Butyrate Treatment Research Articles

Exposure to Microbial Metabolite Butyrate Prolongs the Survival Time and Changes the Growth Pattern of Human Papillomavirus 16 E6/E7-Immortalized Keratinocytes in Vivo

Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. Although cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure may increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. These results suggest that fermentation products of tonsillar bacteria may play an important role in the long-term persistence of high-risk HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.

Butyrate protects endothelial function through PPARδ/miR-181b signaling

Reports of the beneficial roles of butyrate in cardiovascular diseases, such as atherosclerosis and ischemic stroke, are becoming increasingly abundant. However, the mechanisms of its bioactivities remain largely unknown. In this study, we explored the effects of butyrate on endothelial dysfunction and its potential underlying mechanism. In our study, ApoE-/- mice were fed with high-fat diet (HFD) for ten weeks to produce atherosclerosis models and concurrently treated with or without sodium butyrate daily. Thoracic aortas were subsequently isolated from C57BL/6 wild-type (WT), PPARδ-/-, endothelial-specific PPARδ wild-type (EC-specific PPARδ WT) and endothelial-specific PPARδ knockout (EC-specific PPARδ KO) mice were stimulated with interleukin (IL)-1β with or without butyrate ex vivo. Our results demonstrated that butyrate treatment rescued the impaired endothelium-dependent relaxations (EDRs) in thoracic aortas of HFD-fed ApoE-/- mice. Butyrate also rescued impaired EDRs in IL-1β-treated thoracic aorta ring ex vivo. Global and endothelial-specific knockout of PPARδ eliminated the protective effects of butyrate against IL-1β-induced impairment to EDRs. Butyrate abolished IL-1β-induced reactive oxygen species (ROS) production in endothelial cells while the inhibitory effect was incapacitated by genetic deletion of PPARδ or pharmacological inhibition of PPARδ. IL-1β increased NADPH oxidase 2 (NOX2) mRNA and protein expressions in endothelial cells, which were prevented by butyrate treatment, and the effects of butyrate were blunted following pharmacological inhibition of PPARδ. Importantly, butyrate treatment upregulated the miR-181b expression in atherosclerotic aortas and IL-1β-treated endothelial cells. Moreover, transfection of endothelial cells with miR-181b inhibitor abolished the suppressive effects of butyrate on NOX2 expressions and ROS generation in endothelial cells. To conclude, butyrate prevents endothelial dysfunction in atherosclerosis by reducing endothelial NOX2 expression and ROS production via the PPARδ/miR-181b pathway.

Defensin γ-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2

Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin γ-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. The combined treatment of γ-thionin (3.5 µM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while γ-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. The γ-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy. How to cite: Velázquez-Hernández ME, Ochoa-Zarzosa A, López-Meza JE, Defensin γ-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2. Electron J Biotechnol 2021;52. https://doi.org/10.1016/j.ejbt.2021.04.009

The Impact of Microbiome and Microbiota-Derived Sodium Butyrate on Drosophila Transcriptome and Metabolome Revealed by Multi-Omics Analysis.

The host microbiome plays an important role in regulating physiology through microbiota-derived metabolites during host-microbiome interactions. However, molecular mechanism underly host-microbiome interactions remains to be explored. In this study, we used Drosophila as the model to investigate the influence of microbiome and microbiota-derived metabolite sodium butyrate on host transcriptome and metabolome. We established both a sterile Drosophila model and a conventional Drosophila model to demonstrate the role of sodium butyrate. Using multi-omics analysis, we found that microbiome and sodium butyrate could impact host gene expression patterns in both the sterile Drosophila model and the conventional Drosophila model. The analysis of gut microbial using 16S rRNA sequencing showed sodium butyrate treatment also influenced Drosophila bacterial structures. In addition, Drosophila metabolites identified by ultra-high performance liquid chromatography-MS/MS were shown to be affected by sodium butyrate treatment with lipids as the dominant changed components. Our integrative analysis of the transcriptome, the microbiome, and the metabolome data identified candidate transcripts that are coregulated by sodium butyrate. Taken together, our results reveal the impact of the microbiome and microbiota-derived sodium butyrate on host transcriptome and metabolome, and our work provides a better understanding of host-microbiome interactions at the molecular level with multi-omics data.

Selective elimination of tumorigenic hepatic stem cells using hybrid liposomes

To avoid the risk of tumorigenesis after cell transplantation, tumorigenic stem cells should be selectively eliminated from induced pluripotent cells, embryonic stem cells, and somatic stem cells. We previously reported the presence of tumorigenic stem cells in human fetal hepatocyte-induced hepatoblasts after sodium butyrate (SB) treatment. In this study, we aimed to investigate the selective elimination of tumorigenic stem cells in human hepatoblasts using hybrid liposomes (HLs) prepared by sonicating a mixture of 90mol% l-α-dimyristoylphosphatidylcholine and 10mol% polyoxyethylene (n) dodecyl ether (C12 (EO)n, n= 23) in a buffer solution. Flow cytometric analysis revealed that the number of hepatoblasts increased by around 12-18 times in SB-treated cells compared to non-treated cells. In the colony formation assay, colonies of tumorigenic stem cells were observed in a soft agar plate after SB treatment. HL treatment for 48h resulted in a remarkable decrease in the number of colonies. HLs also induced apoptosis of tumorigenic stem cells by activating caspase-3. Flow cytometry showed a significant accumulation of HLs, including fluorescent lipids, in tumorigenic hepatic stem cells. The reappearance of tumorigenic stem cells was suppressed even in subsequent subcultures of HL-treated cells. High CYP3A4 activity was observed in a three-dimensional invitro assay. These results suggest that HL treatment could specifically eliminate tumorigenic hepatic stem cells. Incubation with HLs can be an effective culture method to maintain the quality of stem cells and reduce the risk of tumorigenesis after cell transplantation.

Microbial metabolites in the gut‐brain axis of an amyotrophic lateral sclerosis model

Backgound Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by progressive atrophy of motor neurons. Recent studies have demonstrated gut microbiome-associated contributions to human ALS and ALS experimental mice SOD1­G93A. Treatment with dietary butyrate, a natural bacterial product, rectified intestinal abnormalities and reduced disease progression in ALS mice. The emergence of ALS as a multifactorial disorder is further emphasized by understanding gut-brain mechanisms such as microbial metabolites. Methods Here, we hypothesize that butyrate treatment increases the concentration of anti-inflammatory and neuroprotective metabolites. SOD1­G93A (G93A) mice were used as a model of human ALS. G93A and WT mice classified as treated and untreated groups. Treated groups were given 2% dietary butyrate in their drinking water while untreated were given water ad libitum. Fecal samples were collected at 4, 8 and 14 weeks of age for treated groups while samples for untreated groups were obtained at 4, 7,13, and 17 weeks of age. Metabolites in fecalsamples were prepared using the automated MicroLab STAR® system (Hamilton Company) and measured by Metabolon. Raw data were extracted, peak-identified and QC processed using Metabolon's hardware and software. Statistical comparisons between metabolite concentrations in untreated and treated groups were conducted using two-way ANOVA via R. Results In untreated G93A mice, there was an increase in indole derived metabolites at 13 weeks of age, at onset of ALS symptoms. Specifically, concentration of indole acetate and indole acrylate, which are known to be aryl hydrocarbon receptor (AhR) ligands which indirectly aid in neuroprotection, increased at 13 weeks. Additionally, endocannabinoid concentrations, such as behenoyl ethanolamide (BEA) increased at 13 weeks while linoleoyl ethanolamide (LigEA) increased at 17 weeks. Our results suggest that the increased production of anti-inflammatory and neuroprotective metabolites onset and at 17 weeks could be a compensatory mechanism by the gut to alleviate ALS pathology. We also identify specific microbial metabolites associated with ALS as well as investigate the effect of butyrate on metabolites over the course of disease. Conclusion This study profiled fecal metabolites in G93A mice at different stages of ALS and with butyrate treatment. Four metabolites emerged as metabolic markers with diagnostic potential. Our findings contribute to the notion that the gut microbiome may play an essential role in the pathogenesis and pathophysiology of ALS and have significant translational implications for identifying new biomarkers for ALS. Further investigation is needed to explore whether related genes or enzymes could elucidate biological mechanisms of how ALS develops at the systemic level.

Commensal Bacteria Derived Metabolite Butyrate Restores the Epithelial Mitochondrial Network Disrupted by the Crohn's Disease‐Associated Pathobiont Adherent Invasive E‐coli Infection

Background Mitochondrial dysfunction has been implicated in several autoimmune and inflammatory diseases. Adherent-invasive E. coli (AIEC) is a Crohn's disease-associated pathobiont that triggers pro-inflammatory responses. Previous work done by our group showed that AIEC (strain LF82) infection in gut epithelial cells (T84 human cell line) induces excessive mitochondrial network fragmentation with loss of mitochondrial membrane potential. Commensal bacteria in the gut produce useful metabolites such as short chain fatty acids (SCFA), which have a wide range of benefits including enhancing mitochondrial biogenesis. Aims (1) Determine the ability of sodium butyrate (Na-But) treatment to restore mitochondrial functions disrupted by AIEC (strain LF82) infection in T84 epithelial cells. (2) Identify the mechanism utilized by Na-But to impose its protective effect. Methods Mitochondrial network morphology was assessed via confocal microscopy live cell imaging of Mito-tracker-stained T84 epithelial cells, and images analyzed using ImageJ. We assessed the effect of Na-But (10mM) on the proliferation of bacteria and their invasion of T84-epithelia by growth curve analysis and gentamycin internalization assays. Mitochondrial membrane potential was measured by flow cytometry, and mRNA and protein expression of peroxisome proliferating activator receptor assessed by qPCR and immunoblotting. Results We confirmed that AIEC-infection (strain LF82, 108cfu, 4h) induces massive mitochondrial fragmentation in T84 cells (2x105). Co-treatment with Na-But and LF82 showed increased percentage of fused T84-cell mitochondrial networks compared to LF82-treated cells (Na-But also reduced epithelial mitochondrial fragmentation caused by dinitrophenol (0.1mM, 2h)). The protective effect of Na-But (3-20mM; 0-24hr) was neither due to inhibition of bacterial proliferation nor their invasion of the epithelia. Epithelia infected with E. coli-LF82 displayed reductions in mitochondrial membrane potential and mRNA expression of the master regulator of mitochondrial biogenesis, PGC1α: both effects were partially prevented by Na-But. Conclusions These data indicate that pathogen disruption of the epithelial mitochondrial network is a component of IBD that could identify novel targets to control enteric inflammation. These data underscore the complex bacterial-epithelium interaction: commensal organisms may preserve the mitochondrial network in the face of challenge from pathogens that seek to disrupt mitochondrial form and function.

Microbiota‐derived butyrate is an endogenous inhibitor of HIF prolyl‐hydroxylases

The diverse population of microbial species present in the human intestines, collectively termed the gut microbiota, is an essential component of human health. Microbial supply of fermentation-derived short-chain fatty acids (SCFAs), particularly butyrate, is a well-established component to gut homeostasis and disease resistance. Reaching millimolar luminal concentrations in the colon, butyrate is sequestered and utilized as the favored energy source for intestinal epithelial cells. Beyond energy provision, butyrate broadly regulates gene expression as a histone deacetylase inhibitor and modulates the intestinal environment via signaling through SCFA-specific G-protein coupled receptors. Additionally, increasing oxygen utilization due to SCFA metabolism has been shown to stabilize hypoxia-inducible factor (HIF), a transcription factor that broadly functions as the master regulator of oxygen homeostasis but also specifically maintains intestinal homeostasis and is essential to intestinal barrier function. Under normoxic conditions, HIF is degraded in a hydroxylation-dependent manner mediated by one of three HIF prolyl-hydroxylases (PHDs). When oxygen is limiting, PHDs are inhibited and HIF is stabilized. Given the low O2 conditions of the intestinal microenvironment, it is of interest whether butyrate could more specifically influence HIF. We show that the microbiota-derived butyrate directly binds and inhibits PHD2 to stabilize HIF. Using cultured epithelia and molecular approaches, we found that butyrate treatment stabilized HIF in vitro independent of oxygen metabolism. Utilizing recombinant PHD protein, we identified butyrate as a unique inhibitor of PHD relative to other SCFAs. We also found that butyrate delivered to the colon of mice stabilizes HIF in a specific manner. Our results suggest that the co-evolution of mammals and commensal bacteria have selected for butyrate to impact a crucial gene regulation pathway that could be extended to beyond the intestinal mucosa. PHDs are a major target for drug development, as HIF regulates numerous genes involved in erythropoiesis, angiogenesis, energy metabolism, ischemia, and inflammation. Small-molecule inhibitors of PHDs have been developed in hopes of treating a variety of diseases not limited to chronic kidney disease and inflammatory bowel diseases. As an endogenous, well-tolerated small-molecule metabolite, butyrate holds far-reaching therapeutic potential as a HIF stabilizing agent.

Butyrate Prevents TGF-β1-Induced Alveolar Myofibroblast Differentiation and Modulates Energy Metabolism.

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by excessive collagen matrix deposition and extracellular remodeling. Signaling pathways mediated by fibrotic cytokine transforming growth factor β1 (TGF-β1) make important contributions to pulmonary fibrosis, but it remains unclear how TGF-β1 alters metabolism and modulates the activation and differentiation of pulmonary fibroblasts. We found that TGF-β1 lowers NADH and NADH/NAD levels, possibly due to changes in the TCA cycle, resulting in reductions in the ATP level and oxidative phosphorylation in pulmonary fibroblasts. In addition, we showed that butyrate (C4), a short chain fatty acid (SCFA), exhibits potent antifibrotic activity by inhibiting expression of fibrosis markers. Butyrate treatment inhibited mitochondrial elongation in TGF-β1-treated lung fibroblasts and increased the mitochondrial membrane potential (MMP). Consistent with the mitochondrial observations, butyrate significantly increased ADP, ATP, NADH, and NADH/NAD levels in TGF-β1-treated pulmonary fibroblasts. Collectively, our findings indicate that TGF-β1 induces changes in mitochondrial dynamics and energy metabolism during myofibroblast differentiation, and that these changes can be modulated by butyrate, which enhances mitochondrial function.

Effect of dietary sodium butyrate on the innate immune response of Procambarus clarkii and disease resistance against white spot syndrome virus

Sodium butyrate is a compound used as a feed additive in animal production. Sodium butyrate regulates electrolyte balance, gastrointestinal micro-ecology, intestinal mucosal nutrients, and also acts as an immunostimulant. It has great potential research value. The effect of sodium butyrate on the innate immunity of crustaceans was investigated in this study. Procambarus clarkii (crayfish) was used as a model organism and healthy crayfish were challenged with White spot syndrome virus (WSSV). The results showed that the group treated with sodium butyrate had significantly lower levels of mortality caused by WSSV, and the replication of WSSV was also reduced to a certain extent. Fluorescence quantitative PCR showed that sodium butyrate could up-regulate the expression of two immune-related genes, including C-type-lectin and prophenoloxidase. It was found that sodium butyrate treatment had positive effects on the total number of hemocyte (THC), phenoloxidase activity (PO), superoxide dismutase activity (SOD), and CAT activity of crayfish. In addition, we found that whether infected with WSSV or not, sodium butyrate treatment can significantly reduce the hemocyte apoptosis rate of crayfish. The results show that sodium butyrate can regulate the innate immunity of crayfish and reduce crayfish mortality following WSSV infection. This study provides evidence for the novel use of sodium butyrate to regulate the immunity of crayfish, treat WSSV infection, and provide a new theoretical basis for the research of sodium butyrate on crustaceans as a new additive for aquatic feed.

Butyrate modulates adipose-derived stem cells isolated from polygenic obese and diabetic mice to drive enhanced immunosuppression

Background aimsAdipose-derived stem cells (ASCs) offer promising therapeutic possibilities for immunomodulation. Butyrate (BA) exerts potent anti-inflammatory effects and exhibits multiple regulatory functionalities in adipose tissue (AT). The authors aimed to explore whether BA modulates ASCs to augment their immunosuppressive capabilities. MethodsThe authors examined the potency of BA and ASCs for controlling anti-CD3 plus CD28-stimulated splenocyte proliferation in vitro, both in combination and with pre-treatment. Further, the authors investigated genes specifically upregulated by BA-treated ASCs, which were harvested from ASC-splenocyte co-culture after the removal of floating splenocytes. In addition, the authors investigated the influence of oral BA supplementation on the ex vivo immunosuppressive potency of ASCs from BALB/c and Tsumura, Suzuki, obese, diabetes (TSOD) mice. ResultsBA enhanced the immunosuppressive potency of ASCs when directly added to ASC-splenocyte co-cultures or via pre-conditioning treatment. The percentages of ASC-induced Foxp3+ regulatory T cells increased, whereas the numbers of ASC-suppressed T helper 17 cells further decreased after BA exposure. The messenger RNA expression levels of inducible nitric oxide (NO) synthase (iNOS), chemokines, IL-10 and amphiregulin in ASCs co-cultured with activated splenocytes were upregulated after incubation with BA. This was accompanied by an amplification of iNOS-inducing cytokines, interferon gamma and tumor necrosis factor alpha in the ASC-splenocyte co-culture, triggering ASCs to produce high NO levels under the influence of BA. Mechanistically, the authors detected BA-mediated acetylated histone H3 in ASCs. BA treatment consistently improved the immunosuppressive potency of ASCs derived from both BALB/c and TSOD mice. ConclusionsThe use of BA to counteract metaflammation by restoring the defective immunomodulation of ASCs from dysregulated AT in obese donors is recommended.

URIC ACID-ASSOCIATED CARDIAC TISSUE DAMAGE CAUSED BY HIGH FAT DIET IS REVERSED BY SODIUM BUTYRATE

Objective: Increased global consumption of high-fat/high-calorie diet has led to higher incidence of the multifactorial cardiometabolic syndrome especially among women. The links between glucose deregulation and eventual mortal cardiac diseases are still being investigated. However, several reports have implicated elevated uric acid (UA) in the progression of metabolic disorders especially during high-fructose diet. Also, butyrate (BUT) a short-chain fatty acid is being identified with intriguing therapeutic potentials in metabolic disorders. We therefore hypothesized that high-fat diet-induced glucose deregulation and cardiac tissue damage are associated with elevated UA and attenuated by BUT in female rats. Design and method: Twenty-four 10-week-old female Wistar rats with weights ranging from 135 to 150 g were treated with normal rat chow and distilled water (po) or sodium butyrate (200 mg/kg; po) or high-fat diet and distilled water (po) or high-fat diet and sodium butyrate. Treatments lasted for 6 weeks. Results: Results showed that high-fat diet caused glucose dysmetabolism, elevated plasma triglyceride (TG), total cholesterol (TC), corticosterone, malondialdehyde (MDA), plasma and cardiac UA, and lactate dehydrogenase (LDH). High-fat diet also led to depressed reduced glutathione (GSH). Histological analysis of cardiac tissue showed cellular infarction, infiltration, and fibrosis in high-fat diet-fed rats. However, all these effects were ameliorated by BUT treatment. Conclusions: The findings here showed that high-fat diet resulted in glucose dysmetabolism and cardiac tissue damage through a uric acid (UA) - dependent mechanism and that butyrate (BUT) can protect against high-fat diet-induced cardiometabolic disorders through UA suppression and augmentation of glutathione antioxidant defenses. This study therefore demonstrates that UA is involved in high fat diet-induced cardiometabolic syndrome. However, data here shows that the UA - associated events in high fat diet will chronically engender cardiac tissue damage. Interestingly, with UA suppression, BUT treatment showed plausible beneficial glucoregulatory, anti-inflammatory and antioxidant properties which were potent enough to confer protection on the cardiac tissue preventing overt damage in high fat diet-fed rats. Therefore, BUT may be considered for the development of suitable therapeutic candidate in the treatment of metabolic syndrome and cardiac derangements associated with heightened UA.

Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8+ T cell immunity

Recent studies in both mice and humans have suggested that gut microbiota could modulate tumor responsiveness to chemo- or immunotherapies. However, the underlying mechanism is not clear yet. Here, we found that gut microbial metabolites, especially butyrate, could promote the efficacy of oxaliplatin by modulating CD8+ Tcell function in the tumor microenvironment. Butyrate treatment directly boosted the antitumor cytotoxic CD8+ Tcell responses both invitro and invivo in an ID2-dependent manner by promoting the IL-12 signaling pathway. In humans, the oxaliplatin responder cancer patients exhibited a higher amount of serum butyrate than did non-responders, which could also increase ID2 expression and function of human CD8+ Tcells. Together, our findings suggest that the gut microbial metabolite butyrate could promote antitumor therapeutic efficacy through the ID2-dependent regulation of CD8+ Tcell immunity, indicating that gut microbial metabolites could be effective as a part of cancer therapy.

Impaired Butyrate Induced Regulation of T Cell Surface Expression of CTLA-4 in Patients with Ulcerative Colitis.

Patients with ulcerative colitis (UC) have reduced intestinal levels of short-chain fatty acids (SCFAs), including butyrate, which are important regulators of host–microbiota crosstalk. The aim was therefore to determine effects of butyrate on blood and intestinal T cells from patients with active UC. T cells from UC patients and healthy subjects were polyclonally stimulated together with SCFAs and proliferation, activation, cytokine secretion, and surface expression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) were analyzed. Butyrate induced comparable, dose dependent inhibition of activation and proliferation in blood T cells and activation in intestinal T cells from UC patients and healthy subjects. However, surface expression of the inhibitory molecule CTLA-4 on stimulated blood and intestinal T cells was impaired in UC patients and was not restored following butyrate treatment. Furthermore, unlike intestinal T cells from healthy subjects, butyrate was unable to downregulate secretion of interferon gamma (IFNγ), interleukin (IL)-4, IL-17A, and IL-10 in UC patients. Although seemingly normal inhibitory effects on T cell activation and proliferation, butyrate has an impaired ability to reduce cytokine secretion and induce surface expression of CTLA-4 in T cells from UC patients with active disease. Overall, these observations indicate a dysfunction in butyrate induced immune regulation linked to CTLA-4 signaling in T cells from UC patients during a flare.

A49 BUTYRATE PRESERVES THE EPITHELIAL MITOCHONDRIAL NETWORK DISRUPTED BY THE CROHN’S DISEASE PATHOBIONT ADHERENT-INVASIVE E. COLI

Abstract Background Mitochondria exist in a dynamic network that undergoes continuous cycles of fission and fusion that is tightly controlled. Mitochondrial dysfunction is implicated in several autoimmune and inflammatory diseases. Adherent-invasive E. coli (AIEC) is a pathogenic strain of bacteria associated with Crohn’s disease (CD), that can evoke pro-inflammatory responses. Data from our lab showed that AIEC (strain LF82) infection in gut epithelial cells (T84 human cell line) caused dramatic mitochondrial network fragmentation and loss of mitochondrial membrane potential. Short chain fatty acids (SCFA) produced by commensal bacteria in the gut have a wide range of benefits including enhancing mitochondrial biogenesis. Aims To determine (1) if sodium butyrate (NaB) treatment can protect against mitochondrial dysfunction induced by AIEC (strain LF82) infection in T84 epithelial cells and then (2) to identify the mechanism by which NaB restores mitochondrial functions. Methods We assessed changes in mitochondrial network morphology through confocal microscopy live cell imaging of mitotracker-stained T84 epithelial cells based on unbiased Hoechst-stained nuclei to select the field of view. The effect of NaB on the proliferation of bacteria and their invasion of T84-epithelia was assessed by growth curve analysis and bacterial internalisation assays. Finally, mRNA and protein expression of peroxisome proliferating activator receptor gamma co-activator 1 alpha (PGC1α), a regulator of mitochondrial biogenesis, were assessed by qPCR and Western Blot. Results We confirmed that AIEC infection (strain LF82, 108cfu, 4h) induces massive mitochondrial fragmentation in T84 cells (2x105). We also found that cotreatment of T84 cells with NaB (10mM) and LF82 showed increased percentage of fused mitochondrial networks compared to LF82-treated cells. This result was also seen in cells treated with mitochondrial uncoupler dinitrophenol (DNP; 0.1 mM, 2h) and NaB. Moreover, the protective effect of NaB was not related to inhibition of proliferation of the bacteria as we demonstrated that LF82 growth and its invasive phenotype was not compromised by NaB (3-20mM; 0-24hr). In agreement with the effect of LF82 on mitochondrial functions, LF82 significantly reduced PGC1α mRNA expression in T84 cells, that was prevented by co-treatment with NaB. Conclusions These data suggest the pathogen disruption of the epithelial mitochondrial network is a component of IBD; thus, identifying mitochondrial fission and fusion pathways as novel therapeutic targets to control enteric inflammation. The data underscore the complex interplay between bacteria and the epithelium, such that commensal organisms may preserve the mitochondrial network in the face of challenge from pathogens that seek to disrupt mitochondrial form and function Funding Agencies CIHR

Short-chain fatty acid butyrate: A novel shield against chronic gastric ulcer.

Butyrate is one of the most abundant short-chain fatty acids produced by intestinal bacteria. In the present study, the action of butyrate on chronic gastric mucosa lesions was investigated, as well as its underlying mechanism in mice. Male mice from the Institute of Cancer Research were randomly divided into three groups: Sham, model and butyrate groups. Butyrate was administered intragastrically for 7 days to butyrate group mice following the establishment of a gastric ulcer model. Hematoxylin and eosin staining, immunohistochemical analysis, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction were used to determine the therapeutic effects and molecular mechanism of butyrate treatment. The findings demonstrated that butyrate induced a marked shift in superoxide dismutase and catalase activities, along with a decrease in malondialdehyde levels, thereby attenuating oxidative stress. Furthermore, butyrate decreased the levels of pro-inflammatory cytokines interleukin-1β, tumour necrosis factor-α and leukotriene B4, which helped combat inflammatory responses. Moreover, butyrate treatment exerted remarkable positive influences that mediate an increase in 6-keto-PGF-1α (a degradation product of prostacyclin), trefoil factor 2, MUC5AC and fibroblast growth factor-7 levels to promote gastric mucosal repair. The expression of specific receptor GPR109A for butyrate was upregulated, with no significant difference noted in the expression of GPR43 or GPR41. Overall, the present findings revealed that butyrate exerted therapeutic effects by upregulating mucosal repair factors and stimulating protective responses against oxidation and inflammation. GPR109A may be the key receptor for butyrate therapy.

REGULATION OF HUMAN INTESTINAL ORGANOID REACTIVE OXYGEN SPECIES PRODUCTION AND MITOCHONDRIAL FUNCTION BY DUOX2 GENETIC VARIATION AND MICROBIAL PRODUCTS

Abstract Introduction The DUOX2 intestinal epithelial NADPH oxidase is upregulated in Crohn’s Disease (CD), and DUOX2 mutations are associated with increased CD risk. Oxidative stress and loss of mitochondrial function disrupt the intestinal barrier promoting inflammatory responses to commensals. The relative impact of DUOX2 mutations and microbial products in this regard is poorly understood. Hypothesis We hypothesized that DUOX2 genetic variation would be associated with differences in cellular reactive oxygen species (ROS) production and mitochondrial function in a Human Intestinal Organoid (HIO) model system. Methods Induced pluripotent stem cell lines derived from pediatric CD patients with and without combined DUOX2 missense mutations(R701Q, P982A, and H678R) were used to generate wild type (WT) and DUOX2mut HIOs. Reactive oxygen species (ROS) production was measured using the two-color ROS-ID® Total ROS/Superoxide detection kit, and the mitochondrial membrane potential (MMP) was measured using JC1 staining by flow cytometry in HIO EpCAM+ epithelial cells and CD90+ stromal cells. Expression of inflammatory and mitochondrial genes which varied with DUOX2 mutation carriage in CD patent ileal biopsies was measured by RT-PCR. HIO mitochondrial complex I and II activity was measured using an Oroboros respirometer. Results Epithelial ROS production was reduced in DUOX2mut HIO under basal conditions; this difference was not observed following pyocyanin stimulation (Fig. 1A). A profound suppression of epithelial ROS production was observed following butyrate treatment. Butyrate did not alter stromal cell ROS production. Under these conditions, induction of ROS by pyocyanin was abrogated in WT, but not DUOX2mut HIO epithelial cells (Fig. 1B). Butyrate increased expression of core genes regulating the mitochondrial respiratory chain and DNA synthesis (COX5B, NDUFA1, POLG2, SLC25A27) and HIF1A implicated in barrier function, independent of genotype (p<0.05). The epithelial and stromal cell mitochondrial membrane potential (MMP) (Fig. 2A), and HIO mitochondrial complex I activity (Fig. 2B), were reduced in DUOX2mut HIO under basal conditions. This was specific, as mitochondrial complex II activity did not vary with DUOX2 genotype. Conclusions We confirmed epithelial effects of DUOX2 genotype and butyrate exposure on ROS production in the HIO model system. Genotype dependent effects on basal ROS production were largely abrogated by the microbial products pyocyanin and butyrate, although butyrate inhibition of pyocyanin induced ROS production was dependent on intact DUOX2 function. Data suggest a previously unanticipated effect of DUOX2 genetic variation on the epithelial and stromal cell MMP and cellular respiration. This may have implications for mechanisms by which DUOX2 regulates barrier function and inflammatory responses to commensals in CD.

Role and mechanism of action of butyrate in atherosclerotic diseases: a review.

Butyrate is a bioactive molecule produced by the intestinal flora and plays a major role in a variety of inflammatory diseases. Increasing evidence indicates that butyrate can regulate the occurrence and development of atherosclerosis (AS). Coincidentally, it reduces hyperlipidemia and hyperglycemia, which are major risk factors of AS. However, the mechanism by which butyrate regulates the development of AS remains unclear. In this article, we review the effect of butyrate treatment on AS with a focus on the mechanisms of butyrate-mediated modulation of several atherosclerotic processes. These include the improvement of monocyte-endothelial interactions, macrophage lipid accumulation, smooth muscle cell proliferation and migration, and lymphocyte differentiation and function. The existing research indicates that butyrate treatment may be a potentially effective strategy for the prevention of AS. Identity and underlying mechanisms of the molecular pathways of these interactions should be explored in the future to counter AS effectively.

Evidence for the butyrate metabolism as key pathway improving ulcerative colitis in both pediatric and adult patients

ABSTRACT Accumulating evidence has shown many similarities and differences of gene profiles and pathways between pediatric and adult ulcerative colitis (UC) patients. In this study, we aimed to investigate the shared genes and pathways in intestinal tissues of pediatric and adult UC. Differentially expressed genes (DEGs) between pediatric and adult UC were identified via bioinformatic analysis of Gene Expression Omnibus datasets GSE87473 and GSE126124. Gene Ontology and pathway enrichment were used to analyze overlapped and distinguished DEGs. Gene Set Variation Analysis (GSVA) was utilized for contrast consistency. Mice colitis models were induced by dextran sulfate sodium (DSS) and Citrobacter rodentium. 2616 DEGs were screened out in intestinal tissues of adult UC compared with those of adult healthy controls, and 1195 DEGs in pediatrics. Same pathways between pediatric and adult UC were enriched using overlapped DEGs, mainly related to immune responses and metabolic processes, including butyrate metabolism, which was also identified by GSVA analysis. Of note, butyrate metabolism was the exclusive down-regulated pathway enriched by these two analyses, indicating that butyrate metabolism is one of the key pathways associated with both pediatric and adult UC. In addition, butyrate suppressed DSS-induced and Citrobacter rodentium-induced intestinal inflammation in mice. Therefore, the study revealed that butyrate metabolism was critical in both pediatric and adult UC. And butyrate suppressed colitis in mice, which provided a theoretical basis for the potential treatment of butyrate for UC patients. Abbreviations: UC, Ulcerative colitis; IBD, Inflammatory bowel disease; DEGs, Differentially expressed genes; GEO, Gene Expression Omnibus; SVA, Spatial variant apodization; LIMMA, Linear models for the microarray data; FC, Fold change; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSVA, Gene Set Variation Analysis; MSigDB, Molecular Signatures Database; WT, Wild-type; DSS, Dextran sulfate sodium; HC, Healthy control; SD, Standard deviation; SNHG5, Small nucleolar RNA host gene 5; GLP-2, Glucagon-like peptide 2; GSE, Gene set enrichment; ECM, Extracellular matrix; TCA, Tricarboxylic acid cycle; NA, Not available.

Real-Time Monitoring of DWV-Infected Bee Foraging Behavior Following Histone Deacetylase Inhibitor Treatment

Impairment in learning and memory behaviors in bees due to pesticide exposures and pathogen infections has resulted in massive disappearance of bee populations and subsequent agricultural economic losses. One of the causes of this bee colony collapse disorder (CCD) is the infection of deformed wing virus (DWV). The present study found that sodium butyrate, a histone deacetylase inhibitor (HDACi) previously shown to reverse the detrimental effects of a nicotine-like pesticide on learning behavior and memory in bees, significantly increased the survival of bee after DWV infection and reversed the learning/memory impairment in proboscis extension response (PER) assay. A next-generation sequencing analysis revealed the gene expression profile underwent DWV infection and sodium butyrate treatment and showed that sodium butyrate affected the expression of a wide range of genes involved in diverse biological processes. Particularly, sodium butyrate could up-regulate the expression of genes involved in glycolytic process and memory formation that were down-regulated during DWV infection. As brain physiological analyses presented that sodium butyrate restored the energy supply and the production of neurotransmitter in the infected bees, a large-scale movement tracking was performed by using a wireless sensor network (WSN)-based automatic real-time monitoring system and confirmed that sodium butyrate can improve the ability of DWV-infected bees to return to their hives. In short, we demonstrated the mechanism of how epigenetic regulation can resume the memory function of honeybees and suggested strategies for applying sodium butyrate as possible solutions for reducing the incidence of CCD.