Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection
Tengfang Li, Zhongqiang Zhang, Joe G. Bartolacci, Gaelen K. Dwyer, Quan Liu, Lisa R. Mathews, Murugesan Velayutham, Anna S. Roessing, Yoojin C. Lee, Helong Dai, Sruti Shiva, Martin H. Oberbarnscheidt, Jenna L. Dziki, Steven J. Mullet, Stacy G. Wendell, James D. Wilkinson, Steven A. Webber, Michelle Wood-Trageser, Simon C. Watkins, Anthony J. Demetris, George S. Hussey, Stephen F. Badylak, Hēth R. Turnquist
Tengfang Li, Zhongqiang Zhang, Joe G. Bartolacci, Gaelen K. Dwyer, Quan Liu, Lisa R. Mathews, Murugesan Velayutham, Anna S. Roessing, Yoojin C. Lee, Helong Dai, Sruti Shiva, Martin H. Oberbarnscheidt, Jenna L. Dziki, Steven J. Mullet, Stacy G. Wendell, James D. Wilkinson, Steven A. Webber, Michelle Wood-Trageser, Simon C. Watkins, Anthony J. Demetris, George S. Hussey, Stephen F. Badylak, Hēth R. Turnquist
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Research Article Immunology

Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection

Abstract

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell–derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33–deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix–based materials may be a promising biologic for chronic rejection prophylaxis.

Authors

Tengfang Li, Zhongqiang Zhang, Joe G. Bartolacci, Gaelen K. Dwyer, Quan Liu, Lisa R. Mathews, Murugesan Velayutham, Anna S. Roessing, Yoojin C. Lee, Helong Dai, Sruti Shiva, Martin H. Oberbarnscheidt, Jenna L. Dziki, Steven J. Mullet, Stacy G. Wendell, James D. Wilkinson, Steven A. Webber, Michelle Wood-Trageser, Simon C. Watkins, Anthony J. Demetris, George S. Hussey, Stephen F. Badylak, Hēth R. Turnquist

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Figure 5

IL-33 mediates an FA-dependent poising of macrophages for differentiation into a reparative and regulatory subset by promoting oxidative phosphorylation and negatively regulating iNOS expression.

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IL-33 mediates an FA-dependent poising of macrophages for differentiatio...
(A) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined for B6 bone marrow–derived macrophages (BMDMs) left untreated (M0) or stimulated with LPS plus IFN-γ, or IL-4, or IL-33. One representative experiment of 3 independent experiments is shown. (B) Calculated mitochondrial basal respiration and ATP production for replicates from 1 experiment. (C) Relative concentration (normalized to internal standard [ISTD] and cell number) of TCA metabolites or citrulline in lysates after overnight culture in media alone (M0) or media with LPS plus IFN-γ, or IL-4, or IL-33; or LPS plus IFN-γ and IL-33 determined using liquid chromatography–high-resolution mass spectrometry. n = 6 samples/group. (D and E) Macrophages stimulated as above alone (non-treated, NT) or with 200 μM sulfosuccinimidyl oleate (SSO) for 24 hours were assessed by flow cytometry. (D) Plots of CD45+CD11b+Ly6G–F4/80+-gated cells. (E) Frequency of CD206+CD301+ cells in the CD45+CD11b+Ly6G–F4/80+ gate. n = 3 samples/group. (F) F4/80+ macrophages from WT or St2–/– B6 mice were treated as in G before isolated RNA was assessed for Nos2 by qRT-PCR. (H) WT B6 macrophages were first incubated with IL-33 overnight and then stimulated for 2 hours with LPS plus IFN-γ before RNA isolation and Nos2 qRT-PCR. n = 3 mice/group. Data are from 1 experiment and representative of 2 completed. (H) Macrophages treated as in C were assessed by immunoblotting for iNOS. Quantification of immunoblotting mean pixel density was performed using ImageJ and loading was normalized to β-actin (n = 3). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 by 1-way ANOVA.