Differing roles for short chain fatty acids and GPR43 agonism in the regulation of intestinal barrier function and immune responses

<div><p>Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients.</p></div

Expression of GPR43 in intestinal cells.

<p>(A) The expression of GPR43 mRNA in the indicated cell lines was measured using droplet digital PCR (ddPCR). Parental CHO cells and cells transfected with GPR43 served as the negative and positive controls, respectively. (B, C) GPR43 expression in different regions of the mouse intestinal tissue (B) or purified intestinal epithelial cells (C) was measured using ddPCR. Results were expressed as copies of GPR43 per ng of isolated RNA. Plots represent the average of duplicates/triplicates with standard deviation.</p

Potency of GPR43 agonists.

<p>(A, B) Inhibition of forskolin-induced cAMP production in CHO cells stably expressing human GPR43 was measured in the presence of the SCFA (A) or GPR43 agonists (B). The data was plotted as percent of control (POC), to normalize to the levels of cAMP obtained following stimulation with forskolin alone. (C, D) GPR43 agonist-induced receptor activation in CHO-K1 cells stably expressing human GPR43 was determined by measuring the increase in cytosolic calcium concentrations. This was performed with the indicated GPR43 agonists in the absence (C) or presence (D) of 10% FBS. Plots represent the average, and the results shown are representative of 3 independent experiments.</p

Enhancement of intestinal epithelial barrier function by butyrate.

<p>(A) C2BBe1 cells were cultured in the apical chamber of transwells in the ECIS transwell array. Barrier resistance was measured on the ECIS instrument @ 75 Hz beginning at day 2 post-addition of the test agents with resistance values normalized to those observed just prior to the addition of test agents. (B) Cells were cultured as in A for 2 days. The media was then replaced with media containing 2.5 mM EGTA (both apical and basolateral chambers) for 5 hours prior to washing and replacing with media containing test agents. Cells were monitored over the next 48 hours for barrier reformation and the plots shown are at 75 Hz with resistance values normalized to those observed just prior to the addition of test agents. The results shown are representative of 2 independent experiments.</p

Anti-inflammatory effects of butyrate on LPS-induced cytokine production.

<p>Human PBMC were incubated with the indicated concentrations of butyrate (mM range) or GPR43 agonists (μM range) for 45 minutes prior to the addition of 1 μg/mL LPS. The levels of TNFα, IL-1β and IL-6 production in the supernatants were measured 18 hours later. Inhibition curves were plotted and IC₅₀ concentrations are indicated in parenthesis when applicable. For the panel displaying cell viability, the concentrations of SCFA in mM and agonists in μM were plotted along the same axis for easier viewing. The data plotted are the values obtained following the subtraction of background levels of cytokines obtained without LPS stimulation and represent the average of duplicates with standard deviation. The results shown are representative of 2 independent experiments consisting of a total of 4 donors.</p