IL-4–STAT6 axis amplifies histamine-induced vascular endothelial dysfunction and hypovolemic shock - 27/06/24

Doi : 10.1016/j.jaci.2024.05.009

James Krempski, PhD ^a, Amnah Yamani, PhD ^a,^c, Lakshmi Narasimha Rao Thota, PhD ^a, Sahiti Marella, PhD ^b, Varsha Ganesan, BSc ^a,^b, Ankit Sharma, PhD ^a, Atsunori Kaneshige, PhD ^d,^e,^f, Longchuan Bai, PhD ^d,^e,^f, Haibin Zhou, PhD ^d,^e,^f, Paul S. Foster, PhD ^g, Shaomeng Wang, PhD ^d,^e,^f, Andrea T. Obi, MD ^h, Simon P. Hogan, PhD ^a,^b,^⁎
^a Mary H. Weiser Food Allergy Center, Michigan Medicine, University of Michigan, Ann Arbor, Mich
^b Department of Pathology, Michigan Medicine, University of Michigan, Ann Arbor, Mich
^c Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
^d Department of Internal Medicine, University of Michigan, Ann Arbor, Mich
^e Department of Pharmacology, University of Michigan, Ann Arbor, Mich
^f Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Mich
^g School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, and Immune Health Program, Hunter Medical Research Institute, Newcastle, Australia
^h Conrad Jobst Vascular Research Laboratories, University of Michigan Medical School, Ann Arbor, Mich

^∗Corresponding author: Simon P. Hogan, PhD, Mary H. Weiser Food Allergy Center, Department of Pathology, Michigan Medicine, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200.Mary H. Weiser Food Allergy CenterDepartment of PathologyMichigan MedicineUniversity of Michigan109 Zina Pitcher PlaceAnn ArborMI48109-2200

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Abstract

Background

Mast cell–derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We previously revealed a synergistic interaction between the cytokine IL-4 and the mast cell–derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis is unknown.

Objective

We sought to identify the IL-4–induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylactic reactions.

Methods

RNA sequencing, Western blot, Ca²⁺ imaging, and barrier functional analyses were performed on the VE cell line (EA.hy926). Pharmacologic degraders (selective proteolysis-targeting chimera) and genetic (lentiviral short hairpin RNA) inhibitors were used to determine the roles of signal transducer and activator of transcription 3 (STAT3) and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock.

Results

IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-cadherin degradation, intracellular calcium flux, and phosphorylated Src levels and required transcription and de novo protein synthesis. RNA sequencing analyses of IL-4–stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth, and transcription factor motif analyses revealed a significant enrichment of differential expressed genes with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both serine residue 727 and tyrosine residue 705 phosphorylation of STAT3. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function; however, IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3 independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6 dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4–mediated amplification of histamine-induced hypovolemia.

Conclusions

These studies unveil a novel role of the IL-4/STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.

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Key words : Anaphylaxis, vascular endothelium, histamine, IL-4, transcription

Abbreviations used : [Ca⁺²]_i, ChIP-Seq, CHX, DAVID, DEG, FC, FDR, GO, HC, HUVEC, HRP, IL-4Ra, phospho-Src, PROTAC, R_b, RNA-Seq, S727, shRNA, STAT, TER, TF, VE, WT, Y705