Contribution of Mast Cells in Non-Allergic Ocular Inflammation

This is a competitive renewal application to further investigate the mechanisms by which IgE-independent
activation of mast cells contributes to ocular inflammation. Mast cells have garnered much attention over the
past decade for their diverse IgE-independent effector function in the setting of non-allergic inflammatory
diseases. Nevertheless, fundamental questions remain unanswered regarding the function and immune
mechanisms of mast cells in mediating ‘non-allergic’ ocular pathologies. Our laboratory has made substantial
progress in understanding how ocular mast cells contribute to the non-allergic inflammatory response. Reports
from our lab provide evidence that (i) epithelial cell-derived interleukin-33 activates mast cells in an IgE-
independent manner and that mast cells (ii) as resident immune cells initiate neutrophil infiltration by releasing
CXCL2, and (iii) promote pathological growth of blood and lymph vessels in part by secreting high levels of
VEGFs. Moreover, our robust preliminary findings indicate that mast cells augment the tissue-damaging function
of neutrophils and that deficiency of mast cells results in faster regression of mature pathological corneal vessels.
However, the exact mechanisms by which mast cells interact with neutrophils and vascular cells to promote their
pathological functions are not well defined.
In accordance with our laboratory’s expertise in immunological studies and our well-established murine
models of corneal injury and neovascularization, we propose a series of novel experiments to decipher the
function of mast cells as an orchestrator of inflammation and tissue damage. Our pilot investigations show that
mast cells express high levels of neutrophil (granulocyte)-stimulating factors, GM-CSF and G-CSF. In Aim 1, we
will test the hypothesis that Mast cell-derived granulocyte stimulating factors (GM-CSF and G-CSF) promote the
release of tissue-damaging cytokines and enzymes by neutrophils following corneal injury. Specifically, we will
assess the effect of GM-CSF versus G-CSF deficient mast cells (using CRISPR-Cas9) and neutralizing antibody
treatment on (i) neutrophil effector function and survival using our standardized in vitro co-culture assays and (ii)
neutrophil-mediated tissue damage in the corneal injury model. In Aim 2, based on our preliminary data of mast
cells expressing high levels of vessel stabilizing Angiopoietin 1 (Ang1), we will test the hypothesis that Mast cells
juxtaposing pathological vessels prolong vascular endothelial cell (VEC) survival and maintain vessel integrity
by secreting Ang1. Specifically, we will determine the effect of (i) mast cells and VECs interaction on Ang1-Tie2
ligand-receptor axis, (ii) Ang1-deficient mast cells on the stability of pathological blood vessels. We will also
evaluate the relative therapeutic efficacy of topical application of Ang1 blocked versus general mast cell inhibitor
in accelerating vessel regression. It is anticipated that the completion of these aims will elucidate as-yet-unknown
mechanisms of mast cell function in non-allergic inflammation and provide a framework to develop new
therapeutics for tissue injury and angiogenesis.

Grant Number: 5R01EY029727-07
NIH Institute/Center: NIH
Principal Investigator: Sunil Chauhan