Human neutrophil peptides induce interleukin-8 in intestinal epithelial cells through the P2 receptor and ERK1/2 signaling pathways

Abstract

Human neutrophil peptides (HNPs) are antimicrobial peptides produced predominantly by neutrophils. We have previously reported that HNP 1-3 levels are increased in the sera and plasma of patients with active ulcerative colitis. The increased expression of interleukin-8 (IL-8) has also been demonstrated in the colonic mucosa of patients with active ulcerative colitis. HNPs induce IL-8 in lung epithelial cells and monocytes through the P2Y6 signaling pathway. However, the association between HNPs and IL-8 in the intestinal mucosa has not yet been investigated. In the present study, we investigated the effects of HNP-1 on the production of IL-8 by human intestinal epithelial cells and the underlying signaling mechanisms. We observed a significant increase in IL-8 expression in the human colon carcinoma cell line, Caco-2, following treatment with HNP-1. The non-selective P2 receptor antagonists, suramin and pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), significantly blocked the HNP-1-induced expression of IL-8 in the Caco-2 cells. The P2Y6-specific antagonist, MRS2578, led to a significant but partial decrease in IL-8 expression, suggesting that P2 receptors in addition to P2Y6 are involved in the HNP-1-induced production of IL-8 by Caco-2 cells. In agreement with this finding, HNP-1 also significantly increased IL-8 production in the P2Y6-negative human colon cancer cell line, HT-29, and this increase was blocked by treatment with suramin and PPADS. HNP-1 significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in the HT-29 cells. However, the HNP-1-induced production of IL-8 was suppressed by the ERK1/2 inhibitor, U0126, but not by the p38 MAPK inhibitor, SB203580. In conclusion, our data demonstrate that HNP-1 induces IL-8 production not only through P2Y6, but also through additional P2 receptors via an ERK1/2-dependent mechanism in intestinal epithelial cells.

Decellularized colorectal cancer matrix as bioactive microenvironment for in vitro 3D cancer research

“…The vessels’ attraction was confirmed also by HE at the end of in ovo incubation (Figure c). In addition, it is reported that the stimulation of tumor cell lines with DEFA3 resulted in the induction of IL‐8 overexpression (Ibusuki et al, ; Khine et al, ). Also in our hands, stimulation of HT‐29 cell line with exogenous DEFA3 (Figure d) or conditioned media obtained through DN and DT matrices disaggregation (Figure e) induced the overexpression of IL‐8 in respect to the untreated cells (Figures d and e), with no significant difference between DN and DT conditioned cells.…”

“…In literature, it has been shown that the release of neutrophils proteins into the inflamed tumor stroma is a strong activator of chemokine CXCL‐8, also known as IL‐8, in resident cells (Khine et al, ). In addition, a recent research work showed that released DEFA proteins are able to induce the secretion of IL‐8 in HT‐29 cell line through the activation of ERK1/2 signaling pathway (Ibusuki et al, ). Also in our hand, HT‐29 cells were able to over‐expressed IL‐8 transcript after conditioning with either exogenous or decellularized matrix‐related DEFA3, but this increase in gene expression was statistically different between acellular tumor and healthy mucosa samples only when the cell line was cultivated in 3D biologic‐derived environment.…”

P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Urate Crystals

“…This is also likely because the ectonucleotidase NTPDase1 (CD39) has been shown to control IL-8 release in human PMNs (108). P2Y6R could also play a role because IL-8 release in epithelial cells induced by neutrophil defensins is blocked by MRS2578 (109). MRS2578 could also switch off NETosis in human PMNs, only to shorten their lifespan and accelerate apoptosis (72).…”

Low concentrations of human neutrophil peptide ameliorate experimental murine colitis

“…High concentrations of HNP-1 reduce the proliferation and viability of intestinal epithelial cells (14). Additionally, HNP-1 induces IL-8 production from intestinal epithelial cells in a dose-dependent manner, which may stimulate additional neutrophil accumulation in the intestine (36). …”