INDOLE ACETIC ACID AS AN INHIBITOR OF MYELOPEROXIDASE-MEDIATED BYSTANDER TISSUE DAMAGE IN INFLAMMATORY BOWEL DISEASE

Abstract Accumulation of polymorphonuclear neutrophils (PMNs) in crypt abscesses is a hallmark of inflammatory bowel disease (IBD), where PMNs play an important role in eliminating invasive pathogens and promoting wound healing. However, excessive neutrophil activity can cause bystander tissue damage and impair resolution of inflammation. Myeloperoxidase (MPO), a key PMN enzyme, produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chlorine, which kills microbes. HOCl is also a strong oxidant that can damage tissue by reacting with tyrosine to produce 3-chlorotyrosine, a marker of MPO-mediated tissue damage. Active IBD is also characterized by gut bacteria imbalance and changes in microbiota-derived metabolites, which impact the intestinal immune response and permeability. Short-chain fatty acids such as butyrate are reduced in IBD, while tryptophan-derived metabolites like indole and its derivatives, are less well studied. Recent studies show reduced indole derivatives in human and murine IBD, and their administration in murine IBD reduces disease severity. Our group recently demonstrated that microbial-derived indole functions as an endogenous inhibitor of MPO and attenuates bystander tissue damage in vitro and in vivo. Based on this, we hypothesize that select indole metabolites can be optimized for maximal inhibition of MPO-mediated bystander tissue damage. To test this, we screened 17 indole derivatives in an in vitro MPO assay using recombinant human MPO (rhMPO) activated by H2O2 and monitored with ABTS. Among the 17 indole derivatives, indole acetic acid (IAA) exhibited the most potent MPO inhibition. To further corroborate the inhibitory activity of MPO, we measured tyrosine chlorination in a cell-free rhMPO system reacted with indole derivatives, H2O2, and free tyrosine, using isocratic reversed-phase high-performance liquid chromatography with electrochemical detection (EC-HPLC). IAA also showed the lowest level of tyrosine chlorination among all derivatives, indicating that it can be a strong candidate for reducing bystander tissue damage. Based on these in vitro findings, we subsequently tested IAA in an in vivo model of DSS-induced colitis. C57BL/6 mice were treated with 3% DSS in the presence or absence of IAA (0.1 mg/ml) for 5 days. Mice received drinking water containing IAA 2 days before DSS administration, continuing through the DSS treatment and 2 days thereafter until euthanasia. Mice treated with DSS and IAA improved disease activity index scores compared to those receiving DSS alone. IAA treatment also reduced colonic MPO and proinflammatory cytokines, such as KC/gro (interleukin-8 related protein in rodents) and tumor necrosis factor-alpha. These findings indicate IAA can serve as a promising therapeutic intervention to diminish MPO-mediated tissue damage and mucosal inflammation in IBD.