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Immune tolerance is maintained in lymphoid organs (LOs). Despite the presence of complex immune cell networks in non-LOs, it is unknown whether self-tolerance is maintained in these tissues. We developed a technique to restrict genetic recombination to regulatory T cells (T regs ) only in skin. Selective depletion of skin T regs resulted in T cell-mediated inflammation of hair follicles (HFs). Suppression did not rely on CTLA-4, but instead on high-affinity interleukin-2 (IL-2) receptor expression by skin T regs , functioning exclusively in a cell-extrinsic manner. In a novel model of HF stem cell (HFSC)-driven autoimmunity, we reveal that skin T regs immunologically protect the HFSC niche. Finally, we used spatial transcriptomics to identify aberrant IL-2 signaling at stromal-HF interfaces in a rare form of human alopecia characterized by HFSC destruction and alopecia areata. Collectively, these results reveal the fundamental biology of T regs in skin uncoupled from the systemic pool and elucidate a mechanism of self-tolerance.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites., Competing Interests: Declaration of interests R.M.L. is a member of the Scientific Resource Board at Genentech and serves on the Advisory Board at Immunity., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inflammatory conditions represent the largest class of chronic skin disease, but the molecular dysregulation underlying many individual cases remains unclear. Single-cell RNA sequencing (scRNA-seq) has increased precision in dissecting the complex mixture of immune and stromal cell perturbations in inflammatory skin disease states. We single-cell-profiled CD45 + immune cell transcriptomes from skin samples of 31 patients (7 atopic dermatitis, 8 psoriasis vulgaris, 2 lichen planus (LP), 1 bullous pemphigoid (BP), 6 clinical/histopathologically indeterminate rashes, and 7 healthy controls). Our data revealed active proliferative expansion of the T reg and Trm components and universal T cell exhaustion in human rashes, with a relative attenuation of antigen-presenting cells. Skin-resident memory T cells showed the greatest transcriptional dysregulation in both atopic dermatitis and psoriasis, whereas atopic dermatitis also demonstrated recurrent abnormalities in ILC and CD8 + cytotoxic lymphocytes. Transcript signatures differentiating these rash types included genes previously implicated in T helper cell (T H 2)/T H 17 diatheses, segregated in unbiased functional networks, and accurately identified disease class in untrained validation data sets. These gene signatures were able to classify clinicopathologically ambiguous rashes with diagnoses consistent with therapeutic response. Thus, we have defined major classes of human inflammatory skin disease at the molecular level and described a quantitative method to classify indeterminate instances of pathologic inflammation. To make this approach accessible to the scientific community, we created a proof-of-principle web interface (RashX), where scientists and clinicians can visualize their patient-level rash scRNA-seq-derived data in the context of our T H 2/T H 17 transcriptional framework.

Over the last decade, we have come to appreciate group 2 innate lymphoid cells (ILC2s) as important players in host and tissue immunity. New studies of ILC2s and their precursors using novel reporter mice, advanced microscopy, and multi-omics approaches have expanded our knowledge on how these cells contribute to tissue physiology and function. This review highlights recent literature on this enigmatic cell, and we organize our discussion across three important paradigms in ILC2 biology: development, divergence, and dispersal. In addition, we frame our discussion in the context of other innate and adaptive immune cells to emphasize the relevance of expanding knowledge of ILC2s and tissue immunity., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell-deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid-sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions.

Innate lymphoid cells (ILCs) are tissue-resident effectors poised to activate rapidly in response to local signals such as cytokines. To preserve homeostasis, ILCs must employ multiple pathways, including tonic suppressive mechanisms, to regulate their primed state and prevent inappropriate activation and immunopathology. Such mechanisms remain incompletely characterized. Here we show that cytokine-inducible SH2-containing protein (CISH), a suppressor of cytokine signaling (SOCS) family member, is highly and constitutively expressed in type 2 innate lymphoid cells (ILC2s). Mice that lack CISH either globally or conditionally in ILC2s show increased ILC2 expansion and activation, in association with reduced expression of genes inhibiting cell-cycle progression. Augmented proliferation and activation of CISH-deficient ILC2s increases basal and inflammation-induced numbers of intestinal tuft cells and accelerates clearance of the model helminth, Nippostrongylus brasiliensis, but compromises innate control of Salmonella typhimurium. Thus, CISH constrains ILC2 activity both tonically and after perturbation, and contributes to the regulation of immunity in mucosal tissue., (© 2021. The Author(s).)

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare but serious disease with poorly understood mechanisms. Here, we report that patients with EGPA have elevated levels of TSLP, IL-25, and soluble ST2, which are well-characterized cytokine "alarmins" that activate or modulate type 2 innate lymphoid cells (ILC2s). Patients with active EGPA have a concurrent reduction in circulating ILC2s, suggesting a role for ILC2s in the pathogenesis of this disease. To explore the mechanism of these findings in patients, we established a model of EGPA in which active vasculitis and pulmonary hemorrhage were induced by IL-33 administration in predisposed, hypereosinophilic mice. In this model, induction of pulmonary hemorrhage and vasculitis was dependent on ILC2s and signaling through IL4Rα. In the absence of IL4Rα or STAT6, IL-33-treated mice had less vascular leak and pulmonary edema, less endothelial activation, and reduced eotaxin production, cumulatively leading to a reduction of pathologic eosinophil migration into the lung parenchyma. These results offer a mouse model for use in future mechanistic studies of EGPA, and they suggest that IL-33, ILC2s, and IL4Rα signaling may be potential targets for further study and therapeutic targeting in patients with EGPA.

Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals. ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines 1 . In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis. However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions. Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state. Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology. Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum-even at steady state-reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state. Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories. We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping. Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and, left unchecked, drive pathological remodelling.

Inflammatory response heterogeneity has impeded high-resolution dissection of diverse immune cell populations during activation. We characterize mouse cutaneous immune cells by single-cell RNA sequencing, after inducing inflammation using imiquimod and oxazolone dermatitis models. We identify 13 CD45 + subpopulations, which broadly represent most functionally characterized immune cell types. Oxazolone pervasively upregulates Jak2 / Stat3 expression across T cells and antigen-presenting cells (APCs). Oxazolone also induces Il4 / Il13 expression in newly infiltrating basophils, and Il4ra and Ccl24, most prominently in APCs. In contrast, imiquimod broadly upregulates Il17 / Il22 and Ccl4 / Ccl5 . A comparative analysis of single-cell inflammatory transcriptional responses reveals that APC response to oxazolone is tightly restricted by cell identity, whereas imiquimod enforces shared programs on multiple APC populations in parallel. These global molecular patterns not only contrast immune responses on a systems level but also suggest that the mechanisms of new sources of inflammation can eventually be deduced by comparison to known signatures., Competing Interests: A.J.S. and S.C.B. are employees of ImmunityBio Inc, S.C.B. is an equity holder of ImmunityBio Inc. The remaining authors disclose no conflicts.

Group 2 innate lymphoid cells (ILC2s) are tissue-resident cells prominent at barrier sites. Although precursors are found in blood, mature ILC2s can enter the circulation after small intestinal perturbation by migratory helminths and move to distant tissues to influence the local reparative response. Using fate-mapping and methods to bypass the lung or intestinal phases of Nippostrongylus brasiliensis infection, we show that blood ILC2s comprise heterogeneous populations derived from distinct tissues that are dependent on alarmins matched to the receptor profile of the specific tissue ILC2s. Activation of local ILC2s by tissue-specific alarmins induced their proliferation, lymph node migration, and blood dissemination, thus systemically distributing type 2 cytokines. These studies uncover a possible mechanism by which local innate responses transition to systemic type 2 responses by extrusion of activated sentinel ILC2s from tissue into the circulation., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Ricardo-Gonzalez et al.)

Skin is the largest barrier organ and an important interface between the body and the outside environment. Immune surveillance and homeostatic regulation of skin function are governed by complex interactions between resident lymphoid and myeloid cells and their communications with the surrounding parenchyma. Recent studies have provided exciting insights about the unique characteristics of skin-resident innate lymphoid cells (ILCs). Here, we discuss advances demonstrating how skin ILCs contribute to tissue homeostasis by regulating microbiome balance in steady-state and how their dysregulation can trigger and promote inflammatory skin diseases such as atopic dermatitis and psoriasis. We review the phenotypic and functional similarities and differences of ILCs between the skin and other organs and highlight future areas of investigation for this field., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this issue of JEM , Rana et al. (https://doi.org/10.1084/jem.20190689) report that adipose tissue multipotent stromal cells (MSCs) provide multifaceted support for adipose tissue-resident ILC2s through contact-mediated proliferation and IL-33-mediated stress-induced activation., (© 2019 Ricardo-Gonzalez and Locksley.)

The perinatal period is a critical window for distribution of innate tissue-resident immune cells within developing organs. Despite epidemiologic evidence implicating the early-life environment in the risk for allergy, temporally controlled lineage tracing of group 2 innate lymphoid cells (ILC2s) during this period remains unstudied. Using complementary fate-mapping approaches and reporters for ILC2 activation, we show that ILC2s appeared in multiple organs during late gestation like tissue macrophages, but, unlike the latter, a majority of peripheral ILC2 pools were generated de novo during the postnatal window. This period was accompanied by systemic ILC2 priming and acquisition of tissue-specific transcriptomes. Although perinatal ILC2s were variably replaced across tissues with age, the dramatic increases in tissue ILC2s following helminth infection were mediated through local expansion independent of de novo generation by bone marrow hematopoiesis. We provide comprehensive temporally controlled fate mapping of an innate lymphocyte subset with notable nuances as compared to tissue macrophage ontogeny., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Group 2 innate lymphoid cells (ILC2s) are distributed systemically and produce type 2 cytokines in response to a variety of stimuli, including the epithelial cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Transcriptional profiling of ILC2s from different tissues, however, grouped ILC2s according to their tissue of origin, even in the setting of combined IL-25-, IL-33-receptor-, and TSLP-receptor-deficiency. Single-cell profiling confirmed a tissue-organizing transcriptome and identified ILC2 subsets expressing distinct activating receptors, including the major subset of skin ILC2s, which were activated preferentially by IL-18. Tissue ILC2 subsets were unaltered in number and expression in germ-free mice, suggesting that endogenous, tissue-derived signals drive the maturation of ILC2 subsets by controlling expression of distinct patterns of activating receptors, thus anticipating tissue-specific perturbations occurring later in life.

The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of T regs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The inflammatory infiltrate seen in biopsy specimens obtained from patients with subcutaneous fat necrosis of the newborn (SCFN) has classically been described as consisting mostly of histiocytes. However, we encountered patients with SCFN whose biopsy specimens revealed mostly neutrophils, prompting infection to be an initial consideration., Objectives: We sought to describe cases of SCFN in which neutrophils formed the majority of the infiltrate at our institution and in the literature., Methods: We performed a retrospective analysis of patients with SCFN reported at our institution and a literature review of SCFN., Results: Thirteen cases of SCFN were identified at our institution. In 2 of 13 cases, neutrophils composed >75% of the inflammatory infiltrate, and both lesions were 1 day old. From the literature review, neutrophils were mentioned as a component of the infiltrate in 10 of 124 cases, but in none were neutrophils described as forming the majority of the infiltrate., Limitations: This study is limited by its retrospective nature and small sample size., Conclusions: Neutrophils can comprise most of the inflammatory cells in patients with SCFN, especially early in the course of the disease. This variant of SCFN can be easily mistaken for infection., (Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Bladder plasmacytoid carcinoma is an invasive urothelial carcinoma subtype that is emphasized for its morphological overlap with plasma cells and metastatic carcinoma. Our experience suggests frequent intraperitoneal spread that is not typical of conventional urothelial carcinoma., Materials and Methods: We identified cases of plasmacytoid urothelial carcinoma diagnosed on radical cystectomy. Patient age, gender, American Joint Committee on Cancer (7th edition) stage, metastatic spread/recurrence sites and clinical disease status at last followup were recorded., Results: A total of 10 male and 5 female patients 42 to 81 years old were identified. One tumor was pT2, 11 pT3 and 3 pT4. Six of 15 patients (40%) presented with lymph node metastasis and 5 (33%) had intraperitoneal metastasis at cystectomy. These initial sites of metastatic spread included the prerectal space, ovary and vagina, ovary and fallopian tube, bowel serosa, and omentum and bowel serosa in 1 case each. Three patients had subsequent metastasis involving the prerectal space, pleural fluid and small bowel serosa, and bowel serosa in 1 each. Eight patients had followup information available, including 3 who died of disease, 3 with disease and 2 with no evidence of disease., Conclusions: Of the patients 33% with the plasmacytoid variant of urothelial carcinoma presented with intraperitoneal disease spread and 20% had subsequent metastasis involving serosal surfaces. The possibility of noncontiguous intraperitoneal spread involving serosal surfaces should be recognized to ensure proper intraoperative staging and clinical followup for patients with plasmacytoid carcinoma., (Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Eosinophils are associated with helminth immunity and allergy, often in conjunction with alternatively activated macrophages (AAMs). Adipose tissue AAMs are necessary to maintain glucose homeostasis and are induced by the cytokine interleukin-4 (IL-4). Here, we show that eosinophils are the major IL-4-expressing cells in white adipose tissues of mice, and, in their absence, AAMs are greatly attenuated. Eosinophils migrate into adipose tissue by an integrin-dependent process and reconstitute AAMs through an IL-4- or IL-13-dependent process. Mice fed a high-fat diet develop increased body fat, impaired glucose tolerance, and insulin resistance in the absence of eosinophils, and helminth-induced adipose tissue eosinophilia enhances glucose tolerance. Our results suggest that eosinophils play an unexpected role in metabolic homeostasis through maintenance of adipose AAMs.

Immune cells take residence in metabolic tissues, providing a framework for direct regulation of nutrient metabolism. Despite conservation of this anatomic relationship through evolution, the signals and mechanisms by which the immune system regulates nutrient homeostasis and insulin action remain poorly understood. Here, we demonstrate that the IL-4/STAT6 immune axis, a key pathway in helminth immunity and allergies, controls peripheral nutrient metabolism and insulin sensitivity. Disruption of signal transducer and activator of transcription 6 (STAT6) decreases insulin action and enhances a peroxisome proliferator-activated receptor α (PPARα) driven program of oxidative metabolism. Conversely, activation of STAT6 by IL-4 improves insulin action by inhibiting the PPARα-regulated program of nutrient catabolism and attenuating adipose tissue inflammation. These findings have thus identified an unexpected molecular link between the immune system and macronutrient metabolism, suggesting perhaps the coevolution of these pathways occurred to ensure access to glucose during times of helminth infection.

Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and the silent disposal of apoptotic cells is unknown. Here we show that peroxisome proliferator-activated receptor-delta (PPAR-delta) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-delta decreases expression of opsonins such as complement component-1qb (C1qb), resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific Ppard(-/-) mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-delta has a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.

Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator-activated receptor delta (PPARdelta) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARdelta(-/-) (Ppard(-/-)) bone marrow into wild-type mice diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned medium from PPARdelta(-/-) macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes.

Obesity and insulin resistance, the cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation. In adipose tissue chronic overnutrition leads to macrophage infiltration, resulting in local inflammation that potentiates insulin resistance. For instance, transgenic expression of Mcp1 (also known as chemokine ligand 2, Ccl2) in adipose tissue increases macrophage infiltration, inflammation and insulin resistance. Conversely, disruption of Mcp1 or its receptor Ccr2 impairs migration of macrophages into adipose tissue, thereby lowering adipose tissue inflammation and improving insulin sensitivity. These findings together suggest a correlation between macrophage content in adipose tissue and insulin resistance. However, resident macrophages in tissues display tremendous heterogeneity in their activities and functions, primarily reflecting their local metabolic and immune microenvironment. While Mcp1 directs recruitment of pro-inflammatory classically activated macrophages to sites of tissue damage, resident macrophages, such as those present in the adipose tissue of lean mice, display the alternatively activated phenotype. Despite their higher capacity to repair tissue, the precise role of alternatively activated macrophages in obesity-induced insulin resistance remains unknown. Using mice with macrophage-specific deletion of the peroxisome proliferator activated receptor-gamma (PPARgamma), we show here that PPARgamma is required for maturation of alternatively activated macrophages. Disruption of PPARgamma in myeloid cells impairs alternative macrophage activation, and predisposes these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings suggest that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes.