IL-8/CXC Ligand 8 Survives Neonatal Gastric Digestion as a Result of Intrinsic Aspartyl Proteinase Resistance

Abstract

The human fetus and neonate swallow biologically significant quantities of IL-8/CXC ligand 8 (CXCL8) in amniotic fluid and breast milk, and this remains measurable through simulated neonatal gastric and proximal intestinal digestions. We sought to confirm the structural and functional integrity of IL-8/CXCL8 in digestates and determine the mechanisms underlying this protease resistance. We observed that in comparison with BSA, IL-8/CXCL8 is highly resistant to pepsin and can be detected intact in assays for structural, immunologic, and functional integrity. In a computational molecular docking simulation, IL-8/ CXCL8 was observed to fit poorly in the pepsin active site. On the basis of simulated mutation analyses, we hypothesized that this protease resistance is due to disulfide bond-related tertiary folding in IL-8/CXCL8. This was confirmed on chemical reduction of these groups. IL-8/CXC ligand 8 (CXCL8), the prototypal neutrophilspecific CXC chemokine in humans, is a key mediator of acute inflammation (1). In the developing intestine, elevated serum and tissue IL-8/CXCL8 concentrations are associated with inflammatory conditions such as necrotizing enterocolitis (2,3). It seems anomalous that the human fetal/neonatal gastrointestinal lumen is normally exposed to biologically significant IL-8/CXCL8 concentrations in swallowed amniotic fluid (4,5) and breast milk (6 -9).In an in vitro model of neonatal gastric and proximal intestinal conditions, IL-8/CXCL8 remains undigested, as tested by enzyme immunoassay (8). Recombinant human IL-8/CXCL8 promotes cellular migration, proliferation, and differentiation in intestinal epithelial cell (IEC) lines, besides protecting these cells from chemical injury (8). For assessing the physiologic relevance of these effects, the intact survival of IL-8/CXCL8 through gastric digestion needs confirmation in assays for structural and functional integrity. We hypothesized that IL-8/ CXCL8 survives neonatal gastric digestion as a result of an intrinsic resistance to aspartyl proteinase activity. We treated recombinant human IL-8/CXCL8 (rhIL-8/CXCL8) with an established gastric fluid model (Simulated Gastric Fluid; USP, 10) (10) and measured IL-8/CXCL8 concentration and bioactivity in a variety of assays. IL-8/CXCL8 -pepsin interaction was studied further in computational simulations involving molecular docking. The protease resistance of IL-8/CXCL8 was identified to be due to its disulfide bond-related tertiary folding and confirmed in actual chemical reduction experiments. METHODSSimulated gastric fluid (SGF) was prepared with 3.2 mg/mL of porcine pepsin (3200 -4500 units/mg protein; Sigma Chemical Co., St. Louis, MO) in 0.2% NaCl/0.7% HCl (pH 1.2; US Pharmacopoeia) (10). Carrier-free rhIL-8/ CXCL8 (R&D Systems, Minneapolis, MN) and BSA (control; Sigma Chemical Co.) were freshly reconstituted in PBS as 0.25-mg/mL solutions, which provided a pepsin:protein ratio of 50:1.

Maternal country of birth and previous pregnancies are associated with breast milk characteristics

“…High levels of breast milk IL‐8 may have a systemic influence on immune function in offspring. However, this exposure may be particularly relevant to immune function of the gut, as IL‐8 remains measurable throughout simulated neonatal gastric and proximal intestinal digestion (41). When human fetal intestinal cells are stimulated with rhIL‐8 in vitro , there is an increase in cell migration, proliferation, and differentiation (42).…”

High-dose erythropoietin inhibits apoptosis and stimulates proliferation in neonatal rat intestine

“…For example, epidermal growth factor (EGF) is developmentally regulated, and has an important role in preserving gut barrier function, increasing intestinal enzyme activity, and improving nutrient transport, and supplementation with EGF improves outcome from NEC in rat models [16][17][18][19]. Interleukin-8 increases intestinal epithelial cell migration and proliferation, and protects against tumor necrosis factor alpha-induced cytotoxicity [20,21]. The trefoil peptides are also interesting because they protect against hypoxia-induced NEC in rats [22,23] and because they are downregulated in human intestine from NEC patients [24].…”

Transforming growth factor-β2 and endotoxin interact to regulate homeostasis via interleukin-8 levels in the immature intestine

“…In newborn mice, intestinal exposure to LPS stimulates TLR-4 signaling and NF-B activation leading to NEC development (11). Furthermore, in NEC-affected tissue, IL-8, a member of the CXC chemokine family with chemotactic activity, is found in excessive amounts and has been speculated to be associated with severe inflammatory conditions (5,21). However, in the fetal intestine, IL-8 exerts other effects, stimulating maturation, but without associated inflammation (23).…”