The gene regulatory basis of bystander activation in CD8+ T cells.

CD8+ T cells are classically recognized as adaptive lymphocytes based on their ability to recognize specific foreign antigens and mount memory responses. However, recent studies indicate that some antigen-inexperienced CD8+ T cells can respond to innate cytokines alone in the absence of cognate T cell receptor stimulation, a phenomenon referred to as bystander activation. Here, we demonstrate that neonatal CD8+ T cells undergo a robust and diverse program of bystander activation, which corresponds to enhanced innate-like protection against unrelated pathogens. Using a multi-omics approach, we found that the ability of neonatal CD8+ T cells to respond to innate cytokines derives from their capacity to undergo rapid chromatin remodeling, resulting in the usage of a distinct set of enhancers and transcription factors typically found in innate-like T cells. We observed that the switch between innate and adaptive functions in the CD8+ T cell compartment is mediated by changes in the abundance of distinct subsets of cells. The innate CD8+ T cell subset that predominates in early life was also present in adult mice and humans. Our findings provide support for the layered immune hypothesis and indicate that the CD8+ T cell compartment is more functionally diverse than previously thought.

T Helper 17-Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis.

BACKGROUND: Infiltration of cluster of differentiation (CD) 3+ (CD3+) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets. HYPOTHESIS: An imbalance of the ratio of regulatory T cells and T helper 17 cells would be associated with disease progression in posttraumatic osteoarthritis, suggesting opportunities for immunomodulatory therapy. STUDY DESIGN: Descriptive laboratory study. METHODS: Synovial fluid was aspirated from the joints of equine clinical patients undergoing arthroscopic surgery for posttraumatic osteoarthritis resulting from intra-articular fragmentation. Joints were classified as having mild or moderate posttraumatic osteoarthritis. Synovial fluid was also obtained from nonoperated horses with normal cartilage. Peripheral blood was obtained from horses with normal cartilage and those with mild and moderate posttraumatic osteoarthritis. Synovial fluid and peripheral blood cells were analyzed by flow cytometry, and native synovial fluid was analyzed by enzyme-linked immunosorbent assay. RESULTS: CD3+ T cells represented 81% of lymphocytes in synovial fluid, which increased in animals with moderate posttraumatic osteoarthritis to 88.3% (P = .02). CD14+ macrophages were doubled in those with moderate posttraumatic osteoarthritis compared with mild posttraumatic osteoarthritis and controls (P < .001). Less than 5% of CD3+ T cells found within the joint were forkhead box P3 protein+ (Foxp3+) regulatory T cells, but a 4- to 8-times higher percentage of nonoperated and mild posttraumatic osteoarthritis joint regulatory T cells secreted interleukin (IL)-10 than peripheral blood Tregs (P < .005). T regulatory-1 cells that secreted IL-10 but did not express Foxp3 accounted for approximately 5% of CD3+ T cells in all joints. T helper 17 cells and Th17-like regulatory T cells were increased in those with moderate posttraumatic osteoarthritis (P < .0001) compared with mild and nonoperated patients. IL-10, IL-17A, IL-6, chemokine (C-C motif) ligand (CCL) 2 (CCL2), and CCL5 concentrations detected by enzyme-linked immunosorbent assay in synovial fluid were not different between groups. CONCLUSIONS: An imbalance of the ratio of regulatory T cells and T helper 17 cells and an increase in T helper 17 cell-like regulatory T cells in synovial fluid from joints with more severe disease provide novel insights into immunological mechanisms that are associated with posttraumatic osteoarthritis progression and pathogenesis. CLINICAL RELEVANCE: Early and targeted use of immunotherapeutics in the mitigation of posttraumatic osteoarthritis may improve patient clinical outcomes.

Interleukin-6 neutralization and regulatory T cells are additive in chondroprotection from IL-1ß-induced inflammation.

Anti-inflammatory Regulatory T cells (Tregs) are enriched in the joints of patients with osteoarthritis (OA) compared to healthy joints. Tregs maintain homeostasis through secretion of anti-inflammatory cytokines and cell-to-cell interactions including immune checkpoint signaling. Interleukin-6 (IL-6) is a pleiotropic cytokine secreted by inflamed synoviocytes and chondrocytes that can inhibit or alter Treg function. This study tested the hypothesis that neutralization of IL-6 would enable Treg anti-inflammatory function to resolve inflammation and catabolism elicited by IL-1ß in an equine chondrocyte/synoviocyte/Treg tri-culture OA model. Synoviocyte/chondrocyte co-cultures were stimulated with IL-1ß, and treated with αIL-6 neutralizing antibody. Activated Tregs secreting IL-10 were added in direct contact with synoviocytes to create a tri-culture. Neutralization of IL-6 partially restored Treg anti-inflammatory functions and, in combination, reduced IL-1ß-stimulated synoviocyte MMP13 expression to control levels and restored Acan expression in chondrocytes. IL-6 neutralization alone decreased Il6 expression in chondrocytes and synoviocytes, mitigating IL-6 positive feedback loop. Although Tregs were the primary producers of anti-inflammatory IL-10 and IL-4, they also produced pro-inflammatory IL-17A, as detected by ELIA, which may have been responsible for incomplete rescue of synoviocyte/chondrocyte homeostasis following IL-1ß stimulation. Treg secretion of IL-10, IL-4, and IL-17A was not altered by tri-culture conditions or presence of αIL-6, therefore, it was unlikely that Treg phenotype instability occurred. The significant effect of chondrocyte/synoviocyte donor, but not Treg donor, on gene expression and IL-6 concentration in conditioned media, indicated that personalized therapy considering the patient's OA status might be needed for successful implementation of immunotherapy in the context of OA.

TSLP, IL-33, and IL-25: Not just for allergy and helminth infection.

The release of cytokines from epithelial and stromal cells is critical for the initiation and maintenance of tissue immunity. Three such cytokines, thymic stromal lymphopoietin, IL-33, and IL-25, are important regulators of type 2 immune responses triggered by parasitic worms and allergens. In particular, these cytokines activate group 2 innate lymphoid cells, TH2 cells, and myeloid cells, which drive hallmarks of type 2 immunity. However, emerging data indicate that these tissue-associated cytokines are not only involved in canonical type 2 responses but are also important in the context of viral infections, cancer, and even homeostasis. Here, we provide a brief review of the roles of thymic stromal lymphopoietin, IL-33, and IL-25 in diverse immune contexts, while highlighting their relative contributions in tissue-specific responses. We also emphasize a biologically motivated framework for thinking about the integration of multiple immune signals, including the 3 featured in this review.

E-Protein Inhibition in ILC2 Development Shapes the Function of Mature ILC2s during Allergic Airway Inflammation.

E-protein transcription factors limit group 2 innate lymphoid cell (ILC2) development while promoting T cell differentiation from common lymphoid progenitors. Inhibitors of DNA binding (ID) proteins block E-protein DNA binding in common lymphoid progenitors to allow ILC2 development. However, whether E-proteins influence ILC2 function upon maturity and activation remains unclear. Mice that overexpress ID1 under control of the thymus-restricted proximal Lck promoter (ID1tg/WT) have a large pool of primarily thymus-derived ILC2s in the periphery that develop in the absence of E-protein activity. We used these mice to investigate how the absence of E-protein activity affects ILC2 function and the genomic landscape in response to house dust mite (HDM) allergens. ID1tg/WT mice had increased KLRG1- ILC2s in the lung compared with wild-type (WT; ID1WT/WT) mice in response to HDM, but ID1tg/WT ILC2s had an impaired capacity to produce type 2 cytokines. Analysis of WT ILC2 accessible chromatin suggested that AP-1 and C/EBP transcription factors but not E-proteins were associated with ILC2 inflammatory gene programs. Instead, E-protein binding sites were enriched at functional genes in ILC2s during development that were later dynamically regulated in allergic lung inflammation, including genes that control ILC2 response to cytokines and interactions with T cells. Finally, ILC2s from ID1tg/WT compared with WT mice had fewer regions of open chromatin near functional genes that were enriched for AP-1 factor binding sites following HDM treatment. These data show that E-proteins shape the chromatin landscape during ILC2 development to dictate the functional capacity of mature ILC2s during allergic inflammation in the lung.

Helminths make themselves at home.

Drurey et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20211140) show that excretory/secretory products from the parasitic helminth Heligmosomoides polygyrus suppress the host-protective small intestinal epithelial response. These findings establish that helminths directly modulate the tissue in which they live, shining new light on the host-parasite interaction.

Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types.

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells, and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely cotranscribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.NEW & NOTEWORTHY In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs.

Prostaglandin regulation of type 2 inflammation: From basic biology to therapeutic interventions.

Type 2 immunity is critical for the protective and repair responses that mediate resistance to parasitic helminth infection. This immune response also drives aberrant inflammation during atopic diseases. Prostaglandins are a class of critical lipid mediators that are released during type 2 inflammation and are integral in controlling the initiation, activation, maintenance, effector functions, and resolution of Type 2 inflammation. In this review, we explore the roles of the different prostaglandin family members and the receptors they bind to during allergen- and helminth-induced Type 2 inflammation and the mechanism through which prostaglandins promote or suppress Type 2 inflammation. Furthermore, we discuss the potential role of prostaglandins produced by helminth parasites in the regulation of host-pathogen interactions, and how prostaglandins may regulate the inverse relationship between helminth infection and allergy. Finally, we discuss opportunities to capitalize on our understanding of prostaglandin pathways to develop new therapeutic options for humans experiencing Type 2 inflammatory disorders that have a significant prostaglandin-driven component including allergic rhinitis and asthma.

Plasmacytoid Dendritic Cells Facilitate Th Cell Cytokine Responses throughout Schistosoma mansoni Infection.

Plasmacytoid dendritic cells (pDCs) are potent producers of type I IFN (IFN-I) during viral infection and respond to IFN-I in a positive feedback loop that promotes their function. IFN-I shapes dendritic cell responses during helminth infection, impacting their ability to support Th2 responses. However, the role of pDCs in type 2 inflammation is unclear. Previous studies have shown that pDCs are dispensable for hepatic or splenic Th2 responses during the early stages of murine infection with the trematode Schistosoma mansoni at the onset of parasite egg laying. However, during S. mansoni infection, an ongoing Th2 response against mature parasite eggs is required to protect the liver and intestine from acute damage and how pDCs participate in immune responses to eggs and adult worms in various tissues beyond acute infection remains unclear. We now show that pDCs are required for optimal Th2 cytokine production in response to S. mansoni eggs in the intestinal-draining mesenteric lymph nodes throughout infection and for egg-specific IFN-γ at later time points of infection. Further, pDC depletion at chronic stages of infection led to increased hepatic and splenic pathology as well as abrogated Th2 cell cytokine production and activation in the liver. In vitro, mesenteric lymph node pDCs supported Th2 cell responses from infection-experienced CD4+ T cells, a process dependent on pDC IFN-I responsiveness, yet independent of Ag. Together, these data highlight a previously unappreciated role for pDCs and IFN-I in maintaining and reinforcing type 2 immunity in the lymph nodes and inflamed tissue during helminth infection.

PGD2 and CRTH2 counteract Type 2 cytokine-elicited intestinal epithelial responses during helminth infection.

Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.

Regulatory T cells provide chondroprotection through increased TIMP1, IL-10 and IL-4, but cannot mitigate the catabolic effects of IL-1ß and IL-6 in a tri-culture model of osteoarthritis.

Objective: To gain insight into Treg interactions with synovial tissues in early OA, an equine tri-culture model of OA was used to test the hypothesis that Tregs, in the absence of T Helper 17 â€‹cells, are sufficient to resolve inflammation elicited by IL-1ß. Methods: To model normal and OA joints, synoviocytes were co-cultured with chondrocytes in a transwell system and ± stimulated with IL-1ß. Tregs were activated and enriched, then added to co-cultures, creating tri-cultures. At culture end, synoviocytes and chondrocytes were analyzed for gene expression, Treg Foxp3 expression was reexamined by flow cytometry, and conditioned media were evaluated by ELISA. Results: Tregs increased IL-10 and IL-4 in tri-culture media and increased TIMP1 gene expression in synoviocytes and chondrocytes. Tregs increased IL-6 in conditioned media and Il6 gene expression in synoviocytes, which was additive with IL-1ß. In chondrocytes, addition of Tregs decreased Col2b gene expression while Acan gene expression was decreased by IL-1ß and addition of Tregs. IL-17A was detected in tri-cultures. CCL2 and CCL5 were increased in tri-cultures. Conclusions: In a tri-culture model of OA, addition of Tregs resulted in conditions conducive to chondroprotection including increased concentration of IL-10 and IL-4 in conditioned media and increased gene expression of TIMP1 in both chondrocytes and synoviocytes. However, there was increased concentration of the catabolic cytokine IL-6, and decreased gene expression of Col2b and Acan in IL-1ß-stimulated chondrocytes. These results suggest that blocking IL-6 could enhance Treg function in mitigating OA progression.

Astrocytes promote a protective immune response to brain Toxoplasma gondii infection via IL-33-ST2 signaling.

It is of great interest to understand how invading pathogens are sensed within the brain, a tissue with unique challenges to mounting an immune response. The eukaryotic parasite Toxoplasma gondii colonizes the brain of its hosts, and initiates robust immune cell recruitment, but little is known about pattern recognition of T. gondii within brain tissue. The host damage signal IL-33 is one protein that has been implicated in control of chronic T. gondii infection, but, like many other pattern recognition pathways, IL-33 can signal peripherally, and the specific impact of IL-33 signaling within the brain is unclear. Here, we show that IL-33 is expressed by oligodendrocytes and astrocytes during T. gondii infection, is released locally into the cerebrospinal fluid of T. gondii-infected animals, and is required for control of infection. IL-33 signaling promotes chemokine expression within brain tissue and is required for the recruitment and/or maintenance of blood-derived anti-parasitic immune cells, including proliferating, IFN-γ-expressing T cells and iNOS-expressing monocytes. Importantly, we find that the beneficial effects of IL-33 during chronic infection are not a result of signaling on infiltrating immune cells, but rather on radio-resistant responders, and specifically, astrocytes. Mice with IL-33 receptor-deficient astrocytes fail to mount an adequate adaptive immune response in the CNS to control parasite burden-demonstrating, genetically, that astrocytes can directly respond to IL-33 in vivo. Together, these results indicate a brain-specific mechanism by which IL-33 is released locally, and sensed locally, to engage the peripheral immune system in controlling a pathogen.

Exercising Immunity: Interleukin-13 Flexes Muscle.

Endurance exercise drives physiological changes in the muscle to optimize performance. In a recent study in Science, Knudsen et al. report a role for the type 2 cytokine interleukin-13 in orchestrating metabolic reprogramming that drives adaptation to endurance exercise.

Elevated circulating Th2 but not group 2 innate lymphoid cell responses characterize canine atopic dermatitis.

Atopic dermatitis (AD) is an allergic skin disease that causes significant morbidity and affects multiple species. AD is highly prevalent in companion dogs, and the clinical management of the disease remains challenging. An improved understanding of the immunologic and genetic pathways that lead to disease could inform the development of novel treatments. In allergic humans and mouse models of AD, the disease is associated with Th2 and group 2 innate lymphoid cell (ILC2) activation that drives type 2 inflammation. Type 2 inflammation also appears to be associated with AD in dogs, but gaps remain in our understanding of how key type 2-associated cell types such as canine Th2 cells and ILC2s contribute to the pathogenesis of canine AD. Here, we describe previously uncharacterized canine ILC2-like cells and Th2 cells ex vivo that produced type 2 cytokines and expressed the transcription factor Gata3. Increased circulating Th2 cells were associated with chronic canine AD. Single-cell RNA sequencing revealed a unique gene expression signature in T cells in dogs with AD. These findings underline the importance of pro-allergic Th2 cells in orchestrating AD and provide new methods and pathways that can inform the development of improved therapies.

The Prostaglandin D2 Receptor CRTH2 Promotes IL-33-Induced ILC2 Accumulation in the Lung.

Group 2 innate lymphoid cells (ILC2s) are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 inflammation. ILC2s express ST2, the receptor for the cytokine IL-33, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D2 (PGD2). The IL-33-ST2 and the PGD2-CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. In this study, we show that ILC2 accumulation in the murine lung in response to systemic IL-33 treatment was partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. Whereas donor wild-type ILC2s preferentially accumulated in the lungs compared with CRTH2-deficient ILC2s following transfer into IL-33-treated recipients, wild-type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD2-CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation.

Cytokines and beyond: Regulation of innate immune responses during helminth infection.

Parasitic helminth infection elicits a type 2 cytokine-mediated inflammatory response. During type 2 inflammation, damaged or stimulated epithelial cells exposed to helminths and their products produce alarmins and cytokines including IL-25, IL-33, and thymic stromal lymphopoietin. These factors promote innate immune cell activation that supports the polarization of CD4+ T helper type 2 (Th2) cells. Activated innate and Th2 cells produce the cytokines IL-4, -5, -9, and -13 that perpetuate immune activation and act back on the epithelium to cause goblet cell hyperplasia and increased epithelial cell turnover. Together, these events facilitate worm expulsion and wound healing processes. While the role of Th2 cells in this context has been heavily studied, recent work has revealed that epithelial cell-derived cytokines are drivers of key innate immune responses that are critical for type 2 anti-helminth responses. Cutting-edge studies have begun to fully assess how other factors and pathways, including lipid mediators, chemokines, Fc receptor signaling, danger-associated molecular pattern molecules, and direct cell-cell interactions, also participate in shaping innate cell-mediated type 2 inflammation. In this review, we discuss how these pathways intersect and synergize with pathways controlled by epithelial cell-derived cytokines to coordinate innate immune responses that drive helminth-induced type 2 inflammation.

Lung Innate Lymphoid Cell Composition Is Altered in Primary Graft Dysfunction.

Rationale: Primary graft dysfunction (PGD) is the leading cause of early morbidity and mortality after lung transplantation, but the immunologic mechanisms are poorly understood. Innate lymphoid cells (ILC) are a heterogeneous family of immune cells regulating pathologic inflammation and beneficial tissue repair. However, whether changes in donor-derived lung ILC populations are associated with PGD development has never been examined.Objectives: To determine whether PGD in chronic obstructive pulmonary disease or interstitial lung disease transplant recipients is associated with alterations in ILC subset composition within the allograft.Methods: We performed a single-center cohort study of lung transplantation patients with surgical biopsies of donor tissue taken before, and immediately after, allograft reperfusion. Donor immune cells from 18 patients were characterized phenotypically by flow cytometry for single-cell resolution of distinct ILC subsets. Changes in the percentage of ILC subsets with reperfusion or PGD (grade 3 within 72 h) were assessed.Measurements and Main Results: Allograft reperfusion resulted in significantly decreased frequencies of natural killer cells and a trend toward reduced ILC populations, regardless of diagnosis (interstitial lung disease or chronic obstructive pulmonary disease). Seven patients developed PGD (38.9%), and PGD development was associated with selective reduction of the ILC2 subset after reperfusion. Conversely, patients without PGD exhibited significantly higher ILC1 frequencies before reperfusion, accompanied by elevated ILC2 frequencies after allograft reperfusion.Conclusions: The composition of donor ILC subsets is altered after allograft reperfusion and is associated with PGD development, suggesting that ILCs may be involved in regulating lung injury in lung transplant recipients.

Enteroendocrine Progenitor Cell-Enriched miR-7 Regulates Intestinal Epithelial Proliferation in an Xiap-Dependent Manner.

BACKGROUND & AIMS: The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis. METHODS: We leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice. RESULTS: First, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling. CONCLUSIONS: This study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling.

Notch Signaling Orchestrates Helminth-Induced Type 2 Inflammation.

Infection with helminth parasites poses a significant challenge to the mammalian immune system. The type 2 immune response to helminth infection is critical in limiting worm-induced tissue damage and expelling parasites. Conversely, aberrant type 2 inflammation can cause debilitating allergic disease. Recent studies have revealed that key type 2 inflammation-associated immune and epithelial cell types respond to Notch signaling, broadly regulating gene expression programs in cell development and function. Here, we discuss new advances demonstrating that Notch is active in the development, recruitment, localization, and cytokine production of immune and epithelial effector cells during type 2 inflammation. Understanding how Notch signaling controls type 2 inflammatory processes could inform the development of Notch pathway modulators to treat helminth infections and allergies.

The Notch signaling pathway promotes basophil responses during helminth-induced type 2 inflammation.

Type 2 inflammation drives the clearance of gastrointestinal helminth parasites, which infect over two billion people worldwide. Basophils are innate immune cells that support host-protective type 2 inflammation during murine infection with the helminth Trichuris muris However, the mechanisms required for basophil function and gene expression regulation in this context remain unclear. We show that during T. muris infection, basophils localized to the intestine and up-regulated Notch receptor expression, rendering them sensitive to Notch signals that rapidly regulate gene expression programs. In vitro, Notch inhibition limited basophil cytokine production in response to cytokine stimulation. Basophil-intrinsic Notch signaling was required for T. muris-elicited changes in genome-wide basophil transcriptional programs. Mice lacking basophil-intrinsic functional Notch signaling had impaired worm clearance, decreased intestinal type 2 inflammation, altered basophil localization in the intestine, and decreased CD4+ T helper 2 cell responses following infection. These findings demonstrate that Notch is required for basophil gene expression and effector function associated with helminth expulsion during type 2 inflammation.