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The intestinal macrophage pool represents the largest population of macrophages present within the body. Nevertheless, flow cytometry analysis of intestinal macrophages remains challenging due to historical lack of consensus on surface markers, variations in sample preparation, and a certain capriciousness of the isolation procedure itself. Furthermore, recent studies have uncovered a hitherto unknown heterogeneity of intestinal macrophages, accompanied by a vast increase of subset-identifying surface markers. Here, the isolation procedure for intestinal tissue for flow cytometry analysis is laid out, with particular attention toward the procedures for isolated intestinal layers, and a trouble-shooting section with strategies to avoid common pitfalls and mistakes., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Muscularis macrophages, as the most abundant immune cells in the intestinal muscularis externa, exhibit tissue protective phenotype in the steady state. Owing to tremendous advances in technology, we now know the fact that muscularis macrophages are a heterogeneous population of cells which could be divided into different functional subsets depending on their anatomic niches. There is emerging evidence showing that these subsets, through molecular interactions with their neighbours, take part in a wide range of physiological and pathophysiological processes in the gut. In this review, we summarize recent progress (particularly over the past 4 years) on distribution, morphology, origin and functions of muscularis macrophages and, where possible, the characteristics of specific subsets in response to the microenvironment they occupy, with particular emphasis on their role in muscular inflammation. Furthermore, we also integrate their role in inflammation-related gastrointestinal disorders, such as post-operative ileus and diabetic gastroparesis, in order to propose future therapeutic strategies., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Interactions between the peripheral nervous system and resident macrophages (MMs) modulate intestinal homeostatic functions. Activation of β2-adrenergic receptors on MMs has been shown to reduce bacterial challenges. These MMs are also crucial for the development of bowel inflammation in postoperative ileus (POI), an iatrogenic, noninfectious inflammation-based motility disorder. However, the role of the sympathetic nervous system (SNS) in the immune modulation of these MMs during POI or other noninfectious diseases is largely unknown. By employing 6-OHDA-induced denervation, we investigated the changes in the muscularis externa by RNA-seq, quantitative PCR, and flow cytometry. Further, we performed transcriptional phenotyping of sorted CX3CR1 + MMs and ex vivo LPS/M-CSF stimulation on these MMs. By combining denervation with a mouse POI model, we explored distinct changes on CX3CR1 + MMs as well as in the muscularis externa and their functional outcome during POI. Our results identify SNS as an important mediator in noninfectious postoperative inflammation. Upon denervation, MMs anti-inflammatory genes were reduced, and the muscularis externa profile is shaped toward a proinflammatory status. Further, denervation reduced MMs anti-inflammatory genes also in the early phase of POI. Finally, reduced leukocyte infiltration into the muscularis led to a quicker recovery of bowel motility in the late phase of POI.

Background: Intestinal resident macrophages play a crucial role in homeostasis and have been implicated in numerous gastrointestinal diseases. While historically believed to be largely of hematopoietic origin, recent advances in fate-mapping technology have unveiled the existence of long-lived, self-maintaining populations located in specific niches throughout the gut wall. Furthermore, the advent of single-cell technology has enabled an unprecedented characterization of the functional specialization of tissue-resident macrophages throughout the gastrointestinal tract., Purpose: The purpose of this review was to provide a panorama on intestinal resident macrophages, with particular focus to the recent advances in the field. Here, we discuss the functions and phenotype of intestinal resident macrophages and, where possible, the functional specialization of these cells in response to the niche they occupy. Furthermore, we will discuss their role in gastrointestinal diseases., (© 2020 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Competing Interests: Competing interests: None declared.

A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days postinjection, animals were euthanized and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n = 154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/tertiary plexus. Very few CSMG neurons projected exclusively (i.e., ∼100% of an arbor's varicose branches) to myenteric plexus (∼2%) or smooth muscle (∼14%). With less stringent inclusion criteria (i.e., ≥85% of an axon's varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (∼13%) or smooth muscle (∼27%). The majority (i.e., ∼60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that coinnervated extensively (>15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ∼87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively. J. Comp. Neurol. 524:2577-2603, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background & Aims: Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI., Methods: We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI., Results: Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1β before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1β were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1β stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus., Conclusions: IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)