# Molecular mechanism and preclinical translation of beta2 integrin auto-inhibition on neutrophil arrest and inflammation

> **NIH NIH R01** · UNIVERSITY OF CONNECTICUT SCH OF MED/DNT · 2020 · $406,608

## Abstract

Neutrophils are the most abundant population of leukocytes in humans and play essential roles in
innate immunity and inflammation, including cardiovascular inflammatory diseases, such as
ischemia-reperfusion injury (IRI), post-myocardial infarction inflammation and atherosclerosis.
Neutrophil adhesion is a key step in neutrophil recruitment from blood vessels to inflamed tissues.
Human neutrophils arrest on activated endothelium under flow using the beta2 integrins. My previous
work confirmed the known pathway of beta2 integrin activation (extension E followed by high-affinity H;
E-H- to E+H- to E+H+) and discovered a new pathway where the headpiece opens before the integrin
extends during arrest of primary human neutrophils (E-H- to E-H+ to E+H+). The newly identified
bent-open (E-H+) beta2 integrin binds ligands (ICAMs) expressed on neutrophils in cis. I showed that
this auto-inhibition limits neutrophil adhesion in vitro and in vivo. The proposed work will, for the first
time in this field, interrogate beta2 integrin activation by super-resolution microscopy. I refined the
preliminary data points by molecular modeling and achieved single molecule resolution. My data show
that E-H+ integrins are not randomly oriented, but show a molecular pattern consistent with a
`Face-to-Face' orientation. In specific aim 1, I will test the hypothesis that this `Face-to-Face' pattern is
caused by pairwise in-cis interactions of E-H+ integrins binding to ICAM dimers. If so,
function-blocking ICAM antibodies should disrupt this `Face-to-Face' pattern. Non-blocking ICAM
antibody will be used to test whether ICAMs are co-localized with E-H+ beta2 integrins as expected. In
specific aim 2, I will screen small molecule allosteric inhibitors that keep beta2 integrins in the
auto-inhibited E-H+ conformation. In my preliminary experiments, I already developed a
flow-cytometry-based screening assay of integrin activation (E+ and H+). I will test compounds in two
libraries to find candidates. I propose to confirm the efficacy of successful candidate molecules in
primary neutrophils using flow cytometry and in established microfluidic adhesion assays. Specific aim
3 is to directly test the physiologic significance of E-H+ integrins. I will test the hypothesis that
auto-inhibition of E-H+ beta2 integrins protects cardiomyocytes from IRI. I will use mice transplanted
with ICAM-1 and ICAM-2 double knockout bone marrow, which I have previously shown to eliminate
the auto-inhibition of beta2 integrin on neutrophils. I expect these chimeric mice to show more severe
myocardial IRI and tissue loss. Successful inhibitors from aim 2 will be tested in this IRI model in vivo.
After completion of these experiments, we will know the molecular details of beta2 integrin activation
during arrest of primary human neutrophils (aim 1), find candidate small molecule inhibitors that
stabilize the E-H+ beta2 integrin conformation (aim 2), and know the in vivo relevance of E-H+ beta2
integrins (...

## Key facts

- **NIH application ID:** 10062651
- **Project number:** 7R01HL145454-02
- **Recipient organization:** UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
- **Principal Investigator:** Zhichao Fan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $406,608
- **Award type:** 7
- **Project period:** 2019-11-26 → 2024-03-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10062651

## Citation

> US National Institutes of Health, RePORTER application 10062651, Molecular mechanism and preclinical translation of beta2 integrin auto-inhibition on neutrophil arrest and inflammation (7R01HL145454-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10062651. Licensed CC0.

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