Resident Eosinophils Research Articles

Eosinophils are sparse in homeostatic rectal tissue which impedes studying resident eosinophils

Eosinophils are sparse in homeostatic rectal tissue which impedes studying resident eosinophils

Phenotypic Shift of an Inflammatory Eosinophil Subset into a Steady-State Resident Phenotype after 2 Years of Vaccination against IL-5 in Equine Insect Bite Hypersensitivity

Eosinophils play a key role in allergic diseases such as insect bite hypersensitivity (IBH). Together with Th2 cells, they shape the course of inflammation in associated type I/IVb allergies. Therefore, a virus-like particle (VLP)-based vaccine targeting equine interleukin-5 (eIL-5), eIL-5-CuMV-TT, was developed to interfere with the IL-5 dependency of eosinophils by inducing the production of anti-self-IL-5 antibodies and alleviating clinical signs in IBH-affected horses. A previous study highlighted the presence of two eosinophil subsets, steady-state resident eosinophils (rEos) and inflammatory eosinophils (iEos), circulating in the blood of healthy and IBH-affected horses, distinguishable by the expression of integrin CD49f. Furthermore, eIL-5-CuMV-TT 1st year vaccination showed a significant decrease of total eosinophils and, in particular, iEos. Nevertheless, the very few remaining eosinophils still shared an iEos phenotype, reflected by bigger size and higher granularity. The aim of this study was to follow up on the phenotype of eosinophils in the 2nd year of vaccination of IBH-affected horses with eIL-5-CuMV-TT. Using flow cytometry analysis of the blood of healthy, IBH, IBH-placebo, and IBH-vaccinated horses, the percentage and count of cells were compared between groups with a focus on pair analysis of eosinophils in 1st and 2nd year vaccinated horses. Our data showed comparably low levels of iEos and a significant increase of rEos in 2nd year compared to 1st year vaccinated horses, suggesting a phenotypic shift toward a resident-like eosinophil population, primarily associated with the phenotype of healthy horses. The reduction of size, granularity, and expression of integrin CD49f in the 2nd year suggests a benefit of long-term treatment with the eIL-5-CuMV-TT vaccine.

A162 DEFINING INNATE IMMUNE CELL MECHANISMS UPON HELICOBACTER PYLORI MEDIATED TUMORIGENESIS

Abstract Aims Hypothesis: H. pylori interacts with eosinophils to inhibit degranulation, alter eosinophil gene expression and promote gastric tumorigenesis Aim 1: Elucidate the mechanisms underlying the interactions between eosinophils and H. pylori. Aim 2: Determine the role of eosinophils during H. pylori mediated gastric tumorigenesis. Methods Histological and immunofluorescent staining. Hemotoxilin and eosin staining is important to analyze gastric gland structure and parietal cell loss. Immunofluorescent staining is used to detect and localize specific antigens present in cells and cell membranes. Gastric organoids: Gastric organoids are an important tool for modelling dissease. Gastric organoids generated from adult stem cells from gastric glands depend on growth factors that mimic tissue regeneration. Bone marrow derived eosinophils: Bone marrow progenitor cells isolated from murine femur and tibia are easily converted into maturer eosinophils with the addition of cytokine interleukin-5. Results Addition of mouse derived eosinophils to gastric organoids significantly increases number of organoids. To understand the relationship between eosinophils and gastric epithelial cell (GEC) growth, I designed a co-culture experiment that involved incubating mature eosinophils with gastric glands in an extracellular matrix. These findings revealed a significant increase in the number of gastric organoids per well when eosinophils were present compared to the gastric organoids without eosinophils. This observation supports the hypothesis that eosinophils may play a crucial role in promoting gastric tumorigenesis. Exposure of mouse derived eosinophils to H. pylori significantly increases gastric organoid proliferation. To explore the impact of stimulating eosinophils prior to incubation with GECs I incubated the eosinophils with H. pylori. As expected, there was a significant increase in the number of organoids per well with the addition of uninfected eosinophils. Interestingly, when H. pylori incubated eosinophils were added to the gastric glands, there was a significant increase in the number of gastric organoids per well compared to uninfected eosinophils. Conclusions My preliminary findings have indicated that eosinophils can promote gastric epithelial stem cell proliferation. These results led me to ask how treatment of eosinophils with H. pylori subsequently impacts gastric organoid growth and gastric stem cell proliferation. Interestingly, I found H. pylori treated eosinophils increase growth and gastric stem cell proliferation. These results support my hypothesis as increased proliferation is considered a hallmark of tumorigenesis. There little known about resident and inflammatory GI eosinophils and the role of eosinophils during tumorigenesis in unknown. A better understanding of the role eosinophils play can uncover novel mechanisms to be targeted for therapeutic intervention. Funding Agencies CIHR

Acute HDM exposure shows time-of-day and sex-based differences in the severity of lung inflammation and circadian clock disruption.

Asthma is a chronic inflammatory disease that shows a time-of-day response to variations in symptoms/severity. However, how the lung circadian clock influences time-of-day response and sex-based differences in house dust mite (HDM)-induced airway inflammation and remodeling has not been thoroughly investigated. We sought to determine whether acute HDM exposure in wild-type mice shows time-of-day response and sex-based differences in allergic airway inflammation and circadian clock disruption in the lungs. Wild-type (C57BL/6J) and Rev-erbα knockout (KO) mice were exposed to either PBS or HDM (for 10 days) intranasally at Zeitgeber time (ZT0: 6 am; ZT12: 6 pm) and euthanized 48 hours after the last exposure. Acute HDM-induced time-of-day response and sex-based differences in lung inflammation, gated cytokines/chemokines, humoral and hormonal responses, and circadian clock gene expression were analyzed. Acute HDM-exposed mice showed a time-of-day response and sex-based differences in exaggerated lung inflammation (inflammatory eosinophils and interstitial macrophages) at ZT12 when compared with ZT0. HDM-exposed female mice showed increased inflammatory response at ZT12, but HDM-exposed male mice showed comparatively lower inflammation with no time-of-day response. HDM-exposed female and male mice showed augmented IgE levels at ZT12 when compared with ZT0. Myeloid innate immunity panel, cytokines/chemokines, and mucin genes showed a time-of-day gating response at ZT0 and ZT12 in the HDM group. In addition, HDM exposure altered the expression of circadian clock genes in the lung, which was evident in female mice at ZT12. Overall, female mice showed significant time-of-day responses to all these parameters compared with male mice. Rev-erbα KO mice exposed to acute HDM showed exaggerated lung inflammation associated with increased IgE and proinflammatory cytokines in bronchoalveolar lavage fluid. Interestingly, HDM exposure causes reduced expression of clock genes in flow-sorted resident eosinophils but not alveolar macrophages. Acute HDM exposure reduced the nocturnal locomotor activity in mice 5 days post-HDM exposure until day10. This study shows a time-of-day response to acute HDM exposure and sex-based differences in the severity of lung inflammation and humoral immune response associated with circadian clock disruption. Our findings support the use of separate female and male mice cohorts for preclinical studies to understand the molecular heterogeneity in asthma pathophysiology.

Influenza breakthrough infection in vaccinated mice is characterized by non-pathological lung eosinophilia.

Eosinophils are important mediators of mucosal tissue homeostasis, anti-helminth responses, and allergy. Lung eosinophilia has previously been linked to aberrant Type 2-skewed T cell responses to respiratory viral infection and may also be a consequence of vaccine-associated enhanced respiratory disease (VAERD), particularly in the case of respiratory syncytial virus (RSV) and the formalin-inactivated RSV vaccine. We previously reported a dose-dependent recruitment of eosinophils to the lungs of mice vaccinated with alum-adjuvanted trivalent inactivated influenza vaccine (TIV) following a sublethal, vaccine-matched H1N1 (A/New Caledonia/20/1999; NC99) influenza challenge. Given the differential role of eosinophil subset on immune function, we conducted the investigations herein to phenotype the lung eosinophils observed in our model of influenza breakthrough infection. Here, we demonstrate that eosinophil influx into the lungs of vaccinated mice is adjuvant- and sex-independent, and only present after vaccine-matched sublethal influenza challenge but not in mock-challenged mice. Furthermore, vaccinated and challenged mice had a compositional shift towards more inflammatory eosinophils (iEos) compared to resident eosinophils (rEos), resembling the shift observed in ovalbumin (OVA)-sensitized allergic control mice, however without any evidence of enhanced morbidity or aberrant inflammation in lung cytokine/chemokine signatures. Furthermore, we saw a lung eosinophil influx in the context of a vaccine-mismatched challenge. Additional layers of heterogeneity in the eosinophil compartment were observed via unsupervised clustering analysis of flow cytometry data. Our collective findings are a starting point for more in-depth phenotypic and functional characterization of lung eosinophil subsets in the context of vaccine- and infection-induced immunity.

Eosinophils Sub-Types in Asthmatic and COPD Patients.

There is a differential response to eosinophilic modulation between patients with asthma and chronic obstructive pulmonary disease (COPD). There is also evidence of different subtypes of eosinophils in murine models. However, no study has compared eosinophil subtypes in COPD and in asthmatic patients. We studied 10 stable subjects in each of 4 groups: COPD, asthmatics, non-COPD smokers (NCS) and healthy volunteers (V). COPD and asthmatics were matched by age, sex and FEV1%. The following variables were determined: anthropometrics, smoking, exacerbation history, medication use, lung function, and comorbidities. Using flow cytometry and confocal microscopy from blood samples, we determined differences in eosinophils surface proteins and classified them as: 1.- Resident eosinophils [rEos (Siglec-8+CD62L+IL-3Rlo)] or 2. -Inflammatory eosinophils [iEos (Siglec-8+CD62LloIL-3Rhi)]. IL-5 receptor was also determined. Findings were validated in 59 COPD and in 17 asthmatic patients. Asthmatics had a higher proportion (%) of iEos (25 ± 15) compared to COPD patients (0,5 ± 1), NCS (0,14 ± 0,24), and V (0,67 ± 1,72). In asthmatics, the proportion of iEos was independent of total eosinophil number. iEos had higher IL5 receptor than the rEos (777,02 ± 124,55 vs. 598,35 ± 318,69, p<0,01). In COPD patients there was no relation between iEos numbers with ICS use, disease severity or exacerbations rate. The findings in COPD and asthma patients were confirmed in validation cohorts. There are differences in the sub-types of circulating eosinophils between asthmatic and COPD patients. This could have clinical implications in the interpretation of eosinophil significance and approach to therapy in those patients.

Immunophenotypic and Functional Characterization of Eosinophil and Migratory Dendritic Cell Subsets during Filarial Manifestation of Tropical Pulmonary Eosinophilia.

The role of eosinophil and migratory dendritic cell (migDC) subsets during tropical pulmonary eosinophilia (TPE), a potentially fatal complication of lymphatic filariasis, has not been explored. We show that the onset of TPE is characterized by the accumulation of ROS and anaphylatoxins and a rapid influx of morphologically distinct Siglec-Fint resident eosinophils (rEos) and Siglec-Fhi inflammatory eosinophils (iEos) in the lungs, BAL fluid, and blood of TPE mice. While rEos display regulatory behavior, iEos are highly inflammatory cells, as evident in upregulated expression of activation markers CD69 and CD101, anaphylatoxin receptor C5AR1, alarmins s100a8 and s100a9, components of NADPH oxidase, and copious secretion of TNF-α, IFN-γ, IL-6, IL-1β, IL-4, IL-10, IL-12, and TGF-β. Importantly, iEos exhibited heightened ROS generation, higher phagocytic and increased antigen presentation capacity, elevated Ca2+ influx, and increased F-actin polymerization but downregulated negative regulators of the immune response, i.e., Cd300a, Anaxa1, Runx3, Lilrb3, and Serpinb1a, underlining their essential role in promoting lung damage during TPE. Interestingly, TPE mice also showed significant expansion of CD24+CD11b+ migDCs, which showed upregulated expression of maturation and costimulatory markers CD40, CD80, CD83, CD86, and MHCII, increased antigen presentation capacity, and higher migratory potential as evidenced by increased expression of cytokine receptors CCR4, CCR5, CXCR4, and CXCR5. CD24+CD11b+ migDCs also upregulated the expression of immunoregulators PD-L1 and PD-L2 and secreted proinflammatory cytokines, suggesting their significant involvement during TPE. Taken together, we document important morphological, immunophenotypic, and functional characteristics of eosinophil and migDC subsets in the lungs of TPE mice and suggest that they contribute to worsening lung histopathological conditions during TPE.

Subsets of Eosinophils in Asthma, a Challenge for Precise Treatment

The existence of eosinophils was documented histopathologically in the first half of the 19th century. However, the term "eosinophils" was first used by Paul Ehrlich in 1878. Since their discovery and description, their existence has been associated with asthma, allergies, and antihelminthic immunity. Eosinophils may also be responsible for various possible tissue pathologies in many eosinophil-associated diseases. Since the beginning of the 21st century, the understanding of the nature of this cell population has undergone a fundamental reassessment, and in 2010, J. J. Lee proposed the concept of "LIAR" (Local Immunity And/or Remodeling/Repair), underlining the extensive immunoregulatory functions of eosinophils in the context of health and disease. It soon became apparent that mature eosinophils (in line with previous morphological studies) are not structurally, functionally, or immunologically homogeneous cell populations. On the contrary, these cells form subtypes characterized by their further development, immunophenotype, sensitivity to growth factors, localization, role and fate in tissues, and contribution to the pathogenesis of various diseases, including asthma. The eosinophil subsets were recently characterized as resident (rEos) and inflammatory (iEos) eosinophils. During the last 20 years, the biological therapy of eosinophil diseases, including asthma, has been significantly revolutionized. Treatment management has been improved through the enhancement of treatment effectiveness and a decrease in the adverse events associated with the formerly ultimately used systemic corticosteroids. However, as we observed from real-life data, the global treatment efficacy is still far from optimal. A fundamental condition, "sine qua non", for correct treatment management is a thorough evaluation of the inflammatory phenotype of the disease. We believe that a better understanding of eosinophils would lead to more precise diagnostics and classification of asthma subtypes, which could further improve treatment outcomes. The currently validated asthma biomarkers (eosinophil count, production of NO in exhaled breath, and IgE synthesis) are insufficient to unveil super-responders among all severe asthma patients and thus give only a blurred picture of the adepts for treatment. We propose an emerging approach consisting of a more precise characterization of pathogenic eosinophils in terms of the definition of their functional status or subset affiliation by flow cytometry. We believe that the effort to find new eosinophil-associated biomarkers and their rational use in treatment algorithms may ameliorate the response rate to biological therapy in patients with severe asthma.

C5aR1 activation in mice controls inflammatory eosinophil recruitment and functions in allergic asthma.

Pulmonary eosinophils comprise at least two distinct populations of resident eosinophils (rEOS) and inflammatory eosinophils (iEOS), the latter recruited in response to pulmonary inflammation. Here, we determined the impact of complement activation on rEOS and iEOS trafficking and function in two models of pulmonary inflammation. BALB/c wild-type and C5ar1-/- mice were exposed to different allergens or IL-33. Eosinophil populations in the airways, lung, or mediastinal lymph nodes (mLN) were characterized by FACS or immunohistochemistry. rEOS and iEOS functions were determined in vivo and in vitro. HDM and IL-33 exposure induced a strong accumulation of iEOS but not rEOS in the airways, lungs, and mLNs. rEOS and iEOS expressed C3/C5 and C5aR1, which were significantly higher in iEOS. Initial pulmonary trafficking of iEOS was markedly reduced in C5ar1-/- mice and associated with less IL-5 production from ILC2 cells. Functionally, adoptively transferred pulmonary iEOS from WT but not from C5ar1-/- mice-induced airway hyperresponsiveness (AHR), which was associated with significantly reduced C5ar1-/- iEOS degranulation. Pulmonary iEOS but not rEOS were frequently associated with T cells in lung tissue. After HDM or IL-33 exposure, iEOS but not rEOS were found in mLNs, which were significantly reduced in C5ar1-/- mice. C5ar1-/- iEOS expressed less costimulatory molecules, associated with a decreased potency to drive antigen-specific T cell proliferation and differentiation into memory T cells. We uncovered novel roles for C5aR1 in iEOS trafficking and activation, which affects key aspects of allergic inflammation such as AHR, ILC2, and T cell activation.

Immune Modulation of Adipocyte Mitochondrial Metabolism.

Immune cells infiltrate adipose tissue as a function of age, sex, and diet, leading to a variety of regulatory processes linked to metabolic disease and dysfunction. Cytokines and chemokines produced by resident macrophages, B cells, T cells and eosinophils play major role(s) in fat cell mitochondrial functions modulating pyruvate oxidation, electron transport and oxidative stress, branched chain amino acid metabolism, fatty acid oxidation, and apoptosis. Indeed, cytokine-dependent downregulation of numerous genes affecting mitochondrial metabolism is strongly linked to the development of the metabolic syndrome, whereas the potentiation of mitochondrial metabolism represents a counterregulatory process improving metabolic outcomes. In contrast, inflammatory cytokines activate mitochondrially linked cell death pathways such as apoptosis, pyroptosis, necroptosis, and ferroptosis. As such, the adipocyte mitochondrion represents a major intersection point for immunometabolic regulation of central metabolism.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Benralizumab Used for Eosinophilic Inflammation in a Lung Transplant Patient

<h3>Introduction</h3> Eosinophilic inflammation in lung transplant (LTX) is challenging. Eosinophils may be found in transplant grafts during rejection episodes; however, it is unclear if they are bystanders or pathogenic. We did not find any prior use of biologics to disrupt the interleukin-5 (IL-5) pathway in the transplant literature. We discuss the novel use of benralizumab, a monoclonal antibody that targets IL-5 receptor. <h3>Case Report</h3> 19 year-old female with cystic fibrosis presented 18 months post-LTX with cough and chest pain and was treated with amoxicillin-clavulanate for community acquired pneumonia. She had mild leukocytosis and elevated absolute eosinophil count. She worsened and developed respiratory distress and fever, requiring bilevel positive airway pressure (BiPAP). Computed tomography showed total opacification of right lung. Bronchoscopy identified mild acute cellular rejection, low-grade small airway inflammation, chronic airway rejection, and marked eosinophilic inflammation in tissue and bronchoalveolar lavage (18%). Broad infectious work up was unrevealing. Peripheral eosinophils decreased to undetectable after methylprednisolone pulse, but rebounded one week later. She remained in intensive care unit on BiPAP. Due to favorable side effect profile and deteriorating clinical picture, we initiated benralizumab. Peripheral eosinophilia resolved, and she was weaned off respiratory support. Her two-year post-LTX surveillance bronchoscopy demonstrated resolution of eosinophilic inflammation. <h3>Summary</h3> Eosinophilic inflammation with acute respiratory failure justified the use of benralizumab, which successfully treated eosinophilic associated rejection. We selected benralizumab over other IL-5 disrupters because it theoretically targets IL-5 receptors in the tissue, thereby impacting tissue resident eosinophils. Further study of the role of eosinophils in LTX and the efficacy of IL-5 blockade should be considered.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) drives the resolution of allergic asthma

SummaryRANTES is implicated in allergic asthma and in T cell-dependent clearance of infection. RANTES receptor family comprises CCR1, CCR3, and CCR5, which are G-protein-coupled receptors consisting of seven transmembrane helices. Infections with respiratory viruses like Rhinovirus cause induction of RANTES production by epithelial cells. Here, we studied the role of RANTES in the peripheral blood mononuclear cells in cohorts of children with and without asthma and validated and extended this study to the airways of adults with and without asthma. We further translated these studies to a murine model of asthma induced by house dust mite allergen in wild-type RANTES and CCR5-deficient mice. Here we show an unpredicted therapeutic role of RANTES in the resolution of allergen-induced asthma by orchestrating the transition of effector GATA-3+CD4+ T cells into immune-regulatory-type T cells and inflammatory eosinophils into resident eosinophils as well as increased IL-10 production in the lung.

76938 Does Decreased Adipose Tissue Eosinophil Content Impair Adipocyte Biology in Human Subjects with Obesity and Insulin Resistance?

ABSTRACT IMPACT: Extrapolating from mouse data we explored eosinophil content in human adipose tissue and its effect on adipocyte biology potentially leading to the discovery of novel therapeutic targets for treatment of obesity and insulin resistance. OBJECTIVES/GOALS: The interaction between immune cells and adipose tissue (AT) in obesity has not been fully elicited. Mouse models of diet-induced-obesity show AT resident eosinophils (EOS) help preserve insulin sensitivity (IS). As data in human obesity are lacking, here we explored AT-EOS content and their role in AT metabolism in subjects with and without obesity. METHODS/STUDY POPULATION: We recruited lean (L) subjects and patients with obesity (Ob) to undergo abdominal subcutaneous AT biopsy and evaluation of insulin resistance (IR) by determination of HOMA-IR. Circulating EOS were isolated from all participants under fasting conditions and exposed to high glucose (HG) or high lipids (HL) for 4 hrs. AT EOS number was assessed via FACS analysis. Circulating EOS and AT mRNA was assessed by qPCR for multiple genes involved in inflammation and cell migration. To evaluate the effect of EOS on primary human adipocytes, in vitro cultures were exposed for 4 days to either interleukin-4 (IL-4), interleukin-13 (IL-13) or to human EOS. Adipocytes mRNA levels were evaluated for genes involved in adipogenesis and lipid metabolism. RESULTS/ANTICIPATED RESULTS: 16 lean, IS subjects (BMI 22.5+ 0.4kg/m2) and 22 age-matched IR patients with obesity (BMI: 38.9 + 1.0kg/m2) participated. We observed a ratio of 2:1 in AT EOS content of L vs Ob subjects (P&lt;0.03). To assess the reduced AT-EOS content in obesity, we evaluated expression of Chemokine-C receptor 3 (CCR3) in circulating EOS. We show decreased CCR3 mRNA levels in Ob vs L subjects (P=0.006). We expect HL in vitro experiments on peripheral EOS of L subjects to affect CCR3 mRNA levels. In AT of Ob subjects, we found a significant decreased expression of Eotaxin 2, the main EOS chemokine binding CCR3 expressed on EOS. Preliminary data from in vitro primary adipocytes culture suggest for IL-4 and IL-13 to increase mRNA level of Peroxisome Proliferator Activated Receptor Gamma (PPARG), the master regulator of adipogenesis. DISCUSSION/SIGNIFICANCE OF FINDINGS: Comparable to animal studies, we found a decrease of AT-EOS content in patients with obesity. Alterations in CCR3/Eotaxin 2 signaling may be involved. IL-4 &amp;IL-13 are secreted predominantly by EOS and appear to directly regulate gene expression in human adipocytes. These data represent the first evidence for a novel role of EOS in human AT biology.

Monitoring eosinophils to guide therapy with biologics in asthma: does the compartment matter?

Eosinophils, since their initial description by Thomas Wharton Jones in 1846, and staining characteristics by Paul Ehrlich in 1879, have been associated with asthma1 . They are produced in the bone marrow, from pluripotential stem cells, whichfirst differentiate, largely regulated by a transcription factor GATA-1, into a hybrid precursor for both basophils and eosinophils, and then into a separate eosinophil lineage. The eosinophilopoietins IL-3, GM-CSF and notably IL-5 regulatetheir further expansion and migration out of the bonemarrow into the circulation.Circulating eosinophils subsequently interactwith the endothelium by processes involving rolling, adhesion, and diapedesis. Depending on the targetorgan, eosinophils cross the endothelium into tissues by a regulated process involving the coordinatedinteraction between networks involving cytokines such as IL-13, the chemokine eotaxin-1, eosinophil adhesion molecules (α4β1, α4β7, αmβ2, αLβ2), and adhesion receptors on the endothelium (MAdCAM-1, VCAM-1, and ICAM-1). Under homeostatic conditions, eosinophils traffic into the thymus, mammary gland, uterus, and most prominently into the gastrointestinal tract.

A FACS-based approach to obtain viable eosinophils from human adipose tissue

Eosinophils have been widely investigated in asthma and allergic diseases. More recently, new insights into the biology of these cells has illustrated eosinophils contribute to homeostatic functions in health such as regulation of adipose tissue glucose metabolism. Human translational studies are limited by the difficulty of obtaining cells taken directly from their tissue environment, relying instead on eosinophils isolated from peripheral blood. Isolation techniques for tissue-derived eosinophils can result in unwanted cell or ribonuclease activation, leading to poor cell viability or RNA quality, which may impair analysis of effector activities of these cells. Here we demonstrate a technique to obtain eosinophils from human adipose tissue samples for the purpose of downstream molecular analysis. From as little as 2 g of intact human adipose tissue, greater than 104 eosinophils were purified by fluorescence-activated cell sorting (FACS) protocol resulting in ≥ 99% purity and ≥ 95% viable eosinophils. We demonstrated that the isolated eosinophils could undergo epigenetic analysis to determine differences in DNA methylation in various settings. Here we focused on comparing eosinophils isolated from human peripheral blood vs human adipose tissue. Our results open the door to future mechanistic investigations to better understand the role of tissue resident eosinophils in different context.

OR04-05 MYOD1 Is Associated with Eosinophil-Mediated Browning of Subcutaneous Adipose Tissue

A novel role for adipose tissue (AT)-resident eosinophils (EOS) in metabolism has been suggested. These data were obtained using genetic animal models with either whole-body overexpression of Interleukin-5 (IL-5Tg) leading to eosinophilia or gene ablation resulting in complete lack of EOS. These models limit the specificity of the findings. We hypothesized that AT-resident EOS play a specific role in whole-body metabolism. To this end, we generated a transgenic mouse model overexpressing human eotaxin-2 (hE2) under the control of a fat-specific aP2 promoter, which would exclusively recruit circulating EOS into AT (hEo2Tg), without any changes in the overall EOS numbers. Compared with wild type (WT) mice, AT-EOS numbers were markedly increased in multiple fat depots from hEo2Tg mice, including subcutaneous white adipose tissue (sWAT). After 12 weeks of high-fat diet (HFD), hEo2Tg mice showed significantly less body weight gain and fat mass compared with WT littermates. These changes were associated with an improvement in glucose tolerance. We also found increased oxygen consumption and heat production in hEo2Tg mice under room temperature conditions. The increased thermogenesis was accompanied with an increased expression of browning genes such as Ucp1, Prdm16, Dio2 in sWAT from hEo2Tg HFD mice. So far, our data suggest that AT-resident EOS promote browning of sWAT. This, in turn, protects our animals against development of HFD-induced obesity and insulin resistance. Next, whole transcriptome mRNA sequencing and bioinformatic analyses were performed and showed a significant increase of myogenic differentiation 1 (Myod1) gene expression in hEo2Tg sWAT mice. This was confirmed by qRT-PCR. Myod1 is a key regulator of skeletal muscle differentiation. Given the shared features between brown fat and skeletal muscle, we speculate that by increasing Myod1 gene expression, (AT)-resident EOS mediate sWAT browning. Additional studies are needed to determine the molecular mechanism(s) underling the regulation of Myod1 gene expression by AT-resident EOS and its effect on sWAT browning.

Siglec-F-negative resident eosinophils represent an unexpectedly dominant population in homeostatic murine bone marrow

Abstract Bone marrow, while known to be a hematopoietic site for eosinophils, also harbors resident populations of these cells. Identification of such resident eosinophils typically requires flow cytometry-assisted sorting, with Siglec-F as a key marker for identification of murine eosinophils. At baseline, we found that bone marrow resident eosinophils described as Siglec-F(+) constitute about 5% of CD45 cells. Although this identifies mature eosinophils, it may not be sufficient to represent all resident phenotypes. Using sequencing and FACS sorting approaches, we found that the Siglec-F(+) population represented only a minority of the resident eosinophil pool. Rather, we identified a lineage-positive population of IL-5Ra(+)Ly6G(+) but Siglec-F(−) cells distinct from lineage-negative EoPs (eosinophil progenitors). At 40% of all CD45(+) hematopoietic cells in adult mice, this bone marrow resident population dwarfs the Siglec-F(+) eosinophil pool. When sorted from bone marrow homogenates and treated with IL-5, these resident cells acquired all characteristics of mature eosinophils, including expression of Siglec-F and granular proteins (EPX, MPO). These tissue residents may play critical homeostatic roles in normal murine bone marrow and should be accounted for in studies of eosinophil biology.

Resolvin D2 Promotes the Resolution of Allergen-Induced Lung Inflammation

Abstract Background Asthma is a chronic inflammatory disease without complete resolution. The production of specialized pro-resolving mediators (SPMs) is defective in asthma, so they represent a potential therapeutic opportunity. We hypothesize that docosahexaenoic acid derived resolvin D2 (RvD2) can promote the resolution of asthma. Methods BALB/c mice were challenged intra-tracheally with house dust mite (HDM). Bronchoalveolar lavage (BAL) fluid was collected at predefined protocol endpoints for peak inflammation and during resolution. To determine impact on resolution, some mice were given RvD2 (100 ng, intra-nasally) for 2 days after the last HDM challenge, and the resolution index for eosinophils and other markers were calculated. Results Airway HDM challenge led to marked increases in BAL eosinophils and BAL fluid pro-inflammatory cytokine and chemokine levels. Histology demonstrated changes in airway inflammation and mucous cell metaplasia in HDM-challenged lungs. NF-κB and Nrf2 expression were increased the lungs of HDM allergic mice. Compared to vehicle, treatment with RvD2 significantly decreased BAL inflammatory macrophage and eosinophil counts, and accelerated the resolution of HDM-evoked inflammation with a marked decrease in the resolution index for BAL eosinophils from 5 days to 1 day. Interestingly, RvD2 attenuation of eosinophils specifically targeted the inflammatory eosinophil population, but not the resident eosinophils. The impact of RvD2 on cytokine expression and NF-kB and Nrf2 activity is under current investigation to determine mechanisms for RvD2’s pro-resolving actions. Conclusion RvD2 promotes resolution of allergen-induced inflammation in a murine model of allergic asthma.