RATIONALE:Eosinophilic gastritis (EoG) is a chronic inflammatory disease characterized by infiltration of eosinophils and mast cells, epithelial remodeling, and fibrosis. Although EoG is increasingly recognized as a distinct type 2 inflammatory disease, the cellular and molecular events that drive disease pathogenesis remain poorly understood. This is due in part to the absence of robust and physiologically relevant experimental models that recapitulate human disease METHODS: Experimental EoG was induced in wild-type and Il13ra1-/- mice by repeated intragastric oxazolone challenges in skin-sensitized mice. IL-4Rα was neutralized using antibodies. Gastric histopathology was determined by H&E, anti-Ki67, chloroacetate esterase, and anti-MBP staining. Gastric RNA was subjected to RNA sequencing. RESULTS:Experimental EoG resulted in robust gastric eosinophilia, mastocytosis, epithelial remodeling, and subepithelial fibrosis. Transcriptomic profiling of gastric tissue revealed broad upregulation of type 2 cytokine and epithelial-remodeling genes, including Il4ra, Il4i1, Ccl5, Muc4, Mmp10, Mcpt1/2, Areg, Pparg, and Tff2. The transcriptome profile of experimental EoG was markedly distinct from that of experimental EoE despite both models being initiated by oxazolone, suggesting that identical inflammatory triggers elicit tissue-specific and context-dependent transcriptional programs. Blockade of IL-4Rα signaling abrogated both eosinophil and mast cell infiltration and attenuated epithelial remodeling, whereas genetic deletion of Il13ra1 selectively suppressed epithelial remodeling without affecting inflammatory cell recruitment. CONCLUSION:These findings establish experimental EoG as a robust model for dissecting the cellular and molecular mechanisms driving gastric type 2 inflammation. They further define receptor-specific roles for IL-4Rα and IL-13Rα1 in coordinating immune infiltration and epithelial remodeling in EoG.

Type 2 inflammatory diseases are often grouped together as a single immunological entity, driven by shared cytokines and overlapping pathways. This framework has led to the development of biologic therapies targeting upstream epithelial-derived “alarmins”, including thymic stromal lymphopoietin (TSLP), interleukin (IL)-33, and interleukin (IL)-25. These mediators promote downstream cytokine responses, most notably IL-4, IL-5, and IL-13, which orchestrate type 2 inflammation across multiple tissues. Despite these shared molecular features, type 2-associated diseases exhibit substantial clinical heterogeneity and variable responses to targeted therapies. A notable example is TSLP blockade, which has shown significant clinical efficacy in asthma but failed to demonstrate similar benefit in atopic dermatitis. From a clinical immunology perspective, we explore the complexity of type 2 inflammation. We discuss the dual pro- and anti-inflammatory roles of TSLP isoforms (long form and short form), the contribution of compensatory inflammatory pathways, and the impact of the tissue microenvironment on disease-specific immune responses. These factors may underlie divergent therapeutic outcomes despite targeting a shared upstream mediator. A better understanding of this complexity may help explain the limitations of a linear, cytokine-centered model of type 2 inflammation and support the development of more precise therapeutic strategies, including targeting shared downstream signaling components and combination approaches.

Eosinophilic esophagitis (EoE) is a chronic, food antigen-driven, type 2 immune-mediated disease of the esophagus characterized by eosinophil-predominant mucosal inflammation, epithelial remodeling, and subepithelial fibrosis. Although patient biopsies have established the EoE transcriptome and identified key cellular and molecular mediators, biopsy-based research is inherently correlative and cannot resolve causal disease mechanisms, temporal disease progression or the functional hierarchy of immune cell interactions. Thus, animal models are indispensable tools for addressing these limitations. In this Review, we examine the landscape of experimental EoE models. We assess the capacity of each model to recapitulate key disease features including lamina propria and intraepithelial eosinophilia, subepithelial fibrosis, basal cell hyperplasia, epithelial barrier dysfunction, and angiogenesis. We further map these models to human EoE transcriptomic overlap and disease endotype relevance. Across this analysis, we highlight mechanistic insights that were obtained from these models including the eosinophil-independence of IL-13-driven esophageal remodeling, the respective roles of thymic stromal lymphopoietin and IL-33, and the critical role of epithelial-expressed IL-13Rα1. We further highlight the profibrotic functions of amphiregulin-producing T helper 2 cells and colony-stimulating factor 1-dependent macrophages. We discuss the anatomical, genetic and functional limitations of current models and outline directions for the next generation of EoE preclinical systems.

Introduction Oral immunotherapy (OIT) induces desensitization in IgE-mediated food allergy, yet the role of myeloid cells in acquisition of tolerance is unclear. CD300 receptors regulate activation of myeloid cells, with CD300f acting as an inhibitory receptor and CD300b as an activating receptor. Objective To investigate whether the modulation of CD300 receptors on myeloid cells during OIT may reflect effector cell reprogramming and serve as biomarkers of treatment response. Methods Thirty-five patients undergoing OIT were prospectively enrolled. Peripheral blood was collected at baseline and during up-dosing; 19 patients completed sampling upon reaching maintenance. CD300b and CD300f expression in eosinophils, monocytes, and neutrophils was analyzed by flow cytometry. Allergen-specific IgE and IgG4 were measured by ImmunoCAP. Associations with clinical parameters were assessed using logistic regression. Results Baseline CD300b was higher in patients with lower starting doses (p≤0.05). CD300f expression was lower in those with atopic dermatitis or multiple food allergies (p≤0.03). A significant downregulation of CD300b expression in the surface of eosinophils, monocytes, and neutrophils, was noted early in treatment (p≤0.05). Longitudinally, the expression of CD300f increased on eosinophils, whereas CD300b expression decreased on the surface of monocytes and neutrophils. Specific IgE reduction correlated with downregulation of CD300b expression in monocytes (R=0.51, p=0.02) and higher CD300f expression in eosinophils (R=-0.45, p=0.05). Conclusions Downregulation of CD300b and upregulation of CD300f during OIT suggests myeloid cell reprogramming toward a less inflammatory phenotype. These dynamic changes in expression suggest CD300b and CD300f as candidate biomarkers for understanding and monitoring OIT response.

Abstract Tuft cells initiate intestinal type 2 immunity, yet the mechanisms that restrain excessive tuft cell activation remain poorly understood. Here, we identify the receptor tyrosine phosphatase CD45 ( Ptprc ), previously considered a hematopoietic marker, as a regulator of intestinal tuft cell function. CD45 expression is restricted to a subset of tuft cells and is induced by helminth infection and IL-13. Epithelial-specific deletion of Ptprc activated a tuft cell inflammatory program, promoted an epithelial inflammatory state, and increased eosinophil accumulation at homeostasis. During Heligmosomoides polygyrus bakeri infection, CD45 deficiency enhanced ILC2 and Th2 responses and reduced parasite burden, demonstrating that epithelial CD45 limits type 2 immunity in vivo . Accordingly, in intestinal organoids, CD45 was dispensable for IL-13-driven tuft cell differentiation but restrained IL-13-responsive transcriptional programs. Mechanistically, CD45-deficient tuft cells exhibited altered protein abundance of STAT5 and IL17RB, implicated in tuft cell immune regulation. Together, these findings identify CD45 as a tuft cell-intrinsic regulatory checkpoint that restrains intestinal type 2 immunity through an IL-13-induced negative-feedback circuit.

RATIONALE:Thymic stromal lymphopoietin (TSLP) and IL-33 are alarmins implicated in eosinophilic esophagitis (EoE) pathogenesis by activating multiple cells, including mast cells (MCs). Whether TSLP or IL-33 have a role in EoE and whether their activities are distinct requires further investigation. METHODS:Experimental EoE was induced in wild type (WT) Il33-/- and Crlf2-/- mice. TSLP or IL-5 were neutralized using antibodies. Esophageal histopathology was determined by H&E, anti-Ki67, anti-CD31, and anti-MBP staining. Esophageal RNA was subjected to RNA sequencing. Bone marrow-derived MCs were activated with TSLP and IL-13 was determined (ELISA). RESULTS:TSLP and IL-33 were overexpressed in human and experimental EoE. Human and mouse esophageal MCs displayed the highest level of Crlf2 (TSLPR) compared to other immune cells. Crlf2-/- mice were nearly completely protected from EoE, and TSLP neutralization resulted in decreased basal cell proliferation, eosinophilia, lamina propria thickening, and vascularization. Induction of experimental EoE in Il33-/- mice resulted in reduced eosinophilia, but no alterations in tissue remodeling were observed compared to WT mice. RNA sequencing revealed that TSLP regulates the expression of key genes associated with human EoE (e.g., eotaxins, Il19, Klk5, Flg, Il36rn, Il1r2) and suggests a role for TSLP in regulating IL-1 signaling, barrier integrity, and epithelial cell differentiation. Experimental EoE was characterized by a MC-associated gene signature and elevated MCs. Activation of MCs with TSLP resulted in the secretion of IL-13. CONCLUSION:TSLP and IL-33 have non-redundant functions in experimental EoE. This study highlights TSLP as an upstream regulator of IL-13 and a potential therapeutic target for EoE.

Generating mammalian gametes with a skewed sex ratio has thus far eluded empirical confirmation. The utilization of such genetically engineered organisms would offer the potential to curtail the necessity for culling animals of undesirable sex, mitigate resource wastage, and alleviate superfluous labor burdens. In this study, we introduce a transgenic male mouse lineage, which consistently yields predominantly female progeny (comprising up to ∼94% of the total offspring). This accomplishment was made possible by integrating a controllable genetic cassette onto the Y chromosome. The cassette encodes dCas9 and RNA guides that selectively silence a spermatid maturation gene. After the separation of X and Y gametes during meiosis, gametes containing an X chromosome develop normally, while those harboring the engineered Y chromosome, subjected to dCas9 silencing of the spermatid maturation gene, do not mature properly. Indeed, some spermatozoa from the transgenic mice exhibit a unique morphology, associated with the absence of the maturation gene. Notably, the resultant female offspring do not inherit the genetically engineered Y chromosome and are thus not genetically modified. Importantly, the litter size of the transgenic mice remains unchanged compared to the wild type. These findings represent a groundbreaking demonstration of genetic engineering’s potential to yield sex-biased litters of full size without compromising genetic integrity, marking a pioneering advancement in this field of study. ![Graphical Abstract.][1] Graphical Abstract. A proof of concept for the first mammal producing sexed semen may revolutionize the way for breeding animals such as cows for the dairy industry and chickens for the eggs industry. One sentence summary Y chromosome manipulation yields mostly female mice, preserving litter size without genetic alteration in females. ### Competing Interest Statement I.Y., M.G., and U.Q. have submitted a patent request on a related technology in January 2019. I.Y., A.M., M.G, and U.Q. have submitted a patent request on the described technology in August 2023. [1]: pending:yes

The thymus is a primary lymphoid organ that is essential for the establishment of adaptive immunity through generation of immunocompetent T cells. In response to various stress signals, the thymus undergoes acute but reversible involution. However, the mechanisms governing its recovery are incompletely understood. Here, we used a dexamethasone-induced acute thymic involution mouse model to investigate how thymic hematopoietic cells (excluding T cells) contribute to thymic regeneration. scRNA-seq analysis revealed marked transcriptional and cellular changes in various thymic populations and highlighted thymus-resident innate lymphoid cells type 2 (ILC2) as a key cell type involved in the response to damage. We identified that ILC2 are activated by the alarmins IL-25 and IL-33 produced in response to tissue damage by thymic tuft cells and fibroblasts, respectively. Moreover, using mouse models deficient in either tuft cells and/or IL-33, we found that these alarmins are required for effective thymus regeneration after dexamethasone-induced damage. We also demonstrate that upon their damage-dependent activation, thymic ILC2 produce several effector molecules linked to tissue regeneration, such as amphiregulin and IL-13, which in turn promote thymic epithelial cell differentiation. Collectively, our study elucidates a previously undescribed role for thymic tuft cells and fibroblasts in thymus regeneration through activation of the type 2 immune response.

The RNA-binding proteins LIN28A and LIN28B contribute to a variety of developmental biological processes. Dysregulation of Lin28A and Lin28B expression is associated with numerous types of tumors. This study demonstrates that Lin28A overexpression in the mouse nephrons leads to severe inflammation and kidney damage rather than to tumorigenesis. Notably, Lin28A overexpression causes inflammation only when expressed in nephrons, but not in the stromal cells of the kidneys, highlighting its cell context-dependent nature. The nephron-specific Lin28A-induced inflammatory response differs from previously described Lin28B-mediated inflammatory feedback loops as it is IL-6 independent. Instead, it is associated with the rapid upregulation of cytokines like Cxcl1 and Ccl2. These findings suggest that the pathophysiological effects of Lin28A overexpression extend beyond cell transformation. Our transgenic mouse model offers a valuable tool for advancing our understanding of the pathophysiology of acute kidney injury, where inflammation is a key factor.

T cell inhibitory mechanisms prevent autoimmune reactions, while cancer immunotherapy aims to remove these inhibitory signals. Chronic ultraviolet (UV) exposure attenuates autoimmunity through promotion of poorly understood immune-suppressive mechanisms. Here we show that mice with subcutaneous melanoma are not responsive to anti-PD1 immunotherapy following chronic UV irradiation, given prior to tumor injection, due to the suppression of T cell killing ability in skin-draining lymph nodes. Using mass cytometry and single-cell RNA-sequencing analyzes, we discover that skin-specific, UV-induced suppression of T-cells killing activity is mediated by upregulation of a Ly6ahigh T-cell subpopulation. Independently of the UV effect, Ly6ahigh T cells are induced by chronic type-1 interferon in the tumor microenvironment. Treatment with an anti-Ly6a antibody enhances the anti-tumoral cytotoxic activity of T cells and reprograms their mitochondrial metabolism via the Erk/cMyc axis. Treatment with an anti-Ly6a antibody inhibits tumor growth in mice resistant to anti-PD1 therapy. Applying our findings in humans could lead to an immunotherapy treatment for patients with resistance to existing treatments.

Eosinophils have been mainly studied in allergic diseases and parasitic infections. Nonetheless, eosinophils accumulate in a variety of solid tumors, including colorectal cancer, where their presence is associated with improved prognosis. Eosinophils can promote antitumor immunity through various mechanisms, including direct cytotoxicity toward tumor cells and promoting T-cell activation. However, the mechanisms by which tumor cells regulate eosinophil activities are largely unknown. Herein, we characterized the potential interactions between eosinophils and colorectal cancer cells using an unbiased transcriptomic and proteomic analyses approach. Human eosinophils were stimulated with colorectal cancer cell conditioned media, containing tumor cell secreted factors from multiple cancer cell lines. RNA sequencing analysis identified a "core" signature consisting of 101 genes that characterize a baseline transcriptional program for the response of human eosinophils to colorectal cancer cells. Among these, the increased expression of IL-3R alpha and its beta c chain was identified and validated at the protein level. Secreted factors from tumor cells potentiated IL-3-induced expression of the adhesion molecule CD11a in eosinophils. Combining proteomics analysis of tumor cell secreted factors with RNA sequencing revealed potential ligand-receptor pairs between tumor cells and eosinophils and the potential involvement of the adhesion molecule CD18 and F2RL3/PAR4. Subsequent functional analyses demonstrated that F2RL3/PAR4 suppresses eosinophil migration in response tumor cell secreted factors. These findings add to the growing body of evidence that eosinophils are conditioned by their local microenvironment. Identifying mechanisms by which eosinophils interact with tumor cells could lead to the development of new immunotherapies for colorectal cancer and other solid tumors. Colorectal cancer cells interact with eosinophils by secreting factors that potentiate IL-3-induced expression of the adhesion molecule CD11a and stimulate eosinophil migration in a process negatively regulated by activation of PAR4.

Eosinophilic esophagitis (EoE) is an emerging chronic T helper type 2 (Th2)-associated, allergic, and immune-mediated disease, characterized histologically by eosinophil-predominant mucosal inflammation and clinically by esophageal dysfunction. Over the past years, the prevalence of EoE has dramatically increased globally. Until recently, most studies of EoE focused on using human biopsies, which are also used for diagnostic purposes, or esophageal epithelial cell lines, which led to major advances in the understanding of EoE. Despite this, a robust mouse model that mimics human disease is still crucial for both understanding disease pathogenesis and as a preclinical model for testing future therapeutics. Herein, we describe a highly reproducible and robust model of EoE that can be performed using wild-type mice by ear sensitization with oxazolone (OXA) followed by intraesophageal challenges. Experimental EoE elicited by OXA mimics the main histopathological features of human EoE, including intraepithelial eosinophilia, epithelial and lamina propria thickening, basal cell hyperplasia, and fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Induction of EoE in mice using oxazolone Support Protocol 1: Preparing the mouse esophagus for histological analysis Support Protocol 2: Assessment of epithelial and lamina propria thickness using H&E staining Support Protocol 3: Assessment of eosinophilic infiltration using anti-MBP and basal cell proliferation using anti-Ki-67 staining Support Protocol 4: Flow cytometry of mouse esophageal samples Support Protocol 5: ELISA on protein lysates of esophageal samples.

utotaxin (ATX) produces lysophosphatidic acid (LPA), which directly promotes pancreatic ductal adenocarcinoma (PDAC) growth, but the role of the tumor microenvironment (TME) in ATX-driven tumor growth is unclear. ATX–LPA signaling in PDAC is now shown to shape the TME by inhibiting eosinophil recruitment, resulting in increased tumor growth.

Eosinophils are bone marrow-derived granulocytes that are traditionally associated with type 2 immune responses, such as those that occur during parasite infections and allergy. Emerging evidence demonstrates the remarkable functional plasticity of this elusive cell type and its pleiotropic functions in diverse settings. Eosinophils broadly contribute to tissue homeostasis, host defence and immune regulation, predominantly at mucosal sites. The scope of their activities primarily reflects the breadth of their portfolio of secreted mediators, which range from cytotoxic cationic proteins and reactive oxygen species to multiple cytokines, chemokines and lipid mediators. Here, we comprehensively review basic eosinophil biology that is directly related to their activities in homeostasis, protective immunity, regeneration and cancer. We examine how dysregulation of these functions contributes to the physiopathology of a broad range of inflammatory diseases. Furthermore, we discuss recent findings regarding the tissue compartmentalization and adaptation of eosinophils, shedding light on the factors that likely drive their functional diversification within tissues. This Review by Arnold and Munitz discusses the diverse roles of eosinophils in the settings of tissue homeostasis, infection, allergy and cancer. The authors explain the molecular mechanisms that enable eosinophils to adapt to diverse tissue types and conditions, and they consider the therapeutic potential of eosinophil-depleting drugs in the clinic.

Immune checkpoint blockade (ICB) has revolutionized the landscape of cancer treatment. Nevertheless, most cancer patients still do not respond to ICB. In this issue of Cancer Cell, Blomberg et al. illustrate a critical cooperation between T cells and eosinophils, which jointly enhance effectiveness of ICB in breast cancer.

Supplementary Methods and Data- Changes are unmarked

Chronic inflammation is a hallmark charataristic of various inflammatory diseases including inflammatory bowel disease. Subsequently, current therapeutic approaches target immune-mediated pathways as means for therapeutic intervention and promotion of mucosal healing and repair. Emerging data demonstrate important roles for CD300 receptor family members in settings of innate immunity as well as in allergic and autoimmune diseases. One of the main pathways mediating the activities of CD300 family members is via promotion of resolution through interactions with ligands expressed by viruses, bacteria, or dead cells (e.g., phospholipids such as PtdSer and/or ceramide). We have recently shown that the expression of CD300a, CD300b and CD300f were elevated in patients with IBD and that CD300f (but not CD300a) regulates colonic inflammation in response to dextran sodium sulphate (DSS)-induced colitis. Whether CD300b has a role in colitis or mucosal healing is largely unknown. Herein, we demonstrate a central and distinct role for CD300b in colonic inflammation and subsequent repair. We show that Cd300b-/- mice display defects in mucosal healing upon cessation of DSS treatment. Cd300b-/- mice display increased weight loss and disease activity index, which is accompanied by increased colonic histopathology, increased infiltration of inflammatory cells and expression of multiple pro-inflammatory upon cessation of DSS cytokines. Furthermore, we demonstrate that soluble CD300b (sCD300b) is increased in the colons of DSS-treated mice and establish that CD300b can bind mouse and human epithelial cells. Finally, we show that CD300b decreases epithelial EpCAM expression, promotes epithelial cell motility and wound healing. These data highlight a key role for CD300b in colonic inflammation and repair processes and suggest that CD300b may be a future therapeutic target in inflammatory GI diseases.