ABSTRACT:
Necrotizing enterocolitis (NEC) is a deadly disease of the newborn that affects 7% of patients admitted to the
neonatal intensive care unit and is a leading cause of gram negative, neonatal sepsis. Sepsis is the second
major cause of death among neonates, accounting for one million global deaths per year. Although we have
made progress understanding the effect of maternal colonization patterns on neonatal sepsis, there is a gap in
the understanding of the regulatory processes that regulate intestinal barrier function that contribute to
susceptibility to sepsis. Indeed, the intestine is the largest immune organ in the body and is responsible for
maintaining a barrier to the microbial world within its lumen. Therefore, it is a high priority for the development of
medical interventions to prevent and treat neonatal sepsis. Rho kinases (ROCK) are serine/ threonine kinases
and are involved in multiple cellular processes including regulating tight junction function, actin cytoskeleton
contraction, inflammatory cytokines and cell death. We found that Rho-associated protein kinase (ROCK) is
activated during NEC, and inhibition of ROCK confers protection against intestinal barrier injury and disruption
of normal TJ homeostasis. Our data suggest that TJ regulation, intestinal barrier function and apoptosis are
regulated by a ROCK dependent, caveolin-1 mediated pathway. Furthermore, we have identified differences in
TJ subcellular distribution and caveolin-1 both in patients with and without sepsis and NEC, and in those of
different gestational ages. The objective of our proposed study is to define the mechanism by which barrier loss
occurs in NEC and to leverage this in order to understand the potential of epithelial-specific ROCK inhibition in
preventing NEC and neonatal sepsis. Our central hypothesis is that the response of the intestinal barrier in
premature infants to injury is altered and is characterized by changes in epithelial ROCK signaling, aberrant TJ
regulation, increased mucosal and systemic inflammation, sepsis and epithelial cell death. We will test this
hypothesis by determining whether inhibition of TJ protein expression and redistribution or caveolin-1-mediated
endocytosis prevent paracellular permeability increases, mucosal and systemic inflammatory responses, and
epithelial cell death in experimental sepsis and NEC. The use of intestinal organoids generated from human
infants and from ROCK and caveolin-1 knockout mice, will provide tremendous translational power and allow us
to probe epithelial functions at molecular, cellular, tissue, and organismal levels. Using pharmacological and
genetic approaches, we will determine if and how TJ responsiveness and ROCK signaling increases cell death
to worsen outcomes in premature infants and whether our interventions prevent or induce sepsis. These findings
have a significant positive impact on human health by providing a new understanding of the mechanisms
governing epithelial intestinal barrier function of the premature infant during NEC and neonatal sepsis.
