Abstract
Necrotizing enterocolitis (NEC) is a life-threatening disease in premature infants, causing severe inflammation in the gastrointestinal tract, specifically the ileo-cecal region. Mechanistically, NEC inflammation largely occurs due to the activation of lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) signaling pathway. Upon activation of this upstream signaling pathway, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is activated, which leads to the downstream secretion of key proinflammatory cytokines, including interleukin-8 (IL-8). Human milk (HM) has been found to reduce NEC risk, and recent studies indicated that certain digestion-released HM peptides may play a role in this protective effect. However, successful delivery of these peptides to the ileo-cecal region is a great challenge.
In this study, the potentials of type 1 secretion system (T1SS)-engineered Escherichia coli DH5α were explored to secrete three digestion-released HM small peptides (SP2, SP5, and SP6). Immunomodulating effects of the secreted peptides in the bacterial supernatants were then evaluated in human intestinal epithelial cell line-6 (HIEC-6). The secretion of the peptides was examined through dot blot analysis, followed by image-based quantification. The functional activity was assessed by measuring the IL-8 concentrations in HIEC-6 supernatants under LPS-stimulated and unstimulated conditions, with or without treatments with SP-containing bacterial supernatants collected at 3, 6, 9, and 12 hours.
All three peptides were successfully secreted, with SP5 exhibiting both the highest signal intensity and cumulative outcomes, followed by SP2 and SP6. The SP-containing supernatants consistently increase the IL-8 concentrations compared to controls without SP, suggesting a strong pro-inflammatory pattern. Overall, this research shows that T1SS-engineered bacteria can successfully secrete HM-derived anti-inflammatory peptides, demonstrating the potential of using non-pathogenic bacterial secretion systems to deliver anti-inflammatory peptides. However, the SP-containing supernatants induced inflammation in this study, requiring additional investigation to establish this as a delivery mechanism.