Evolution of In Vitro Cow's Milk Protein–specific Inflammatory and Regulatory Cytokine Responses in Preterm Infants With Necrotising Enterocolitis

Abstract

We have previously reported evidence of in vitro sensitisation to cow's milk protein in peripheral blood mononuclear cells (PBMCs) in preterm infants with necrotising enterocolitis (NEC). In the present study, we document the changes in the PBMC responses to stimulation with mitogen (phytohaemagglutinin) and cow's milk proteins β-lactoglobulin (β-lg) and casein over time: from the acute presentation of NEC, to initial recovery (reinitiation of enteral feeds), to full recovery (full feeding). Of the 14 preterm infants recruited with acute NEC, 12 were followed until fully enterally fed (2 died during the acute phase). Cytokine secretion (interferon-γ [IFN-γ], interleukin 4, [IL-4], IL-10, and transforming growth factor-β1 [TGF-β1]) by PBMCs in response to stimulation by phytohaemagglutinin, β-lg, and casein was measured by enzyme-linked immunospot in the acute phase and subsequently at recovery and full recovery. The high levels of cytokine secretion (IFN-γ, IL-4, IL-10, and TGF-β1) observed in response to β-lg and casein in the acute phase increased by a further 50% to 100% at recovery (P < 0.005). At full recovery (full feeding), however, IFN-γ, IL-4, and IL-10 secretion response had returned to, or below, acute-phase levels, whereas the augmented TGF-β1 response was maintained (P = 0.005 vs acute level). This response pattern was similar for casein, and did not appear to be influenced by the nature of the feed used following NEC (breast milk/formula/hydrolysed formula). The evolution of the cytokine response profile in parallel with the clinical recovery from NEC is consistent with a putative role for TGF-β1 in regulation of inflammation, and possibly also oral tolerance.