Control of Inflammatory Acidity in Mucosal Inflammation

Within the gastrointestinal (GI) tract the primary function of the mucosal surface is to provide a selective barrier
to the outside. It is widely understood that the GI tract exists in a state of low-grade inflammation, as a result of
constant processing of luminal antigenic material. The GI tract is home to trillions of commensal bacteria which
contribute significantly to GI homeostasis, but can also initiate and drive the progression of mucosal diseases.
Acute intestinal inflammation involves early accumulation of neutrophils (PMN) followed by either resolution or
progression to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can
accumulate and contribute to chronic inflammatory conditions, including inflammatory bowel disease. The
ongoing studies outline in this proposal have revealed that PMN transepithelial migration (TEM) results in a
significant decrease in extracellular pH, deemed inflammatory acidification. Extensions of these studies have
shown that this acidic microenvironment impacts barrier function and gene transcription in intestinal epithelial
cells (IEC). Guided by an unbiased RNAseq of IEC exposed to low pH in vitro and tissue metabolomics in vivo,
we identified a gene signature that includes induction of both TNF and members of the DUSP and NR4A
families. We also identified a role of IEC GPR31 in sensing extracellular pH and the loss of microbial-derived
indoles during active inflammation. Extending these studies, we observed significant improvement in IEC
barrier function under acidic conditions in T84 cells which had GPR31 knocked down. Additionally, we
observed that indole treatment significantly decreased inflammatory acidification following PMN TEM and in
K12 E. coli acidic conditions induce the production of indole. Based on these observations, we hypothesize
that inflammatory acidification promotes inflammatory signaling in IEC and that the microbiota-derived indoles
have a significant role in promoting pH homeostasis at sites of inflammation. In vitro functional assays will
define the impact of extracellular acidosis and pH sensing on PMN TEM, IEC barrier function, and IEC
adaptation to chronic extracellular acidosis. These studies will be extended to examine the influence of
microbial/host interactions on IEC responses to extracellular acidification. Finally, utilizing in vivo murine colitis
and ileitis models we will profile inflammatory acidification. We will extend these studies to investigate the
impact of the microbiota and microbiota-derived metabolites on inflammation-associated acidosis. The career
development and training plan outlined in this application is designed to guide and promote my transition to
independence as a VA funded researcher. The proposed combination of didactic courses, professional
conferences, and mentorship committee have been designed to strengthen my knowledge of mucosal
immunology and mucosal inflammation. Technically, I will gain experience in the generation of murine intestinal
enteroids, genetic modification of enteroids, both chemically induced and spontaneous murine models of ileitis
and colitis, and expand my knowledge of microbial/host response and technical experience in manipulating the
microbiota. Upon completion of this proposal I will be well prepared to establish myself as an expert in pH
regulation and inflammation-associated acidification. Furthermore, it is our hope that the studies outline in this
proposal will lay the groundwork for the identification of novel therapeutic avenues in IBD, directly benefiting
the veteran population.

Grant Number: 5IK2BX005710-04
NIH Institute/Center: VA
Principal Investigator: Ian Cartwright