype.

Cooked common beans (Phaseolus vulgaris) improve intestinal health in lean mice and attenuate intestinal dysbiosis and inflammation when consumed concurrent with obesity development. We determined the effects of a high-fat (HF) bean supplemented diet in mice with established obesity (induced by 12 weeks of HF diet (60% fat as kcal)) compared to obese mice consuming a HF or low-fat (LF) weight loss control diet. Obese C57BL/6 male mice remained consuming HF for eight weeks or were randomly switched from HF to an isocaloric HF with 15.7% cooked navy bean powder diet (HF→HFB) or LF (11% fat as kcal; HF→LF) (n = 12/group). HF→HFB improved the obese phenotype, including (i) fecal microbiome (increased Prevotella, Akkermansia muciniphila, and short-chain fatty acid levels), (ii) intestinal health (increased ZO-1, claudin-2, Muc2, Relmβ, and Reg3γ expression), and (iii) reduced adipose tissue (AT) inflammatory proteins (NFκBp65, STAT3, IL-6, MCP-1, and MIP-1α), versus HF (p < 0.05). Conversely, HF→LF reduced body weight and circulating hormones (leptin, resistin, and PAI-1) versus HF and HF→HFB (p < 0.05); however, AT inflammation and intestinal health markers were not improved to the same degree as HF→HFB (p < 0.05). Despite remaining on a HF obesogenic diet, introducing beans in established obesity improved the obese phenotype (intestinal health and adipose inflammation) more substantially than weight loss alone.

Inhibition of RELM-β prevents hypoxia-induced overproliferation of human pulmonary artery smooth muscle cells by reversing PLC-mediated KCNK3 decline

AimsAlthough resistin-like molecule β (RELM-β) is involved in the pathological processes of various lung diseases, such as pulmonary inflammation, asthma and fibrosis, its potential roles in hypoxic pulmonary arterial hypertension (PAH) remain largely unknown. The study aims to investigate whether RELM-β contributes to hypoxia-induced excessive proliferation of human pulmonary artery smooth muscle cells (PASMCs) and to explore the potential mechanisms of this process. Main methodsHuman PASMCs were exposed to normoxia or hypoxia (1% O2) for 24 h. siRNA targeting RELM-β was transfected into cells. Protein levels of KCNK3, RELM-β, pSTAT3 and STAT3 were determined by immunoblotting. The translocation of NFATc2 and expression of KCNK3 were visualized by immunofluorescence. 5-ethynyl-2′-deoxyuridine assays and cell counting kit-8 assays were performed to assess the proliferation of PASMCs. Key findings(1) Chronic hypoxia significantly decreased KCNK3 protein levels while upregulating RELM-β protein levels in human PASMCs, which was accompanied by excessive proliferation of cells. (2) RELM-β could promote human PASMCs proliferation and activate the STAT3/NFAT axis by downregulating KCNK3 protein under normoxia. (3) Inhibition of RELM-β expression effectively prevented KCNK3-mediated cell proliferation under hypoxia. (4) Phospholipase C (PLC) inhibitor U-73122 could not only prevent the hypoxia/RELM-β-induced decrease in KCNK3 protein, but also inhibit the enhanced cell viability caused by hypoxia/RELM-β. (5) Both hypoxia and RELM-β could downregulate membrane KCNK3 protein levels by enhancing endocytosis. SignificanceRELM-β activation is responsible for hypoxia-induced excessive proliferation of human PASMCs. Interfering with RELM-β may alleviate the progression of hypoxic PAH by upregulating PLC-dependent KCNK3 expression.

Helicobacter pylori Infection Facilitates the Expression of Resistin-like Molecule Beta in Gastric Carcinoma and Precursor Lesions

Helicobacter pylori (H. pylori) was reported to be associated with gastric carcinogenesis. Resistin-like molecule beta (RELMβ), a recently described goblet cell-specific protein, was demonstrated to aberrantly express in gastric cancer and correlated with its clinicopathological features. This study aimed to examine the association between H. pylori and RELMβ expression in gastric carcinoma and precursor lesions. H. pylori infection and RELMβ expression were immunohistochemically evaluated in gastric biopsies from 230 patients. The biopsies consisted of normal gastric mucosa (n=20), mucosa with chronic gastritis (n=41), intestinal metaplasia (n=42), dysplasia (n=31), intestinal-type adenocarcinoma (n=56), and diffuse-type adenocarcinoma (n=40). RELMβ expression was measured in gastric biopsies after H. pylori eradication therapy in a subgroup of 32 patients. Cultured gastric cancer cell line SGC-7901 was infected with H. pylori strains, and RELMβ expression was detected by reverse transcription PCR, real-time PCR and Western blotting. Higher RELMβ immunoreactivity was observed in H. pylori-positive intestinal metaplasia (P=0.003), dysplasia (P=0.032), intestinal-type (P=0.037) and diffuse-type adenocarcinomas (P=0.001) than in H. pylori-negative specimens. Expression rates of RELMβ in dysplasia (P=0.005), intestinal-type adenocarcinoma (P<0.001), and diffuse-type adenocarcinoma (P=0.001) were significantly correlated with the grade of H. pylori density. In addition, H. pylori eradication reduced the RELMβ intensity in intestinal metaplasia (P=0.001). Infection of gastric cancer SGC-7901 cells with cag pathogenicity island (PAI)-positive H. pylori TN2, but not with its PAI totally deleted mutant (TN2-ΔPAI) for 4-8 h, resulted in enhanced protein and transcript levels of RELMβ (P<0.05). In summary, our study suggested that H. pylori infection facilitated the expression of RELMβ in gastric garcinoma and precursor lesions.

Red lentil supplementation reduces the severity of dextran sodium sulfate-induced colitis in C57BL/6 male mice

Abstract Lentils (Lens culinaris L.) are a protein-rich plant food, also enriched in fibre and phenolic compounds that may reduce intestinal-associated disease risk. Male C57Bl/6 mice were pre-fed a basal diet (BD) or isocaloric 20% red lentil-supplemented diet (LD) for 3 weeks and acute colitis was induced via dextran sodium sulfate (DSS, 2% w/v in drinking water) for 5 days. LD-fed mice exhibited reduced (i) clinical symptoms, (ii) colon histological damage, and (iii) colonic pro-inflammatory cytokine levels. Additionally, biomarkers of improved colon epithelial barrier integrity and mucosal repair mediators were increased in LD mice (e.g. colonic IL-22, Relmβ, and occludin expression, and serum lipopolysaccharide binding protein). Collectively, the severity of the DSS-induced acute colitis phenotype in mice was attenuated by red lentil dietary supplementation, indicating that lentils may serve as a potential adjuvant dietary therapy in patients with colitis-associated diseases to help limit colonic inflammation and restore barrier function.

LPS-squalene interaction on D-galactose intestinal absorption.

The dynamic and complex interactions between enteric pathogens and the intestinal epithelium often lead to disturbances in the intestinal barrier, altered fluid, electrolyte, and nutrient transport and can produce an inflammatory response. Lipopolysaccharide (LPS) is a complex polymer forming part of the outer membrane of Gram-negative bacteria. On the other hand, squalene is a triterpene present in high levels in the extra-virgin olive oil that has beneficial effects against several diseases and it has also anti-oxidant and anti-inflammatory properties. The aim of this work was to study whether the squalene could eliminate the LPS effect on D-galactose intestinal absorption in rabbits and Caco-2 cells. The results have shown that squalene reduced the effects of LPS on sugar absorption. High LPS doses increased D-galactose uptake through via paracellular but also decreased the active sugar transport because the SGLT1 levels were diminished. However, the endotoxin effect on the paracellular way seemed to be more important than on the transcellular route. At the same time, an increased in RELM-β expression was observed. This event could be related to inflammation and cause a decrease in SGLT1 levels. In addition, MLCK protein is also increased by LPS which could lead to an increase in sugar transport through tight junctions. At low doses, the LPS could inhibit SGLT1 intrinsic activity. Bioinformatic studies by docking confirm the interaction between LPS-squalene as well as occur through MLCK and SGLT-1 proteins.

A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection.

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection.

TGF-β1-induced SMAD2/3/4 activation promotes RELM-β transcription to modulate the endothelium-mesenchymal transition in human endothelial cells.

Endothelial-to-mesenchymal transition (EndMT), which is characterized by increased proliferation, migration and invasion of endothelial cells, increased expression of mesenchymal markers and reduced expression of endothelial markers, has been reported to be closely related to the pathogenesis of several diseases, including pulmonary fibrosis. Resistin-like molecule-β (RELM-β), also known as "found in inflammatory zone 2″ (FIIZ2), plays an essential role in airway remodeling and pulmonary fibrosis; however, its role and mechanism in EndMT remain unclear. Herein, we used TGF-β1-induced EndMT cell model in human umbilical vein endothelial cells (HUVECs) and human primary pulmonary artery endothelial cells (HPAECs) to investigate the function and mechanism of RELM-β in TGF-β1-induced EndMT in endothelial cell lines. We found that TGF-β1 stimulation significantly upregulated RELM-β expression; RELM-β knockdown could attenuate TGF-β1-induced cell proliferation and migration of endothelial cell lines and changes in protein levels of EndMT markers. SB432542, an inhibitor of SMADs, could partially reverse TGF-β1-induced RELM-β expression, endothelial cell migration and changes in EndMT marker protein levels. SMADs complex exerted its effects through SMAD2/3/4 complex mediating RELM-β transcription. In conclusion, TGF-β1 induces RELM-β transcription to promote EndMT in HUVECs and HPAECs through activation of SMAD2/3/4; blocking SMADs-mediated RELM-β transcription might ameliorate TGF-β1-induced EndMT in endothelial cells.

Chickpea supplementation prior to colitis onset reduces inflammation in dextran sodium sulfate-treated C57Bl/6 male mice.

The potential for a chickpea-supplemented diet (rich in fermentable nondigestible carbohydrates and phenolic compounds) to modify the colonic microenvironment and attenuate the severity of acute colonic inflammation was investigated. C57Bl/6 male mice were fed a control basal diet or basal diet supplemented with 20% cooked chickpea flour for 3 weeks prior to acute colitis onset induced by 7-day exposure to dextran sodium sulfate (DSS; 2% w/v in drinking water) and colon and serum levels of inflammatory mediators were assessed. Despite an equal degree of DSS-induced epithelial barrier histological damage and clinical symptoms between dietary groups, biomarkers of the ensuing inflammatory response were attenuated by chickpea pre-feeding, including reduced colon tissue activation of nuclear factor kappa B and inflammatory cytokine production (tumor necrosis factor alpha and interleukin (IL)-18). Additionally, colon protein expression of anti-inflammatory (IL-10) and epithelial repair (IL-22 and IL-27) cytokines were increased by chickpea pre-feeding. Furthermore, during acute colitis, chickpea pre-feeding increased markers of enhanced colonic function, including Relmβ and IgA gene expression. Collectively, chickpea pre-feeding modulated the baseline function of the colonic microenvironment, whereby upon induction of acute colitis, the severity of the inflammatory response was attenuated.

Resistin-Like Molecule Beta (RELM-β) Regulates Proliferation of Human Diabetic Nephropathy Mesangial Cells via Mitogen-Activated Protein Kinases (MAPK) Signaling Pathway.

BackgroundResistin-like molecule beta (RELM-β) has been reported to be associated with diabetic nephropathy (DN). However, the role of RELM-β in DN is poorly understood. This study was conducted to delineate the underlying mechanisms of action and to investigate the role of RELM-β in the primitive development of DN via MAPK signaling pathways.Material/MethodsLentivirus-mediated vectors and RNAi technology were used to establish the model of RELM-β up-regulated and down-regulated expression in human mesangial cells (HMCs). The proliferation of HMCs was detected through CCK-8 method. The cell cycle and cell proliferation of HMCs was detected through flow cytometry. The MAPKs pathway protein activity was detected through Western blotting.ResultsThe HMCs with up-regulated and down-regulated expression of RELM-β increased or decreased significantly at 2–3 days. The HMCs with high glucose intervention reversed the proliferation inhibition. The HMCs with exogenous glucose or RELM-β protein intervention partially reversed the cell cycle inhibition. Among the MAPKs pathway, the phosphorylation activity of p38MAPK and JNK increased or decreased and ERK1/2 did not change in the overexpression or inhibition of RELM-β. The p38 MAPK pathway inhibitor SB202190 significantly inhibited the proliferation of HMCs caused by overexpression of RELM-β. Up-regulated expression of RELM-β induced the phosphorylation of p38 MAPK, JNK in HMCs and promoted HMCs proliferation and participated in early DN through the MAPKs pathway.ConclusionsThe results provide evidence that RELM-β is a potential molecular target for the treatment of DN.

Lactobacillus salivarius reverse diabetes-induced intestinal defense impairment in mice through non-defensin protein

Altered intestinal microbiota and subsequent endotoxemia play pathogenic roles in diabetes. We aimed to study the mechanisms of intestinal defense impairment in type 1 diabetes and the effects of Lactobacillus salivarius as well as fructooligosaccharides (FOS) supplementation on diabetes-induced bacterial translocation. Alterations in the enteric microbiome, expression of mucosal antibacterial proteins and bacteria-killing activity of the intestinal mucosa in streptozotocin (STZ)-induced diabetic mice and Ins2Akita mice were investigated. The effects of dead L. salivarius (2×108CFU/ml) and FOS (250 mg per day) supplementation for 1 week on endotoxin levels and Klebsiella pneumoniae translocation were also examined. Finally, germ-free mice were cohoused with wild-type or Ins2Akita mice for 2 weeks to examine the contribution of microbiota on the antibacterial protein expression. STZ-induced diabetic mice developed intestinal defense impairment as demonstrated by decreased mucosal bacteria-killing activity; reduction of non-defensin family proteins, such as Reg3β, Reg3γ, CRP-ductin and RELMβ, but not the defensin family proteins; and increased bacterial translocation. Intestinal bacteria overgrowth, enteric dysbiosis and increased intestinal bacterial translocation, particularly pathogenic K. pneumoniae in STZ-induced diabetic mice and Ins2Akita mice, were noted. Treating diabetic mice with dead L. salivarius or FOS reversed enteric dysbiosis, restored mucosal antibacterial protein and lessened endotoxin levels as well as K. pneumoniae translocation. Moreover, germ-free mice cohoused with wild-type mice demonstrated more intestinal Reg3β and RELMβ expression than those cohoused with Ins2Akita mice. These results indicate that hyperglycemia induces enteric dysbiosis, reduction of non-defensin proteins as well as bacteria-killing activity of the intestinal mucosa and intestinal defense impairment. Reversal of enteric dysbiosis with dead L. salivarius or FOS supplementation decreases diabetes-induced K. pneumoniae translocation and endotoxin levels through the induction of non-defensin proteins.

Involvement of resistin-like molecule β in the development of methionine-choline deficient diet-induced non-alcoholic steatohepatitis in mice

Resistin-like molecule β (RELMβ) reportedly has multiple functions including local immune responses in the gut. In this study, we investigated the possible contribution of RELMβ to non-alcoholic steatohepatitis (NASH) development. First, RELMβ knock-out (KO) mice were shown to be resistant to methionine-choline deficient (MCD) diet-induced NASH development. Since it was newly revealed that Kupffer cells in the liver express RELMβ and that RELMβ expression levels in the colon and the numbers of RELMβ-positive Kupffer cells were both increased in this model, we carried out further experiments using radiation chimeras between wild-type and RELMβ-KO mice to distinguish between the contributions of RELMβ in these two organs. These experiments revealed the requirement of RELMβ in both organs for full manifestation of NASH, while deletion of each one alone attenuated the development of NASH with reduced serum lipopolysaccharide (LPS) levels. The higher proportion of lactic acid bacteria in the gut microbiota of RELMβ-KO than in that of wild-type mice may be one of the mechanisms underlying the lower serum LPS level the former. These data suggest the contribution of increases in RELMβ in the gut and Kupffer cells to NASH development, raising the possibility of RELMβ being a novel therapeutic target for NASH.