Macrophage–endothelial cell crosstalk orchestrates neutrophil recruitment in inflamed mucosa
Xingsheng Ren, … , Edward B. Thorp, Ronen Sumagin
Xingsheng Ren, … , Edward B. Thorp, Ronen Sumagin
Published June 1, 2023
Citation Information: J Clin Invest. 2023;133(15):e170733. https://doi.org/10.1172/JCI170733.
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Research Article Cell biology Inflammation

Macrophage–endothelial cell crosstalk orchestrates neutrophil recruitment in inflamed mucosa

Abstract

Neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires transendothelial migration (TEM). TEM involves several well-studied sequential adhesive interactions with vascular endothelial cells (ECs); however, what initiates or terminates this process is not well-understood. Here, we describe what we believe to be a new mechanism where vessel-associated macrophages through localized interactions primed EC responses to form ICAM-1 “hot spots” to support PMN TEM. Using real-time intravital microscopy of LPS-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we found that macrophage vessel association is critical for the initiation of PMN-EC adhesive interactions, PMN TEM, and subsequent accumulation in the intestinal mucosa. Anti–colony stimulating factor 1 receptor Ab-mediated macrophage depletion in the lamina propria and at the vessel wall resulted in elimination of ICAM-1 hot spots impeding PMN-EC interactions and TEM. Mechanistically, the use of human clinical specimens, TNF-α–KO macrophage chimeras, TNF-α/TNF receptor (TNF-α/TNFR) neutralization, and multicellular macrophage-EC-PMN cocultures revealed that macrophage-derived TNF-α and EC TNFR2 axis mediated this regulatory mechanism and was required for PMN TEM. As such, our findings identified clinically relevant mechanisms by which macrophages regulate PMN trafficking in inflamed mucosa.

Authors

Xingsheng Ren, Laura D. Manzanares, Enzo B. Piccolo, Jessica M. Urbanczyk, David P. Sullivan, Lenore K. Yalom, Triet M. Bui, Connor Lantz, Hinda Najem, Parambir S. Dulai, Amy B. Heimberger, Edward B. Thorp, Ronen Sumagin

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Figure 1

Macrophages promote PMN adhesion and TEM in inflamed intestinal mucosa.

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Macrophages promote PMN adhesion and TEM in inflamed intestinal mucosa. ...
Inflammation of the intestinal mucosa was induced by i.p. administration of LPS (100 μg, 24 hours). (A) Representative whole-mount confocal microscopy images using CX3CR-EGFP reporter mice showing interstitial macrophage recruitment and association with blood vessels during LPS-induced inflammation. Scale bar: 20 μm. (B) Quantification of CX3CR1+ macrophage (MΦ) numbers per field of view and (C) VAMs per vessel length. Blood vessels were visualized using PECAM-1 (CD31, blue) staining. (D–J) Intravital microscopy (IVM) of inflamed intestines was performed on CX3CR1-EGFP mice with and without macrophage depletion with a CSF-1R Ab (400 μg/mouse, every other day for 3 weeks). PMNs were labeled by a low dose of fluorescently labeled anti-Ly6G Ab (2 μg, i.v.). (D) Representative images of VAM depletion and PMN tissue infiltration by IVM. Scale bar: 20 µm. Arrows indicate extravasated PMNs. (E) Quantification of tissue PMNs by IVM and (F) by flow cytometry of digested intestinal lamina propria. A representative flow diagram is shown (right). (G) Representative time-lapse images (based on a real-time acquisition) show decreased adherent PMN and increased displacement (dotted white arrows) of rolling PMNs in macrophage-depleted animals. Solid white arrows denote adherent PMNs in area of VAM-EC contact. Scale bar: 20 μm. (H) Quantification of adherent and (I) rolling PMN (per 30 seconds) and (J) rolling velocities of individual PMNs with and without macrophage depletion. For whole-mount preparation, images are representative of n = 4–5 mice per condition. For IVM, n = 3–5 mice per condition. **P < 0.01, ***P < 0.001. Two-sided Student’s t test. Data represent mean ± SEM.