Insights into the impact of inflammatory acidification on the mucosa

Mucosal acidification is an often-underappreciated aspect of mucosal inflammation. Like other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients, with reports of colonic pH as low as 4. We recently demonstrated that neutrophil (PMN) transepithelial migration rapidly acidifies the microenvironment resulting in inflammatory acidification of the mucosa. Furthermore, we identified an adaptive tissue response in IEC which buffered extracellular acidification during PMN transepithelial migration, suggesting pH homeostasis is important during acute inflammation. Very little is known in regard to the impact of extracellular acidification on the intestinal epithelial cell (IEC) function. To better understand the impact of extracellular pH on IEC cell function, we investigated the impact of extracellular acidosis on IEC gene expression. Guided by an unbiased RNAseq of T84 IEC exposed to low pH, we identified significant induction of several CREB associated genes. Based on this observation, we examined the phosphorylation of CREB under acidic conditions. Under acidic conditions, CREB is rapidly phosphorylated. We observed CREB phosphorylation within 5 minutes of exposure to acidic conditions and peaks at 30 min. Extending these studies we investigated the mechanism(s) involved in acidosis induced CREB phosphorylation. Interestingly, cAMP signaling, a common signaling pathway for CREB phosphorylation, appears to not be involved in acidosis-induced CREB phosphorylation. Under acidic conditions, there was no significant increase in cAMP levels in IEC and acidosis-induced CREB phosphorylation was still observed in T84 IEC treated with cAMP inhibitors. KEGG analysis of the RNAseq data revealed the induction of several MAPK regulatory genes, including members of the NR4A and DUSP families. Based on this observation we examined several key proteins involved in MAPK signaling, ERK1/2 and MSK1. Under acidic conditions ERK1/2 and MSK1 are rapidly phosphorylation and treatment with a MEK1/2 or MSK1 inhibitor significant attenuated CREB phosphorylation under acidic conditions. Further examination revealed CREB phosphorylation signals through the GaI-linked GPCR, GRP31. Finally, we extended our studies in vivo and examined mucosal acidification in a murine model of ileitis, TNFΔARE mice. Using a novel pH reporting E. coli strain, we identified significant mucosal acidification in the distal ileum of TNFΔARE mice when compared to WT mice. Mucosal acidification was positively correlated with the expression of several acidification associated genes; FosB, NR4A1, and CXCL1. These results identify extracellular acidification as a significant signaling mechanism during tissue inflammation. Thus, regulation of GPR31 or MSK1 may serve as new therapeutic targets to limit acidosis signaling during mucosal inflammation.