RSV-induced type 2 immune responses are inhibited by dietary citrulline supplementation during gestation and early life

In the last funding cycle of this AADCRC, Project 1 (Dr. Hartert – Leader) found that increased blood levels of L-citrulline (L-CIT) were significantly associated with decreased odds of infant bronchiolitis (odds ratio (OR) 0.68, 95% confidence interval (CI) 0.48-0.95). These data suggest that higher blood levels of L-CIT at time of birth protected against infant bronchiolitis and may prevent development of asthma later in childhood. We therefore hypothesize that compared to regular diet (RD), an L-CIT supplemented diet (L-CITsd) fed to parents during gestation and their offspring prevents severe RSV bronchiolitis in the offspring. Our preliminary data reveal that the L-CITsd decreased RSV-induced lung IL-13 expression, reduced the frequency of lung IL-13 expression by ILC2, and restrained airways responsiveness. Further, L-CITsd significantly decreased ILC2 expression of IL-13 when the cells were stimulated ex vivo with IL-33, and L-CIT added to ILC2 culture inhibited IL-33-induced IL-13 production. Elevated levels of ILC2, IL-33, and IL-13 are associated with severe RSV infection in infancy. These finding are important because males have a greater risk of severe RSV-induced disease; therefore, L-CITsd provides a possible benefit to the sex most likely to have severe RSV bronchiolitis. Importantly, these protective effects of L-CITsd did not come at a cost of worsening adversely affecting lung anti-viral immunity to RSV. L-CIT is currently not present in prenatal vitamins and the major sources of citrulline (watermelon) is not commonly introduced into the diets of infants until after six months of life, a time after which children are at greatest risk for RSV bronchiolitis. Aim 1 is to determine the mechanism by which L-citrulline dietary supplementation inhibits RSV-induced type 2 immunopathology. In this aim, we will determine: a) the effect of L-CITsd on IL-33-driven ILC2 cytokine production and immunopathology, b) the protective effect of L-CITsd on ILC2 epigenetic regulation, c) the durability of the protection of L-CITsd during the first infection to subsequent infection, and d) the ability of L-CITsd to inhibit human lung ILC2 function and proliferation. Aim 2 is to define the effect of L-CIT on airway epithelial function in response to RSV infection. In this aim, we will determine: a) the ability of L-CITsd to inhibit RSV-induced ROS generation in mice, and b) the effect of L-CIT on ROS generation, cytokine/chemokine production, and membrane barrier function by primary nasal airway epithelial cells obtained from the INSPIRE cohort. Project 2 is also highly innovative because it will be the first to: 1) determine the role of L-CITsd in decreasing RSV-induced immunopathology, 2) define the effect of L-CITsd on ILC2 epigenetic regulation, 3) determine how L-CITsd regulates epithelial cell function in vivo in the setting of respiratory viral infection, and 4) use primary airway epithelial cells from infants to determine the effect of L-CIT on RSV-induced cytokine gene expression.