Eosinophil-deficient ΔdblGATA Research Articles

Eosinophils Contribute to Oral Tolerance via Induction of RORγt-Positive Antigen-Presenting Cells and RORγt-Positive Regulatory T Cells.

Oral tolerance has been defined as the specific suppression of immune responses to an antigen by prior oral administration of the antigen. It has been thought to serve to suppress food allergy. Previous studies have shown that dendritic cells (DCs) and regulatory T cells (Tregs) are involved in the induction of oral tolerance. However, the detailed mechanisms of Treg induction in oral tolerance remain largely unknown. Eosinophils have been recognized as effector cells in allergic diseases, but in recent years, the diverse functions of tissue-resident eosinophils have been reported. Eosinophils in the intestine have been reported to induce Tregs by releasing TGF-β, but the role of eosinophils in oral tolerance is still controversial. In this study, we analyzed the roles of eosinophils in oral tolerance using eosinophil-deficient ΔdblGATA mice (mice lacking a high-affinity GATA-binding site in the GATA1 promoter). ΔdblGATA mice showed impaired antigen-induced oral tolerance compared to wild-type mice. The induction of RORγt+ Tregs in mesenteric lymph nodes (MLNs) by oral tolerance induction was impaired in ΔdblGATA mice compared to wild-type mice. An increase in RORγt+ antigen-presenting cells (APCs), which are involved in RORγt+ Treg differentiation, in the intestine and MLNs was not seen in ΔdblGATA mice. Notably, the expansion of group 3 innate lymphoid cells (ILC3s), a subset of RORγt+ APCs, by oral tolerance induction was seen in wild-type mice but not ΔdblGATA mice. These results suggest that eosinophils are crucial in the induction of oral tolerance, possibly via the induction of RORγt+ APCs and RORγt+ Tregs.

5-lipoxygenase- and Glucocorticoid-dependent eosinophilia in a novel surgical model in mice

BackgroundSubcutaneous implants of heat–coagulated egg white (egg white implants, EWI) induce intense local eosinophilia and prime for hyperreactivity following airway ovalbumin challenge. The roles of allergen sensitization, surgical trauma–induced glucocorticoids, and the 5-lipoxygenase (5-LO) pathway were hitherto unexplored in this model, in which quantitative recovery and large-scale purification of the eosinophils from the inflammatory site for functional and immunopharmacological studies are difficult to achieve. MethodsWe overcame this limitation by shifting the implantation site to the peritoneal cavity (EWIp), thereby enabling quantitative leukocyte retrieval. ResultsBy day 7 post–surgery, eosinophil counts reached ~ 30% of all leukocytes recovered. Eosinophilia was prevented by: a) induction of allergen–specific oral tolerance to ovalbumin, the main allergen in egg white; b) inactivation of the 5–lipoxygenase pathway; c) blockade of endogenous glucocorticoid signaling by pretreatment with metirapone plus mifepristone before surgery. Highly purified eosinophils (~99% pure) could be obtained from the peritoneal exudate of EWIp–carrier mice in 2 simple, antibody–free steps. Preparative-scale yields, suitable for most biochemical, pharmacological, and molecular applications, were routinely obtained, and could be further enhanced through addition of pre–or post–surgery immunization steps (active or adoptive). The recovered eosinophils were fully functional in vivo, as demonstrated by the transfer of purified eosinophils into eosinophil–deficient Δdbl–GATA–1-KO mice, which upon subsequent challenge with eotaxin-1 present secondary accumulation of neutrophils, but not of mononuclear phagocytes. ConclusionThese findings document glucocorticoid−, allergen– and 5–lipoxygenase–dependent eosinophilia, which makes EWIp carriers an abundant source of pure, nontransgenic eosinophils for immunopharmacological studies.

Th2 cells promote eosinophil-independent pathology in a murine model of allergic bronchopulmonary aspergillosis.

Repeated inhalation of airborne conidia derived from the fungus Aspergillus fumigatus (Af) can lead to a severe eosinophil-dominated inflammatory condition of the lung termed allergic bronchopulmonary aspergillosis (ABPA). ABPA affects about 5 million individuals worldwide and the mechanisms regulating lung pathology in ABPA are poorly understood. Here, we used a mouse model of ABPA to investigate the role of eosinophils and Tcell-derived IL-4/IL-13 for induction of allergic lung inflammation. Selective deletion of IL-4/IL-13 in Tcells blunted the Af-induced lung eosinophilia and further resulted in lower expression of STAT6-regulated chemokines and effector proteins such as Arginase 1, Relm-α, Relm-β, and Muc5a/c. Eosinophil-deficient ΔdblGata mice showed lower IL-4 expression in the lung and the number of Th2 cells in the lung parenchyma was reduced. However, expression of the goblet cell markers Clca1 and Muc5a/c, abundance of mucin-positive cells, as well as weight gain of lungs were comparable between Af-challenged ΔdblGata and WT mice. Based on these results, we conclude that Tcell-derived IL-4/IL-13 is essential for Af-induced lung eosinophilia and inflammation while eosinophils may play a more subtle immunomodulatory role and should not simply be regarded as pro-inflammatory effector cells in ABPA.

Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium which causes human Q fever. An acidified citrate cysteine medium (ACCM-2) has been developed which mimics the intracellular replicative niche of C. burnetii and allows axenic growth of the bacteria. To determine if C. burnetii cultured in ACCM-2 retains immunogenicity, we compared the protective efficacies of formalin-inactivated C. burnetii Nine Mile phase I (PIV) and phase II (PIIV) vaccines derived from axenic culture 7, 14, and 28 days postvaccination. PIV conferred significant protection against virulent C. burnetii as early as 7 days postvaccination, which suggests that ACCM-2-derived PIV retains immunogenicity and protectivity. We analyzed the cellular immune response in spleens from PIV- and PIIV-vaccinated mice by flow cytometry at 7 and 14 days postvaccination and found significantly more granulocytes in PIV-vaccinated mice than in PIIV-vaccinated mice. Interestingly, we found these infiltrating granulocytes to be SSChigh CD11b+ CD125+ Siglec-F+ (where SSChigh indicates a high side scatter phenotype) eosinophils. There was no change in the number of eosinophils in PIV-vaccinated CD4-deficient mice compared to the level in controls, which suggests that eosinophil accumulation is CD4+ T cell dependent. To evaluate the importance of eosinophils in PIV-mediated protection, we vaccinated and challenged eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice had significantly worse disease than their wild-type counterparts when challenged 7 days postvaccination, while no significant difference was seen at 28 days postvaccination. Nevertheless, ΔdblGATA mice had elevated serum IgM with decreased IgG1 and IgG2a whether mice were challenged at 7 or 28 days postvaccination. These results suggest that eosinophils may play a role in early vaccine protection against C. burnetii and contribute to antibody isotype switching.

Eosinophils Mediate Basophil-Dependent Allergic Skin Inflammation in Mice

The mechanisms leading to allergic skin inflammation, such as atopic dermatitis or urticaria, are poorly defined. Here we used a mouse model for IgE-dependent chronic allergic inflammation to study the role of basophils and eosinophils for induction of pathology. FcεRI expression in basophils was required for the ear swelling response, and basophils promoted the expression of eosinophil-recruiting chemokines in the ear. The ear swelling response could be prevented by prior infection of mice with helminths in an IgE-dependent manner. Impaired skin eosinophilia and reduced ear swelling was further observed in IL-4/IL-13-deficient and STAT6-deficient mice. In addition, eosinophil-deficient ΔdblGATA mice showed only weak ear swelling response, which could be enhanced by eosinophil transfer. This suggests that IgE-activated basophils orchestrate the recruitment of eosinophils by secretion of IL-4/IL-13, which leads to STAT6-dependent expression of CCL24 from endothelial cells and extravasation of eosinophils into the ear pinna. Eosinophils are then the critical effector cells that cause pathology. Therefore, combined therapeutic approaches that block basophil activation and reduce eosinophil numbers could be efficient strategies to improve treatment of chronic allergic disorders of the skin.

Eosinophils support adipocyte maturation and promote glucose tolerance in obesity

Accumulating data have indicated a fundamental role of eosinophils in regulating adipose tissue homeostasis. Here, we performed whole-genome RNA sequencing of the small intestinal tract, which suggested the presence of impaired lipid metabolism in eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice fed a high-fat diet (HFD) showed reduced body fat mass, impaired enlargement of adipocytes, decreased expression of adipogenic genes, and developed glucose intolerance. HFD induced accumulation of eosinophils in the perigonadal white adipose tissue. Concordantly, adipocyte-differentiated 3T3-L1 cells promoted the migration of eosinophils through the expression of CCL11 (eotaxin-1) and likely promoted their survival through the expression of interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor. HFD-fed ΔdblGATA mice showed increased infiltration of macrophages, CD4+ T-cells, and B-cells, increased expression of interferon-γ, and decreased expression of IL-4 and IL-13 in white adipose tissue. Interferon-γ treatment significantly decreased lipid deposition in adipocyte-differentiated 3T3-L1 cells, while IL-4 treatment promoted lipid accumulation. Notably, HFD-fed ΔdblGATA mice showed increased lipid storage in the liver as compared with wild-type mice. We propose that obesity promotes the infiltration of eosinophils into adipose tissue that subsequently contribute to the metabolic homeostasis by promoting adipocyte maturation.

18 Eosinophils are an integral part of the tumour microenvironment in colorectal cancer exerting potent anti-tumorigenic activities

IntroductionTumor-associated eosinophilia has been first described more than 120 years ago, and is frequently observed in many types of cancer. Yet, fundamental knowledge is missing regarding the roles of tumor-associated eosinophils. Since gastrointestinal (GI) tract is the largest eosinophil reservoir in the body and a main organ for eosinophil-mediated diseases, we focused our studies on colorectal cancer (CRC) as a model system to define the roles of eosinophils in the tumour microenvironment (TME).Material and methodsEosinophil infiltration and function were studied in two independent models of colorectal cancer (CRC), representing the genetically driven and inflammation-driven CRC models (i.e. Apcmin/+ model and AOM+DSS treatment, respectively).Results and discussionsWe report that eosinophils are an integral part of the TME in CRC. Eosinophils are actively and specifically recruited to the TME in both models, where they undergo degranulation. Moreover, we show that a single intravenous injection of eosinophils into eosinophil deficient mice (ΔdblGATA mice) undergoing colitis-associated colorectal cancer (CAC) or ΔdblGATA/Apcmin/+ mice resulted in substantial recruitment of eosinophils to the TME, which fosters prolonged eosinophils survival likely via secretion of GM-CSF, IL-3 and IL-5.Eosinophil-deficient ΔdblGATA mice undergoing CAC displayed significantly decreased survival, which was associated with increased tumour number and size. Similarly, generation of ΔdblGATA/Apcmin/+ mice revealed significantly decreased survival, which was associated with increased tumour burden, thereby suggesting an anti-tumorigenic role for eosinophils. Importantly, the anti-tumorigenic effects of eosinophils were independent of changes in CD8+ T cells or MDSCs and were accompanied with a specific decrease in anti-active caspase 3+ cells. No changes were observed in the numbers of Ki-67+ or CD31+ cells. Thus, supporting eosinophil-mediated cytotoxicity in-vivo. Indeed, eosinophils were capable of killing CRC cell lines in vitro.ConclusionOur data establish key anti-tumorigenic roles for eosinophils in CRC. These findings may facilitate the development of new pharmacological treatments unleashing robust anti-tumour responses by eosinophils and will hopefully lead to the development of innovative eosinophil-oriented ‘checkpoint inhibitors’.

Transcriptome profiling of mouse colonic eosinophils reveals a key role for eosinophils in the induction of s100a8 and s100a9 in mucosal healing

Eosinophils are bone marrow-derived cells that have been largely implicated in Th2-associated diseases. Recent data highlights a key role for eosinophils in mucosal innate immune responses especially in the gastrointestinal (GI) tract, which is one of the largest eosinophil reservoirs in the body. Although eosinophils express and synthesize a plethora of proteins that can mediate their effector activities, the transcriptome signature of eosinophils in mucosal inflammation and subsequent repair has been considerably overlooked. We demonstrate that eosinophils are recruited to the colon in acute inflammatory stages where they promote intestinal inflammation and remain in substantial numbers throughout the mucosal healing process. Microarray analysis of primary colonic eosinophils that were sorted at distinct stages of mucosal inflammation and repair revealed dynamic regulation of colonic eosinophil mRNA expression. The clinically relevant genes s100a8 and s100a9 were strikingly increased in colonic eosinophils (up to 550-fold and 80-fold, respectively). Furthermore, local and systemic expression of s100a8 and s100a9 were nearly diminished in eosinophil-deficient ΔdblGATA mice, and were re-constituted upon adoptive transfer of eosinophils. Taken together, these data may provide new insight into the involvement of eosinophils in colonic inflammation and repair, which may have diagnostic and therapeutic implications.

E Eosinophils have an essential role in cardiac repair following myocardial infarction

BackgroundLow peripheral blood eosinophil count is associated with increased risk of mortality in ischaemic heart disease patients. Eosinophils contain preformed IL-4 within their cytoplasmic granules, which promotes an anti-inflammatory response...

SiglecF+Gr1hi eosinophils are a distinct subpopulation within the lungs of allergen-challenged mice.

Although eosinophils as a group are readily identified by their unique morphology and staining properties, flow cytometry provides an important means for identification of subgroups based on differential expression of distinct surface Ags. Here, we characterize an eosinophil subpopulation defined by high levels of expression of the neutrophil Ag Gr1 (CD45+CD11c-SiglecF+Gr1hi). SiglecF+Gr1hi eosinophils, distinct from the canonical SiglecF+Gr1- eosinophil population, were detected in allergen-challenged wild-type and granule protein-deficient (EPX-/- and MBP-1-/-) mice, but not in the eosinophil-deficient ΔdblGATA strain. In contrast to Gr1+ neutrophils, which express both cross-reacting Ags Ly6C and Ly6G, SiglecF+Gr1hi eosinophils from allergen-challenged lung tissue are uniquely Ly6G+ Although indistinguishable from the more-numerous SiglecF+Gr1- eosinophils under light microscopy, FACS-isolated populations revealed prominent differences in cytokine contents. The lymphocyte-targeting cytokines CXCL13 and IL-27 were identified only in the SiglecF+Gr1hi eosinophil population (at 3.9 and 4.8 pg/106 cells, respectively), as was the prominent proinflammatory mediator IL-13 (72 pg/106 cells). Interestingly, bone marrow-derived (SiglecF+), cultured eosinophils include a more substantial Gr1+ subpopulation (∼50%); Gr1+ bmEos includes primarily a single Ly6C+ and a smaller, double-positive (Ly6C+Ly6G+) population. Taken together, our findings characterize a distinct SiglecF+Gr1hi eosinophil subset in lungs of allergen-challenged, wild-type and granule protein-deficient mice. SiglecF+Gr1hi eosinophils from wild-type mice maintain a distinct subset of cytokines, including those active on B and T lymphocytes. These cytokines may facilitate eosinophil-mediated immunomodulatory responses in the allergen-challenged lung as well as in other distinct microenvironments.

Eosinophils are Required for Immune Responses Induced by Oral Immunization

Eosinophils are multifunctional leukocytes that reside in several tissues, most abundantly in the small intestinal lamina propria under the steady state. To date, the phenotypic and functional characteristics of small intestinal eosinophils have remained poorly understood. In this study, we found that proliferation of ovalbumin (OVA)-specific CD4 + T cells isolated from the mesenteric lymph nodes of eosinophil-deficient ΔdblGATA mice were decreased relative to wild-type mice after oral immunization with OVA and cholera toxin (CT), the typical mucosal adjuvant that induces CD4 + T cell-dependent responses. ΔdblGATA mice showed reduced mucosal secretion of OVA-specific IgA and IgG1 while maintaining a systemic level of anti-OVA IgG1 upon oral immunization with OVA and CT. These findings suggest that eosinophils might have a role in the modulation of T cell-mediated immune responses including mucosal antibody responses in the gastrointestinal tract following oral immunization.

Basophils regulate the recruitment of eosinophils in a murine model of irritant contact dermatitis

Although eosinophils have been detected in several human skin diseases in the vicinity of basophils, how eosinophils infiltrate the skin and the role of eosinophils in the development of skin inflammation have yet to be examined. Using murine irritant contact dermatitis (ICD) as a model, we sought to clarify the roles of eosinophils in ICD andthe underlying mechanism of eosinophil infiltration of the skin. We induced croton oil-induced ICD in eosinophil-deficient ΔdblGATA mice with or without a reactive oxygen species (ROS) inhibitor. We performed cocultivation with fibroblasts and bone marrow-derived basophils and evaluated eosinophil migration using a chemotaxis assay. ICD responses were significantly attenuated in the absence of eosinophils or by treatment with the ROS inhibitor. ROS was produced abundantly by eosinophils, and both basophils and eosinophils were detected in human and murine ICD skin lesions. In coculture experiments, basophils attracted eosinophils, especially in the presence of fibroblasts. Moreover, basophils produced IL-4 and TNF-α in contact with fibroblasts and promoted the expression of eotaxin/CCL11 from fibroblasts invitro. Eosinophils mediated the development of murine ICD, possibly through ROS production. Recruitment of eosinophils into the skin was induced by basophils in cooperation with fibroblasts. Our findings introduce the novelconcept that basophils promote the recruitment of eosinophils into the skin through fibroblasts in the developmentof skin inflammation.

Chemotaxis of bone marrow derived eosinophils in vivo: A novel method to explore receptor‐dependent trafficking in the mouse

Here, we describe a novel method via which ex vivo cultured mouse bone marrow derived eosinophils (bmEos) can be adoptively transferred into recipient mice in order to study receptor-dependent recruitment to lung tissue in vivo. Intratracheal instillation of recombinant human eotaxin-2 (hCCL24) prior to introduction of bmEos via tail vein injection resulted in an approximately fourfold increase in Siglec F-positive/CD11c-negative eosinophils in the lungs of eosinophil-deficient ΔdblGATA recipient mice compared with controls. As anticipated, bmEos generated from CCR3-gene-deleted mice did not migrate to the lung in response to hCCL24 in this model, indicating specific receptor dependence. BmEos generated from GFP-positive BALB/c mice responded similarly to hCCL24 in vitro and were detected in lung tissue of BALB/c WT as well as BALB/c ΔdblGATA eosinophil-deficient recipient mice, at approximately fourfold (at 5 h post-injection) and approximately threefold (at 24 h postinjection) over baseline, respectively. Comparable results were obtained with GFP-positive C57BL/6 bmEos responding to intratracheal hCCL24 in C57BL/6 ΔdblGATA recipient mice. The use of ex vivo cultured bmEos via one or more of these methods offers the possibility of manipulating bmEos prior to transfer into a WT or gene-deleted recipient host. Thus, this chemotaxis model represents a novel and robust tool for pharmacological studies in vivo.

IL-33 induces Th17-mediated airway inflammation via mast cells in ovalbumin-challenged mice

Allergic asthma is characterized by infiltration of eosinophils, elevated Th2 cytokine levels, airway hyperresponsiveness, and IgE. In addition to eosinophils, mast cells, and basophils, a variety of cytokines are also involved in the development of allergic asthma. The pivotal role of eosinophils in the progression of the disease has been a subject of controversy. To determine the role of eosinophils in the progression of airway inflammation, we sensitized and challenged BALB/c wild-type (WT) mice and eosinophil-deficient ΔdblGATA mice with ovalbumin (OVA) and analyzed different aspects of inflammation. We observed increased eosinophil levels and a Th2-dominant response in OVA-challenged WT mice. In contrast, eosinophil-deficient ΔdblGATA mice displayed an increased proportion of mast cells and a Th17-biased response following OVA inhalation. Notably, the levels of IL-33, an important cytokine responsible for Th2 immune deviation, were not different between WT and eosinophil-deficient mice. We also demonstrated that mast cells induced Th17-differentiation via IL-33/ST2 stimulation in vitro. These results indicate that eosinophils are not essential for the development of allergic asthma and that mast cells can skew the immune reaction predominantly toward Th17 responses via IL-33 stimulation.

Eosinophils Contribute to IL-4 Production and Shape the T-Helper Cytokine Profile and Inflammatory Response in Pulmonary Cryptococcosis

Susceptibility to infection with Cryptococcus neoformans is tightly determined by production of IL-4. In this study, we investigated the time course of IL-4 production and its innate cellular source in mice infected intranasally with C. neoformans. We show that pulmonary IL-4 production starts surprisingly late after 6 weeks of infection. Interestingly, in the lungs of infected mice, pulmonary T helper (Th) cells and eosinophils produce significant amounts of IL-4. In eosinophil-deficient ΔdblGATA mice, IL-33 receptor-expressing Th2s are significantly reduced, albeit not absent, whereas protective Th1 and Th17 responses are enhanced. In addition, recruitment of pulmonary inflammatory cells during infection with C. neoformans is reduced in the absence of eosinophils. These data expand previous findings emphasizing an exclusively destructive effector function by eosinophilic granulocytes. Moreover, in ΔdblGATA mice, fungal control is slightly enhanced in the lung; however, dissemination of Cryptococcus is not prevented. Therefore, eosinophils play an immunoregulatory role that contributes to Th2-dependent susceptibility in allergic inflammation during bronchopulmonary mycosis.

Immune regulatory mechanism in mouse IBD model (168.26)

Abstract Inflammatory bowel disease (IBD) in humans, such as Crohn’s disease and ulcerative colitis, is characterized by chronic inflammation with dysregulated mucosal immune tolerance. Some reported that immune responses to intestinal microbiota are critical to develop IBD and NOD-like receptor-mediated inflammasome is related to epithelial integrity. In order to investigate dysregulated mechanism focusing on the relationship between inflammasome and regulatory cells in IBD, we used a 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model in WT-C57BL/6 mice and caspase-1-/- mice. In addition, to observe Th2-dependent immune responses in IBD mouse, we also used WT-Balb/c mice and eosinophil-deficient ΔdblGATA mice. As a result, mice treated TNBS had significant changes in clinical parameters, weight loss, and colonic weights compared with those of sham control mice. Histologic analysis also showed severe inflammation in intestines of TNBS treated mice. We found that deficiency of eosinophil or IL-1β had resistance in initial stage of disease progression, represented by survival score and body weight loss and we also noted that proportion of Foxp3+ cells and myeloid-derived suppressor cells (MDSCs) increased in above TNBS-treated ΔdblGATA and caspase-1-/- mice. These results provide us another mechanism of immune modulation in the pathogenesis of intestinal inflammation.

This Month in Gastroenterology

This Month in Gastroenterology