The Anti-Inflammatory and Immunomodulatory Role of Protein Hyrolysates: Involvement of Epigenetics and Gut Microbiome

Maintaining gut immune system and microbiota homeostasis is crucial to the individual's health. Any disruptions in gut homeostasis can lead to immune dysfunction not only at the gut level, but also at the distant organs from the gut. Bioactive peptides and hydrolysates from natural sources, including eggshell membrane hydrolysate (ESMH), have been tested in animal models of human diseases for potential anti-inflammatory roles. ESMH has been found to reduce inflammation in the gut-lung axis, prevent dysbiosis, and improve gut microbiota and immune response. Puberty is a critical developmental window of life during which substantial changes in immune system and gut microbiota happens. Previous studies have shown puberty as a sensitive period to immune stressor in which uncorrected inflammation and dysbiosis is accompanied by peripheral and central immune dysfunction and long-term changes is brain function. However, acute, and long-term effects of pubertal exposure to an immune stressor on gut-lung axis immunity has not been sufficiently explored. Therefore, in the current study, we used pubertal lipopolysaccharide (LPS)-induced inflammation in female Balb/c mice to simulate inflammation and dysbiosis during puberty. We hypothesized that LPS-induced inflammation during puberty induces dysbiosis and causes acute and/or enduring alterations in selected cytokines, miRNAs profile and DNA methylation of genes related to the Th17 mediated immunity at the gut and lungs level, while ESMH intake can mitigate effects of pubertal LPS on gut microbiota perturbation and epigenetic alterations. The objectives of this project are: 1. To investigate the role of ESMH in mitigating gut microbiota dysbiosis induced by the acute exposure to the LPS during puberty; 2. To study the role of ESMH in mitigating the acute and lasting effects of pubertal exposure to the LPS on cytokines and epigenetics alterations, including miRNAs related to the Th17 mediated immunity at the gut/lung levels. Furthermore, to examine the DNA methylation of the genes in the lungs, related to the Th17, in the enduring phase. Our results revealed that ESMH consumption partially reduced LPS mediated changes in the gut microbiota, by reducing the levels of Alistipes and segmented filamentous bacterium Candidatus arthromitus. and increasing the abundance of Lactobacillus The results revealed an acute effect of LPS in increasing IL-6 and IL-10 levels in the gut, while ESMH intake was associated with an increase in IL-6 and IL-10 levels in the lungs. ESMH intake also mitigated lasting stimulatory effects of LPS on proinflammatory miRNAs, including miR-155, miR-222, and miR-223 at the lung levels. DNA methylation analysis revealed the role of ESMH in modulating LPS-induced alteration in methylation status of several genes related to Th17 mediated immunity in the lungs, including Tnfaip3, Tnip1, Lcn2, Sox2ot, Ermap, Cxcl11, Rorα, Mcc, Prkcb, Socs7 and Nlrc4. These results in mice may suggest ESMH as a potential strategy for alleviating inflammatory response accompanying a challenge by an immune stressor at puberty by maintaining immune and epigenetic homeostasis in the gut-lung axis.