Peptide IDR-1018 attenuates the severity of DSS colitis by enhancing recruitment of non-inflammatory neutrophils with increased bacteriocidal activity

We have recently reported the development of a new class of anti-infective and anti-inflammatory peptides termed innate defense regulators (IDRs). IDRs mediate their anti-infective activity by enhancing leukocyte recruitment to foci of infection while simultaneously suppressing pro-inflammatory cytokine production, thereby limiting harmful inflammation that might otherwise accompany a stronger innate immune response. We previously reported that IDR-peptide 1018 (VRLIVAVRIWRR) was protective in murine DSS colitis, and that the reduction in disease severity was paradoxically associated with elevated levels of tissue MPO, a marker of PMN infiltration. The aim of this study was to further elucidate the correlation between IDR-1018-mediated PMN recruitment and protection from DSS colitis, and to broadly characterize the effect of the peptide on a variety of PMN functions, including killing of inflammation-provoking gut microorganisms. Optimization of peptide dosing frequency was carried out using a standard acute DSS colitis model (2.75% w/v, male C57BL/6 mice, 8 day exposure). To examine the correlation between protection and PMN recruitment, colons were excised on day 9, homogenized, and used to quantify MPO activity and expression of leukocyte-attractant chemokines by qPCR. Immature murine PMNs were obtained from bone marrow (BM-PMNs) and treated with IDR-1018, alone or in combination with LPS. Peptide modulation LPS-induced cytokine/chemokine production and MPO release was measured by ELISA. The effect of peptide on BM-PMN viability, apoptosis and necrosis was measured using Apotox-Glo Triplex assays, and phagocytosis and extracellular bacterial killing was quantified using a lux reporter strain of Escherichia coli. The effect of IDR-1018 on PMN recruitment and bacterial killing was then confirmed in an in vivo E. coli peritonitis infection model, where mice were pre-treated with peptide or saline 4 hr before infection. The protective effect of IDR-1018 in DSS colitis was directly correlative with increased colon levels of MPO and upregulation of leukocyte chemoattractants CXCL1 (4-fold), CCL2 (6-fold), and CCL20 (20-fold). IDR-1018 dose-dependently modulated BM-PMN inflammatory behaviours by decreasing LPS-induced IL-6 and CXCL1 production, increasing LPS-induced IL-10 production, and by inhibiting both basal and LPS-induced release of MPO. In combination with LPS, IDR-1018 significantly increased BM-PMN cell death entirely through apoptosis rather than necrosis. Despite these functional changes, IDR-treated BM-PMNs retained their phagocytic capabilities, and showed enhanced killing of extracellular E. coli. These findings were confirmed in E. coli peritonitis, where IDR-1018 treated mice showed a significant 3-Log reduction of CFU in their spleens and peritoneal lavages, with significant elevations in MPO activity in infiltrating immune cells. These data suggest that the protective effect of IDR-1018 in DSS colitis arises from increased recruitment functionally altered PMNs to inflamed sites in the colon. These PMNs are more efficient at eradicating gut bacteria which drive inflammation in colitis, and are less prone to toxic behaviours such as necrosis and MPO release which normally perpetuate the inflammatory cycle. Further studies on therapeutic approaches to IBD in which PMN function is selectively modulated (rather than suppressed) are therefore warranted.

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