Macrophage–endothelial cell crosstalk orchestrates neutrophil recruitment in inflamed mucosa
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
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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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 7

Evidence of VAM recruitment and VAM-EC-PMN interactions in clinical IBD specimens.

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Evidence of VAM recruitment and VAM-EC-PMN interactions in clinical IBD ...
(A–D) Single-cell RNA-Seq was performed on biopsies from patients with active UC. (A) Uniform manifold approximation and projection (UMAP) analyses of integrated data from 4 patients with IBD. (B) Dot plot showing scaled expression of selected signature genes for PMN, macrophage, and EC clusters. Gene expression in each cluster was scaled across all clusters. Dot size represents the percentage of cells in each cluster, and color indicates expression (number of reads). (C) Outgoing communication pattern analyses by CellChat, specifically focused on network centrality analysis of inferred TNF-α signaling with macrophages defined as senders. Interaction strengths are shown/scaled between annotated seurat clusters. (D) GO analysis of DEGs for the CCL3/4hi macrophage cluster. The top 12 GO enrichment terms are shown. Analyses were performed with a Fisher’s exact test, with P < 0.01. Terms shown in red highlight more relevant terms consistent with observations made in a murine model. (E–J) Multiplex immunofluorescence staining was performed on healthy biopsied tissue and biopsied tissue from patients with active UC (n = 4 patient/conditions). (E) Quantification from multiplex imaging of PMN tissue infiltration and (F) VAM numbers. (G) Representative multiplex immunofluorescence images. White dotted circles highlight VAMs. Scale bar: 50 μm. Zoom-in panels (on the right) depict healthy and inflamed vessels with respective recruitment of VAMs. PMNs leaving the vessels specifically at a region of VAM contact and PMN-macrophage interactions in the interstitial are shown. (H and I) Quantification of VAM and interstitial macrophage interactions with PMNs and (J) the frequency of PMN-EC interactions specifically at regions of EC-VAM contact or ECs remote from VAMs (ECs alone). For all image analyses, 5–8 images per patient were analyzed. Each data point represents a field of view. (K) Representation schematic summarizing the mechanistic VAM regulation of EC function and PMN TEM. ***P < 0.001. Two-sided Student’s t test. Data are presented as mean ± SEM.