Use of probiotics to modulate antimicrobial peptide signaling in the neonatal innate immune response to influenza virus

Project Abstract:
The mortality rate from influenza viral (IV) infection is highest in infants less than age six months, currently an
age group not eligible for the available influenza vaccine, but the mechanisms for this clinical observation are
not well understood. Vulnerability to respiratory viruses during infancy is likely manifested by an immature lung
and immune system. To understand age-specific differences in hematopoietic and epithelial cell function at the
air-blood interface in the developing lung, an age-appropriate pre-clinical neonatal murine IV infection model is
employed. Previously, we demonstrated that murine neonates are exceptionally susceptible to IV infection.
Differences in the dominant airway microbial communities in these first few months of life have been linked to
susceptibility to respiratory infections. Commensal-derived signals establish an activation threshold of the innate
immune system required for optimal antiviral immunity. Therefore, modulating early airway microbial
communities presents a potential therapeutic strategy to prevent or ameliorate respiratory tract infections.
Recently, we demonstrated that intratracheal administration of Lactobacillus rhamnosus (LGG), a probiotic, prior
to IV infection improved neonatal survival. However, the specific mechanisms by which LGG acts, the cells on
which it exerts the most effect and the dominant pattern recognition receptors it modulates remains to be
determined. The goal of this project is to determine mechanisms by which these LGG-derived signals impact the
function of 1. pulmonary epithelial cells, the target of IV infection, and 2. innate immune cells, such as alveolar
macrophages (AMs), sentinel tissue-resident immune cells which drive immune cell recruitment, and neutrophils,
which are pathologic in the developing lung during respiratory viral infection. We have identified an antimicrobial
peptide as a potential signaling mediator between infected epithelial cells and AMs. Our recently published work
demonstrates that murine neonates deficient in Catheliciden-related Antimicrobial Peptide (CRAMP) are
protected during IV infection. Importantly, LGG pretreatment decreases CRAMP production. Here, we seek to
investigate the pathogenic role of CRAMP as a signaling mediator to orchestrate the initial response to IV. The
overarching hypothesis of this proposal is that in IV-infected neonates, LGG reduces CRAMP production by
epithelial cells, which diminishes CRAMP-mediated alveolar macrophage (AM) activation through Toll-like
receptor 2 (TLR2) and subsequent neutrophil chemoattractant production. Neutrophil infiltration is a potent driver
of mortality. To test our hypothesis, we will: (1) Define IV tropism and the primary cell source of CRAMP in LGG
treated and untreated IV-infected murine neonates and adults using single cell sequencing; (2) Evaluate the age-
specific role of LGG and CRAMP-dependent TLR2 signaling in key innate immune cell production of neutrophil
chemoattractants; and (3) Determine if CRAMP acts directly on neutrophils to increase oxidative stress. Our
studies in an innovative neonatal pre-clinical animal IV model will bring forth new understanding of infant mucosal
immunity to develop targeted therapeutics for the infant population.

Grant Number: 1R56AI183675-01A1
NIH Institute/Center: NIH
Principal Investigator: Alison Carey