Neutrophil (PMN) accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. PMN transepithelial migration is orchestrated through a complex series of cell-cell interactions involving bi-directional signaling molecules. Thus we hypothesized that migrating PMN influence the transcriptional profile of epithelia as they transmigrate, priming them for either resolution or chronic inflammation - a concept we term “transcriptional imprinting”. Employing a novel approach to a PMN-transepithelial migration model, we attempted to ascertain the influence of transmigration on epithelial gene expression by microarray analysis.
DNA Microarray was used to identify transcriptional changes in intestinal epithelial cells post-PMN transmigration. Real-time oxygen sensing was performed with an SDR-OxoDish (PreSens). Wild-type C57/B6 mice, ODD-Luciferase (HIF reporter) mice and gp91phox-null (NADPH oxidase deficient) mice were subjected to chemically-induced colitis (TNBS).
Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Real-time oxygen measurements effectively demonstrated that activated PMN rapidly depleted microenvironmental oxygen. Subsequent studies indicated that activated PMNs are sufficient to induce hypoxia-inducible factor (HIF) stabilization and activity in epithelial cells. Intestinal epithelial hypoxia induced by activated PMNs during transmigration was found to be dependent on the PMN respiratory burst. HIF is known to influence the expression of barrier-protective genes in the epithelium, initiate glycolytic metabolism and attenuates the clinical course/disease parameters in murine colitis. To ascertain the clinical relevance of transmigrating PMN and the induction of epithelial hypoxia, we stained biopsies from UC patients for evidence of Glut1 expression. PMN transmigration increased crypt epithelial Glut1 expression (see Figure). We investigated the relative contribution of PMN to “inflammatory hypoxia” in vivo, utilizing a colitis model. Antibody-mediated depletion of PMNs worsened the course of colitis, exhibited reduced tissue hypoxia visualized with Hypoxyprobe-1 staining and attenuated the induction of hypoxia-dependent genes. Patients with chronic granulomatous disease (CGD) lack functional NADPH oxidase and develop IBD-like symptoms. We demonstrated that murine gp91phox−/− mice mirror human CGD, develop a severe colitis, with exaggerated PMN infiltration. However, these infiltrating PMN are incapable of inducing a hypoxic microenvironment. Finally, pharmacological intervention with a HIF-stabilizing compound (PHD inhibitor), resulted in recapitulation of mucosal HIF-signaling concomitant with abrogation of colitis-severity in the gp91phox−/− mice.
In conclusion, transcriptional imprinting of host tissue by infiltrating neutrophils significantly modulates the host response to inflammation. Moreover, the respiratory burst contributes fundamentally to localized oxygen depletion and resultant microenvironmental hypoxia. We propose that this microenvironment established by infiltrating PMN is protective during colitis.
