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 5

Inflamed intestinal ECs express TNFR2 to interact with VAM-derived TNF-α.

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Inflamed intestinal ECs express TNFR2 to interact with VAM-derived TNF-α...
(A and B) Combined in situ and whole-mount staining and confocal microscopy was performed on CX3CR-EGFP reporter mice to examine expression of TNF-α and its receptors TNFR1 and TNFR2. (A) Representative images show TNF-α expression (red) by interstitial macrophages (elevated specifically in VAMs). TNFR2 but not TNFR1 is expressed by gut ECs. Scale bar: 25 μm. (B) Quantification of mean fluorescence intensity (MFI) in interstitial macrophages remote from vessels and in VAMs. (C and D) Flow cytometry–based analyses were performed on LPS-stimulated, digested intestinal mucosa. (C) Quantification of TNF-α expression in CD45+CX3CR1+ gut macrophages and (D) TNFR1/TNFR2 in CD45–LYVE1–CD31+ ECs. For whole-mount preparations, images are representative of n = 4 independent experiments. Each data point represents a field of view. For flow cytometry, n = 3 independent experiments. **P < 0.01, ***P < 0.001. Two-sided Student’s t test. Data are presented as mean ± SEM.