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 2

VAMs prime gut EC activation.

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VAMs prime gut EC activation. (A–D) CD45–LYVE1–CD31+ ECs were FACS-sorte...
(A–D) CD45–LYVE1–CD31+ ECs were FACS-sorted from LPS-stimulated intestinal lamina propria with and without macrophage depletion and subjected to mRNA sequencing. (A) Principal component analysis comparing macrophage-intact/-depleted (MΦ-intact/MΦ-depletion) conditions based on differentially expressed genes (DEGs). (B) Gene ontology (GO) pathway enrichment analysis of DEGs. The top 20 enriched terms are shown. (C) Expression heatmaps of chemokines and (D) cellular adhesion molecules (CAMs) relevant to PMNs indicate macrophage priming of EC responses. Color scales represent percentage change in gene expression. (E–H) CSF-1R Ab–mediated macrophage depletion in control or LPS-stimulated CX3CR1-EGFP mice. To visualize and quantify EC ICAM-1 expression, a low dose of fluorescently labeled anti–ICAM-1 Ab (2 μg, i.v.) was used (red). (E) Representative whole-mount confocal microscopy images show VAM localization to high ICAM-1 regions with LPS stimulation and loss of local ICAM-1 enrichment with macrophage depletion. Scale bar: 20 µm. (F) Quantification of ICAM-1 expression normalized to PECAM-1 staining to account for tissue depth variation. (G) Quantification of the relative ICAM-1 expression per 25 μm vessel segments with and without macrophage contact. (H) Comparison of the relative ICAM-1 expression per 25 μm vessel segments with and without macrophage depletion. Dotted region highlights the loss of ICAM-1 hot spots. (I) Quantification and (J) representative flow diagram of mucosal EC ICAM-1 expression by flow cytometry. (K) Representative flow diagrams and (L) quantification of ICAM-1 expression in cultured mouse (bEnd.3) and human (HUVEC) ECs, respectively, treated with conditional media from murine BM-derived and human THP-1 monocytic cell line, differentiated with IFN-γ/LPS so that they resemble tissue inflammatory macrophages. For whole-mount preparations, images are representative of n = 3–5 mice, with each data point representing a field of view. For flow cytometry, n = 3–4 independent experiments. Spnt, supernatant. *P < 0.05, **P < 0.01, ***P < 0.001. Two-sided Student’s t test and 1-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SEM.