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Butyrate Reduces TNFα‐Induced Cell Toxicity in Rat Colonic Epithelium
Author(s) -
Souders Christopher,
Zubcevic Jasenka,
Martyniuk Christopher
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.04478
Subject(s) - butyrate , viability assay , metabolite , tumor necrosis factor alpha , short chain fatty acid , cytotoxicity , population , chemistry , homeostasis , cell , pharmacology , biology , microbiology and biotechnology , endocrinology , biochemistry , in vitro , medicine , fermentation , environmental health
Hypertension (HTN) remains one of the most concerning preventable conditions for the global human population. The colonic lumen is the site of production and transport of many vasoactive metabolites and neurotransmitters that can modulate the immune system, affect cellular metabolism, and subsequently regulate blood pressure. As a critical interface between the gut bacteria and the host, dysfunctional and inflammed colonic epithelium has been associated with HTN. Here, we investigate the role of microbial metabolites in the regulation of colonic cell homeostasis, with a special emphasis on the interaction between tumor necrosis factor α (TNFα) and a gut bacterial short chain fatty acid (SCFA) metabolite butyrate, both of which have been implicated in HTN. We hypothesized that TNFα and butyrate exert opposing effects on colonic homeostasis through a mitochondrial mechanism, which is intrinsically linked to the pathology of HTN. To investigate this, Rat Primary Colon Epithelial Cells (CECs) (Cell Biologics, Cat# RN‐6047) were seeded at 10,000 cells per well in a 96 well plate at passage #5‐10 and exposed to increasing concentrations of TNFα (50pg/mL to 10ng/mL) and butyrate (50 µM to 25mM) in vitro for 24hrs. Using the multiplexed Alamar Blue (Cell Viability) (Thermofisher Scientific, Cat# DAL1025) and Cytotox Green (Cytotoxicity) (Promega, Cat# G8741) assays, we show that increasing concentrations of TNFα (150pg/mL‐10ng/mL) led to a concentration‐dependent reduction in CEC viability (by 20‐90%) and elevation in CEC cytotoxicity (by 10‐40%). Interestingly, treatment with butyrate alone (500µM‐25mM) also led to a concentration‐dependent reduction in cell viability (by 10‐70%), while an increase in CEC cytotoxicity (by 10‐20%) was observed only with high non‐physiologic concentrations of butyrate (2.5‐25mM). Next, CECs were exposed to TNFα (1 ng/mL) in the presence and absence of the physiologic colonic concentration of butyrate (500 µM). We found that co‐treatment with butyrate led to a significant reduction in TNFα‐induced CEC cytotoxicity (by 15%) and improved TNFα‐reduced CEC viability (by 15%). Interestingly, 24hr in vitro exposure to TNFα (50pg/mL‐10ng/mL) and butyrate (50 µM to 25mM) alone reduced relative ATP levels in a concentration‐dependent fashion (by 15%‐60% and 15%‐90% respectively). Thus, we assessed oxidative phosphorylation using Seahorse and found that both butyrate (500 µM) and TNFα (1 ng/mL) alone reduced ATP‐linked respiration (by 50% each), whereas co‐treatment of CECs with butyrate and TNFα produced an 85% reduction in ATP‐linked mitochondrial respiration. Taken together, these experiments suggest that colonic butyrate may attenuate the epithelial cell toxicity of TNFα by reducing mitochondrial activity. Moreover, while not affecting cell cytotoxicity at physiologic concentrations, butyrate appears to be toxic at higher concentrations in vitro . This may have important implications on gut homeostasis in the context of HTN where TNFα and butyrate levels are reportedly deregulated. Further research is necessary to elucidate the specific role of TNFα on colonic cell homeostasis and how butyrate and other SCFAs may affect this in order to better understand the host‐microbiota interactions in HTN.