Stromal cells in chronic inflammation and tertiary lymphoid organ formation.

Inflammation is an unstable state. It either resolves or persists. Why inflammation persists and the factors that define tissue tropism remain obscure. Increasing evidence suggests that tissue-resident stromal cells not only provide positional memory but also actively regulate the differential accumulation of inflammatory cells within inflamed tissues. Furthermore, at many sites of chronic inflammation, structures that mimic secondary lymphoid tissues are observed, suggesting that chronic inflammation and lymphoid tissue formation share common activation programs. Similarly, blood and lymphatic endothelial cells contribute to tissue homeostasis and disease persistence in chronic inflammation. This review highlights our increasing understanding of the role of stromal cells in inflammation and summarizes the novel immunological role that stromal cells exert in the persistence of inflammatory diseases.

Creeping Fat in Crohn's Disease: Innocuous or innocuum?

In Crohn's disease, the characteristic expansion of mesenteric tissue on sites of intestinal inflammation is known as creeping fat. In a recent issue of Cell, Ha et al. report that translocation of gut bacteria to the mesenteries is associated with the formation of creeping fat.

Stromal Cells Covering Omental Fat-Associated Lymphoid Clusters Trigger Formation of Neutrophil Aggregates to Capture Peritoneal Contaminants.

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs) that collects peritoneal contaminants and provides a first layer of immunological defense within the abdomen. Here, we investigated the mechanisms that mediate the capture of peritoneal contaminants during peritonitis. Single-cell RNA sequencing and spatial analysis of omental stromal cells revealed that the surface of FALCs were covered by CXCL1+ mesothelial cells, which we termed FALC cover cells. Blockade of CXCL1 inhibited the recruitment and aggregation of neutrophils at FALCs during zymosan-induced peritonitis. Inhibition of protein arginine deiminase 4, an enzyme important for the release of neutrophil extracellular traps, abolished neutrophil aggregation and the capture of peritoneal contaminants by omental FALCs. Analysis of omental samples from patients with acute appendicitis confirmed neutrophil recruitment and bacterial capture at FALCs. Thus, specialized omental mesothelial cells coordinate the recruitment and aggregation of neutrophils to capture peritoneal contaminants.

Fat-associated lymphoid clusters: Supporting visceral adipose tissue B cell function in immunity and metabolism.

It is now widely understood that visceral adipose tissue (VAT) is a highly active and dynamic organ, with many functions beyond lipid accumulation and storage. In this review, we discuss the immunological role of this tissue, underpinned by the presence of fat-associated lymphoid clusters (FALCs). FALC's distinctive structure and stromal cell composition support a very different immune cell mix to that found in classical secondary lymphoid organs, which underlies their unique functions of filtration, surveillance, innate-like immune responses, and adaptive immunity within the serous cavities. FALCs are important B cell hubs providing B1 cell-mediated frontline protection against infection and supporting B2 cell-adaptative immune responses. Beyond these beneficial immune responses orchestrated by FALCs, immune cells within VAT play important homeostatic role. Dysregulation of immune cells during obesity and aging leads to chronic pathological "metabolic inflammation", which contributes to the development of cardiometabolic diseases. Here, we examine the emerging and complex functions of B cells in VAT homeostasis and the metabolic complications of obesity, highlighting the potential role that FALCs play and emphasize the areas where further research is needed.

Inflammation-induced formation of fat-associated lymphoid clusters.

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Cell origin and niche availability dictate the capacity of peritoneal macrophages to colonize the cavity and omentum.

The relationship between macrophages of the peritoneal cavity and the adjacent omentum remains poorly understood. Here, we describe two populations of omental macrophages distinguished by CD102 expression and use an adoptive cell transfer approach to investigate whether these arise from peritoneal macrophages, and whether this depends upon inflammatory status, the origin of peritoneal macrophages and availability of the omental niches. We show that whereas established resident peritoneal macrophages largely fail to migrate to the omentum, monocyte-derived resident cells readily migrate and form a substantial component of omental CD102+ macrophages in the months following resolution of peritoneal inflammation. In contrast, both populations had the capacity to migrate to the omentum in the absence of endogenous peritoneal and omental macrophages. However, inflammatory macrophages expanded more effectively and more efficiently repopulated both CD102+ and CD102- omental populations, whereas established resident macrophages partially reconstituted the omental niche via recruitment of monocytes. Hence, cell origin determines the migration of peritoneal macrophages to the omentum and predisposes established resident macrophages to drive infiltration of monocyte-derived cells.

Turning on ILC2s: diet control.

New research by Fali and colleagues shows that peroxisome proliferator-activated receptor gamma is a central metabolic regulator of group 2 innate lymphoid cells, controlling the functional activation of these potent innate immune initiators in lung and adipose tissue.

Lymphotoxin-ß receptor signaling through NF-κB2-RelB pathway reprograms adipocyte precursors as lymph node stromal cells.

Lymph node development during embryogenesis involves lymphotoxin-ß receptor engagement and subsequent differentiation of a poorly defined population of mesenchymal cells into lymphoid tissue organizer cells. Here, we showed that embryonic mesenchymal cells with characteristics of adipocyte precursors present in the microenvironment of lymph nodes gave rise to lymph node organizer cells. Signaling through the lymphotoxin-ß receptor controlled the fate of adipocyte precursor cells by blocking adipogenesis and instead promoting lymphoid tissue stromal cell differentiation. This effect involved activation of the NF-κB2-RelB signaling pathway and inhibition of the expression of the key adipogenic factors Pparγ and Cebpα. In vivo organogenesis assays show that embryonic and adult adipocyte precursor cells can migrate into newborn lymph nodes and differentiate into a variety of lymph node stromal cells. Thus, we propose that adipose tissues act as a source of lymphoid stroma for lymph nodes and other lymphoid structures associated with fat.

Rank signaling links the development of invariant γδ T cell progenitors and Aire(+) medullary epithelium.

The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire(+) mTECs at this key developmental stage, prior to αß T cell repertoire selection, was jointly directed by Rankl(+) lymphoid tissue inducer cells and invariant Vγ5(+) dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire(+) mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5(+) γδ T cells during thymus medulla formation for αß T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire(+) mTEC maturation.

Kidney Single-Cell Atlas Reveals Myeloid Heterogeneity in Progression and Regression of Kidney Disease.

BACKGROUND: Little is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease. METHODS: Integrated droplet- and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney. RESULTS: A single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ macrophage subset acts during repair. CONCLUSIONS: Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.

IL-33 delivery induces serous cavity macrophage proliferation independent of interleukin-4 receptor alpha.

IL-33 plays an important role in the initiation of type-2 immune responses, as well as the enhancement of type 2 effector functions. Engagement of the IL-33 receptor on macrophages facilitates polarization to an alternative activation state by amplifying IL-4 and IL-13 signaling to IL-4Rα. IL-4 and IL-13 also induce macrophage proliferation but IL-33 involvement in this process has not been rigorously evaluated. As expected, in vivo delivery of IL-33 induced IL-4Rα-dependent alternative macrophage activation in the serous cavities. IL-33 delivery also induced macrophages to proliferate but, unexpectedly, this was independent of IL-4Rα signaling. In a filarial nematode infection model in which IL-4Rα-dependent alternative activation and proliferation in the pleural cavity is well described, IL-33R was essential for alternative activation but not macrophage proliferation. Similarly, during Alternaria alternata induced airway inflammation, which provokes strong IL-33 responses, we observed that both IL-4Rα and IL-33R were required for alternative activation, while macrophage proliferation in the pleural cavity was still evident in the absence of either receptor alone. Our data show that IL-33R and IL-4Rα promote macrophage proliferation independently of each other, but both are essential for induction of alternative activation.

The expression of mouse CLEC-2 on leucocyte subsets varies according to their anatomical location and inflammatory state.

Expression of mouse C-type lectin-like receptor 2 (CLEC-2) has been reported on circulating CD11b(high) Gr-1(high) myeloid cells and dendritic cells (DCs) under basal conditions, as well as on a variety of leucocyte subsets following inflammatory stimuli or in vitro cell culture. However, previous studies assessing CLEC-2 expression failed to use CLEC-2-deficient mice as negative controls and instead relied heavily on single antibody clones. Here, we generated CLEC-2-deficient adult mice using two independent approaches and employed two anti-mouse CLEC-2 antibody clones to investigate surface expression on hematopoietic cells from peripheral blood and secondary lymphoid organs. We rule out constitutive CLEC-2 expression on resting DCs and show that CLEC-2 is upregulated in response to LPS-induced systemic inflammation in a small subset of activated DCs isolated from the mesenteric lymph nodes but not the spleen. Moreover, we demonstrate for the first time that peripheral blood B lymphocytes present exogenously derived CLEC-2 and suggest that both circulating B lymphocytes and CD11b(high) Gr-1(high) myeloid cells lose CLEC-2 following entry into secondary lymphoid organs. These results have significant implications for our understanding of CLEC-2 physiological functions.

CLEC-2 is required for development and maintenance of lymph nodes.

The importance of CLEC-2, a natural ligand/receptor for Gp38/Podoplanin, in the formation of the lymphatic vasculature has recently been demonstrated. As the development and maintenance of lymph nodes (LNs) is dependent on the formation of the lymphatic vasculature and the differentiation of Gp38/Podoplanin(+) stromal cells, we investigated the role of CLEC-2 in lymphoneogenesis and LN homeostasis. Using constitutive Clec1b(-/-) mice, we showed that while CLEC-2 was not necessary for initiation of the LN anlage, it was required at late stages of development. Constitutive deletion of CLEC-2 induced a profound defect in lymphatic endothelial cell proliferation, resulting in lack of LNs at birth. In contrast, conditional deletion of CLEC-2 in the megakaryocyte/platelet lineage in Clec1b(fl/fl)PF4-Cre mice led to the development of blood-filled LNs and fibrosis, in absence of a proliferative defect of the lymphatic endothelial compartment. This phenotype was also observed in chimeric mice reconstituted with Clec1b(fl/fl)PF4-Cre bone marrow, indicating that CLEC-2 expression in platelets was required for LN integrity. We demonstrated that LNs of Clec1b(fl/fl)PF4-Cre mice are able to sustain primary immune responses but show a defect in immune cell recirculation after repeated immunizations, thus suggesting CLEC-2 as target in chronic immune response.

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development.

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

Inflammatory regulation of glucocorticoid metabolism in mesenchymal stromal cells.

OBJECTIVE: Tissue glucocorticoid (GC) levels are regulated by the GC-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). This enzyme is expressed in cells and tissues arising from mesenchymal stromal cells. Proinflammatory cytokines dramatically increase expression of 11ß-HSD1 in stromal cells, an effect that has been implicated in inflammatory arthritis, osteoporosis, obesity, and myopathy. Additionally, GCs act synergistically with proinflammatory cytokines to further increase enzyme expression. The present study was undertaken to investigate the mechanisms underlying this regulation. METHODS: Gene reporter analysis, rapid amplification of complementary DNA ends (RACE), chemical inhibition experiments, and genetic disruption of intracellular signaling pathways in mouse embryonic fibroblasts (MEFs) were used to define the molecular mechanisms underlying the regulation of 11ß-HSD1 expression. RESULTS: Gene reporter, RACE, and chemical inhibitor studies demonstrated that the increase in 11ß-HSD1 expression with tumor necrosis factor α (TNFα)/interleukin-1ß (IL-1ß) occurred via the proximal HSD11B1 gene promoter and depended on NF-κB signaling. These findings were confirmed using MEFs with targeted disruption of NF-κB signaling, in which RelA (p65) deletion prevented TNFα/IL-1ß induction of 11ß-HSD1. GC treatment did not prevent TNFα-induced NF-κB nuclear translocation. The synergistic enhancement of TNFα-induced 11ß-HSD1 expression with GCs was reproduced by specific inhibitors of p38 MAPK. Inhibitor and gene deletion studies indicated that the effects of GCs on p38 MAPK activity occurred primarily through induction of dual-specificity phosphatase 1 expression. CONCLUSION: The mechanism by which stromal cell expression of 11ß-HSD1 is regulated is novel and distinct from that in other tissues. These findings open new opportunities for development of therapeutic interventions aimed at inhibiting or stimulating local GC levels in cells of mesenchymal stromal lineage during inflammation.

FRC-derived retinoic acid: The key to unlocking milky spots.

Milky spots of the omentum enable lymphocyte access to the peritoneal cavity. In this issue of JEM, Yoshihara and Okabe (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221813) demonstrate how secretion of retinoic acid by fibroblastic reticular cells allows lymphocyte entry into milky spots and the peritoneal cavity.

IL-17 signalling is critical for controlling subcutaneous adipose tissue dynamics and parasite burden during chronic murine Trypanosoma brucei infection.

In the skin, Trypanosoma brucei colonises the subcutaneous white adipose tissue, and is proposed to be competent for forward transmission. The interaction between parasites, adipose tissue, and the local immune system is likely to drive the adipose tissue wasting and weight loss observed in cattle and humans infected with T. brucei. However, mechanistically, events leading to subcutaneous white adipose tissue wasting are not fully understood. Here, using several complementary approaches, including mass cytometry by time of flight, bulk and single cell transcriptomics, and in vivo genetic models, we show that T. brucei infection drives local expansion of several IL-17A-producing cells in the murine WAT, including TH17 and Vγ6+ cells. We also show that global IL-17 deficiency, or deletion of the adipocyte IL-17 receptor protect from infection-induced WAT wasting and weight loss. Unexpectedly, we find that abrogation of adipocyte IL-17 signalling results in a significant accumulation of Dpp4+ Pi16+ interstitial preadipocytes and increased extravascular parasites in the WAT, highlighting a critical role for IL-17 signalling in controlling preadipocyte fate, subcutaneous WAT dynamics, and local parasite burden. Taken together, our study highlights the central role of adipocyte IL-17 signalling in controlling WAT responses to infection, suggesting that adipocytes are critical coordinators of tissue dynamics and immune responses to T. brucei infection.

Selective effects of NF-κB1 deficiency in CD4⁺ T cells on Th2 and TFh induction by alum-precipitated protein vaccines.

NF-κB1-dependent signaling directs the development of CD4(+) Th2 cells during allergic airway inflammation and protective responses to helminth infection. Here, we show that IL-4 and IL-13 production is NF-κB1-dependent in mouse OVA-specific CD4(+) (OTII) T cells responding to alum-precipitated OVA (alumOVA) immunization. More surprisingly, we found that NF-κB1 deficiency in OTII cells also selectively impairs their CXCR5 induction by alumOVA without affecting upregulation of BCL6, IL-21, OX40 and CXCR4 mRNA and PD-1 protein. This results in functional impairment of follicular helper T cells. Thus, fewer germinal center B cells develop in LN responses to alumOVA in T-cell-deficient mice reconstituted with NF-κB1(-/-) OTII cells as opposed to NF-κB1(+/+) OTII cells, while plasma cell numbers are comparable. Unlike CXCR5 induction in CD4(+) T cells, NF-κB1-deficient recirculating follicular B cells are shown to express normal levels of CXCR5. The selective effects of NF-κB1-deficiency on Th2 and follicular helper T cell induction do not appear to be due to altered expression of the Th2-associated transcription factors - GATA-3, c-Maf and Ikaros. Altogether, these results suggest that NF-κB1 regulates the expression of CXCR5 on CD4(+) T cells primed in vivo, and thus selectively controls the T-cell-dependent germinal center component of B-cell response to alumOVA.

Ontogeny of stromal organizer cells during lymph node development.

The development of secondary lymphoid organs, such as lymph nodes (LNs), in the embryo results from the reciprocal action between lymphoid tissue inducer (LTi) cells and stromal cells. However, the initial events inducing LN anlagen formation before the LTi stromal cells cross-talk interactions take place are not fully elucidated. In this study, we show that the inguinal LN anlagen in mouse embryos developed from mesenchymal cells surrounding the lymph sacs, spherical structures of endothelial cells that bud from veins. Using inguinal and mesenteric LNs (mLNs), we provide evidence supporting a two-step maturation model for stromal cells: first, ICAM-1(-)VCAM-1(-) mesenchymal precursor cells become ICAM-1(int)VCAM-1(int) cells, in a process independent of LTi cells and lymphotoxin beta receptor (LTbetaR) signaling. The second step involves the maturation of ICAM-1(int)VCAM-1(int) cells to ICAM-1(high)VCAM-1(high) mucosal addressin cell adhesion molecule-1(+) organizer cells and depends on both LTi cells and LTbetaR. Addition of alphaLTbetaR agonist to LN organ cultures was sufficient to induce ICAM-1(int)VCAM-1(int) cells to mature. In LtbetaR(-/-) embryos, both inguinal and mLN stromal cells showed a block at the ICAM-1(int)VCAM-1(int) stage, and, contrary to inguinal LNs, mLNs persist longer and contained LTi cells, which correlated with the sustained gene expression of Il-7, Cxcl13, and, to a lesser degree, Ccl21. Taken together, these results highlight the importance of the signals and cellular interactions that induce the maturation of stromal cells and ultimately lead to the formation of lymphoid tissues.

Human embryonic stem cell-derived endothelial cell product injection attenuates cardiac remodeling in myocardial infarction.

Background: Mechanisms contributing to tissue remodeling of the infarcted heart following cell-based therapy remain elusive. While cell-based interventions have the potential to influence the cardiac healing process, there is little direct evidence of preservation of functional myocardium. Aim: The aim of the study was to investigate tissue remodeling in the infarcted heart following human embryonic stem cell-derived endothelial cell product (hESC-ECP) therapy. Materials and methods: Following coronary artery ligation (CAL) to induce cardiac ischemia, we investigated infarct size at 1 day post-injection in media-injected controls (CALM, n = 11), hESC-ECP-injected mice (CALC, n = 10), and dead hESC-ECP-injected mice (CALD, n = 6); echocardiography-based functional outcomes 14 days post-injection in experimental (CALM, n = 13; CALC, n = 17) and SHAM surgical mice (n = 4); and mature infarct size (CALM and CALC, both n = 6). We investigated ligand-receptor interactions (LRIs) in hESC-ECP cell populations, incorporating a publicly available C57BL/6J mouse cardiomyocyte-free scRNAseq dataset with naive, 1 day, and 3 days post-CAL hearts. Results: Human embryonic stem cell-derived endothelial cell product injection reduces the infarct area (CALM: 54.5 ± 5.0%, CALC: 21.3 ± 4.9%), and end-diastolic (CALM: 87.8 ± 8.9 uL, CALC: 63.3 ± 2.7 uL) and end-systolic ventricular volume (CALM: 56.4 ± 9.3 uL, CALC: 33.7 ± 2.6 uL). LRI analyses indicate an alternative immunomodulatory effect mediated via viable hESC-ECP-resident signaling. Conclusion: Delivery of the live hESC-ECP following CAL modulates the wound healing response during acute pathological remodeling, reducing infarct area, and preserving functional myocardium in this relatively acute model. Potential intrinsic myocardial cellular/hESC-ECP interactions indicate that discreet immunomodulation could provide novel therapeutic avenues to improve cardiac outcomes following myocardial infarction.