Induce Eosinophil Apoptosis Research Articles

Retraction Note: Two Mixed-Ligand Co(II)-Based Coordination Polymers: Treatment Activity on Pediatric Asthma Via Inducing Eosinophil Apoptosis through Down-Regulating Bcl-2 Expression

Retraction Note: Two Mixed-Ligand Co(II)-Based Coordination Polymers: Treatment Activity on Pediatric Asthma Via Inducing Eosinophil Apoptosis through Down-Regulating Bcl-2 Expression

Hemogram Derived Clinical Traits Evinced Differential Contribution to Explained Variance of Mortality Risk Model at Index Hospitalization for Sars-Cov-2 Infection across Wuhan/D614G, Delta, Omicron and Omicron BA.x Variants

Introduction SARS-CoV-2 infection promulgates an immune dysregulation that drives an inflammatory cascade therefore inducingmorbidity and mortality across the COVID-19 presentation spectrum. Hemogram (CBC) derived traits associated with COVID-19 severity have been reported (Meizlish ML et al., 2021, Dhinata KS et al., 2021), but their relative impact across SARS-CoV-2 variants is unclear. We employed sequential machine learning approachesto identify significantly contributing traits with attendant portion of explained variance (EV%) in hospital mortality risk models across predominant SARS-CoV-2 variants. Methods Our retrospective design, analyses and interpretations followed constructs detailed in the Strengthening the Reporting of Observational Studies in Epidemiology reporting guideline. Adult patients with laboratory confirmed COVID-19 who underwent index hospitalization during Wuhan/D614G (March 14, 2020- June 18, 2021), Delta (June 19, 2021- December 18, 2021), Omicron (December 19-March 30, 2022) or Omicron BA.x (March 31, 2022 - April 14, 2023) variant waves at an 820-bed academic public health trust hospital in Florida were studied. Demographics, laboratory results (within 24h of presentation), ICD-10-CM-based comorbidity, COVID-19 directed treatment and administrative data were extracted from electronic medical records under IRB exemption. Generalized regression with adaptive LASSO identified hemogram-derived traits significantly associated with mortality in at least one of the variants while controlling for presentation vital signs, age, sex, extant comorbidities and ultimate COVID-19 directed treatment. Boosted Tree modeling computed within-variant proportion contributed by each trait to model's accuracy (R 2) representing EV% of mortality risk. Traits contributing to one or more variants for at least 1% portion of EV were retained in model. A Bonferroni corrected two-tailed p < .0125 was considered significant. Results We included 6490 consecutively discharge patients distributed across Wuhan/D614G (n=2249), Delta (n=1196), Omicron (n=953) and Omicron BA.x (n=2092) variants with respective mortality of 9.3%, 13.0%, 4.4%, and 2.4% (p< .0001). Patient characteristics and on-presentation laboratory medicine clinical traits are in Table 1. Table 2 provides ranked contribution for each of the 7 traits retained in the model with attendant presentation mortality risk accuracy (R 2) across variant waves. Potent synergy can be generated with biomarkers ratios, as seen with Segmented neutrophil/monocyte ratio (SMR) and lymphocyte/monocyte ratio (LMR), compared to studying these variables individually. Conclusion Sequential machine-learning approaches identified differential expression intensity across SARS-CoV-2 variants in presentation hemogram-derived traits associated with hospital mortality risk modeling at index infection. These findings suggest that COVID-19 presentation risk and level of care assessment tools employing hemogram-derived traits may not generalize across pandemic variants. Platelets counts were associated with mortality in Delta, Wuhan/D614G and Omicron variants. Although it was also associated in Omicron Bx.5, the R 2 is lower, this can be explained because in this study the variant of Omicron Bx.5 had the lowest mortality rate. Studies have shown that platelet activation generates a cascade with more thromboxane A2 and platelet thrombofactor A which increases prothrombotic states (Dhinata KS et al., 2021). The absence of eosinophils was seen in Wuhan/D614G and Delta, the cause of which cannot be ascertained. A theory is that the inflammation caused by SARS-CoV-2 infection releases certain cytokines that induce eosinophil apoptosis (Dhinata KS et al., 2021). Immature neutrophils evidenced across variants can be explained by the inflammatory cascade inducing G-CSF and IL-8 production, which induces neutrophil activation (Meizlish ML et al., 2021).Lymphopenia seen throughout variants contributes to an increase in LMR. One presumption is that the SARS-CoV-2 affects T-Lymphocytes through ACE-2 receptor and CD117 spike proteins (Meizlish ML et al., 2021). Overall, our study brings to attention how hemogram-derived traits express differently across SARS-CoV-2 variants, which could guide the stratification of COVID-19 severity and assist in the care of patients before decompensation is noticed.

Myeloid-associated differentiation marker is a novel SP-A-associated transmembrane protein whose expression on airway epithelial cells correlates with asthma severity

Surfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma.

Potent Anti-Inflammatory Effects of Tetracyclines on Human Eosinophils.

Eosinophils are potent pro-inflammatory cells. Not only in allergic diseases but also in other diseases there is a need for treatment strategies to induce resolution of eosinophil-mediated inflammation. During the last years beneficial non-antibiotic activities of tetracyclines (TCNs) have been shown in different diseases in which eosinophils play a role, for example, asthma and bullous pemphigoid. The working mechanism of these effects remains to be clarified. Aim of the present study was to investigate the effects of TCNs on eosinophils. Flow cytometry analysis of apoptosis, mitochondrial membrane potential, activation of caspases, intracellular H2O2 and calcium, surface expression of eosinophil activation markers was performed in highly purified peripheral blood eosinophils of non-atopic donors. Tetracycline hydrochloride, minocycline and doxycycline significantly induced eosinophil apoptosis. All TCNs were able to significantly overcome the strong survival enhancing effects of pro-eosinophilic cytokines and staphylococcus aureus enterotoxins. Tetracycline hydrochloride induced eosinophil apoptosis was accompanied by intracellular production of hydrogen peroxide, loss of mitochondrial membrane potential and activation of caspases. Moreover, tetracycline hydrochloride significantly down regulated eosinophil surface expression of CD9 and CD45, and of the activation markers CD11b and CD69, but not of CD54, CD63, or CD95. Our data, propably for the first time, point to a potent anti-inflammatory role of TCNs on eosinophils.

Engineering of Humanized Antibodies Against Human Interleukin 5 Receptor Alpha Subunit That Cause Potent Antibody-Dependent Cell-Mediated Cytotoxicity

Patients with severe eosinophilic asthma (SEA; characterized by persistent eosinophilia in blood and airway tissues) experience frequent asthma exacerbations with poor clinical outcomes. Interleukin 5 (IL-5) and IL-5 receptor alpha subunit (IL-5α) play key roles in eosinophilia maintenance, and relevant therapeutic strategies include the development of antibodies (Abs) against IL-5 or IL-5α to control eosinophilia. Benralizumab, an anti–IL-5α Ab that depletes eosinophils mainly via Ab-dependent cell-mediated cytotoxicity and through blockage of IL-5 function on eosinophils, has been clinically approved for patients with SEA. Here, we report engineering of a new humanized anti–IL-5Rα Ab with potent biological activity. We first raised murine Abs against human IL-5Rα, humanized a leading murine Ab, and then further engineered the humanized Abs to enhance their affinity for IL-5Rα using the yeast surface display technology. The finally engineered version of the Ab, 5R65.7, with affinity (KD ≈ 4.64 nM) stronger than that of a clinically relevant benralizumab analogue (KD ≈ 26.8 nM) showed improved neutralizing activity toward IL-5–dependent cell proliferation in a reporter cell system. Domain level Ab epitope mapping revealed that 5R65.7 recognizes membrane-proximal domain 3 of IL-5Rα, distinct from domain I epitope of the benralizumab analogue. In ex vivo assays with peripheral eosinophils from patients with SEA and healthy donors, 5R65.7 manifested more potent biological activities than the benralizumab analogue did, including inhibition of IL-5–dependent proliferation of eosinophils and induction of eosinophil apoptosis through autologous natural-killer-cell–mediated Ab-dependent cell-mediated cytotoxicity. Our study provides a potent anti–IL-5Rα Ab, 5R65.7, which is worthy of further testing in preclinical and clinical trials against SEA as a potential alternative to the current therapeutic arsenal.

RETRACTED ARTICLE: Two Mixed-Ligand Co(II)-Based Coordination Polymers: Treatment Activity on Pediatric Asthma Via Inducing Eosinophil Apoptosis Through Down-Regulating Bcl-2 Expression

In this work, two coordination polymers containing Co(II) ions as nodes, namely, [Co(NDC)(L)(H2O)]n (1, H2NDC = 1,4-naphthalene dicarboxylic acid) and [Co(DCTP)(L)·(H2O)2]n (2, H2DCTP = 2,5-dichloroterephthalic acid) were prepared based on 1,4-bis(5,6-dimethylbenzimidazole)butane (L), the flexible N-donor ligand and different dicarboxylate ligand. Their treatment activity on the pediatric asthma was evaluated in the biological research. Firstly, the enzyme linked immunosorbent assay (ELISA) detection was carried out to determine the IL-1β and TNF-α content in the bronchus. The inflammatory response in the bronchial epithelial cells was determined by the real time reverse transcription-polymerase chain reaction (RT-PCR). After that, the eosinophil apoptosis in the airway was measured. Finally, the western was performed for the confirmation of eosinophil apoptosis by detecting Bcl-2 and caspase-3.

Omalizumab does not lead to a distinct alteration in hematological parameters and complete blood count-derived inflammation biomarkers except for basophil count

Purpose Omalizumab is a monoclonal anti-IgE antibody used to treat patients with chronic spontaneous urticaria by decreasing free IgE levels. Omalizumab may have an anti-inflammatory effect by inhibiting T-cell activation and inducing eosinophil apoptosis. In this study, we evaluated the effect of omalizumab on hematological parameters and inflammation biomarkers in patients with chronic spontaneous urticaria. Methods Between July 2018 and November 2019, medical records of 60 patients (44 female, 16 male) with chronic spontaneous urticaria who were treated with omalizumab were reviewed retrospectively. Hematological parameters and inflammation biomarkers including the neutrophil/lymphocyte, monocyte/lymphocyte, platelet/lymphocyte and mean platelet volume/platelet count ratios were compared before and after 12 weeks of omalizumab treatment. Results The absolute count of basophils and percentage of basophils increased significantly after omalizumab treatment (p = 0.04, p = 0.004). The absolute count of eosinophils, percentage of eosinophils, neutrophil/lymphocyte, monocyte/lymphocyte, and mean platelet volume/platelet ratios decreased, while platelet/lymphocyte ratio increased after omalizumab treatment. Nevertheless, these changes were not statistically significant. Conclusions Increased basophil counts suggest that omalizumab has a crucial effect through basophils in chronic spontaneous urticaria. Further studies focussing on basophils may contribute to the literature both to elucidate the etiopathogenesis of urticaria and to improve novel treatment agents for the disease. On the other hand, our study revealed that omalizumab did not have a distinct effect on complete blood count-derived inflammation biomarkers and thus inflammation.

Nanoformulated ABT-199 to effectively target Bcl-2 at mitochondrial membrane alleviates airway inflammation by inducing apoptosis

Elimination of airway inflammatory cells is essential for asthma control. As Bcl-2 protein is highly expressed on the mitochondrial outer membrane in inflammatory cells, we chose a Bcl-2 inhibitor, ABT-199, which can inhibit airway inflammation and airway hyperresponsiveness by inducing inflammatory cell apoptosis. Herein, we synthesized a pH-sensitive nanoformulated Bcl-2 inhibitor (Nf-ABT-199) that could specifically deliver ABT-199 to the mitochondria of bronchial inflammatory cells. The proof-of-concept study of an inflammatory cell mitochondria-targeted therapy using Nf-ABT-199 was validated in a mouse model of allergic asthma. Nf-ABT-199 was proven to significantly alleviate airway inflammation by effectively inducing eosinophil apoptosis and inhibiting both inflammatory cell infiltration and mucus hypersecretion. In addition, the nanocarrier or Nf-ABT-199 showed no obvious influence on cell viability, airway epithelial barrier and liver function, implying excellent biocompatibility and with non-toxic effect. The nanoformulated Bcl-2 inhibitor Nf-ABT-199 accumulates in the mitochondria of inflammatory cells and efficiently alleviates allergic asthma.

Wogonin attenuates nasal polyp formation by inducing eosinophil apoptosis through HIF-1\u03b1 and survivin suppression

Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is an inflammatory sinonasal disorder characterized by eosinophilic inflammation and T-helper 2 skewing. Eosinophil accumulation in sinonasal mucosa comprises a major feature of CRSwNP. The study aimed to investigate the effect of the flavone wogonin in nasal polyposis by assessing its ability to induce eosinophil apoptosis in vitro and attenuate eosinophilic CRSwNP in mice. Double immunofluorescence, immunohistochemistry, flow cytometry, and immunoblotting were performed to evaluate hypoxia-inducible factor (HIF)-1α, survivin, and apoptotic markers in the human eosinophilic EoL-1 cell line or sinonasal tissues from patients with CRS with or without NPs. In sinonasal specimens from patients with CRS, HIF-1α and survivin were up-regulated in eosinophils from patients with NPs compared with levels in patients without NPs. Under hypoxia, HIF-1α and survivin expression was up-regulated in EoL-1 cells. Wogonin down-regulated both HIF-1α and survivin in EoL-1 cells. In addition, overexpression of survivin protected EoL-1 cells against apoptosis in response to wogonin. Moreover, wogonin attenuated nasal polyp formation in a murine model. Our findings suggest that wogonin could induce caspase-3 activation by suppressing HIF-1α and survivin expression in EoL-1 cells. Further studies regarding novel therapeutic options for CRSwNP targeting eosinophil apoptosis are needed.

Frontline Science: Characterization of a novel mouse strain expressing human Siglec-8 only on eosinophils.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a human cell surface protein expressed exclusively on eosinophils, mast cells, and basophils that, when engaged, induces eosinophil apoptosis and inhibits mast cell mediator release. This makes Siglec-8 a promising therapeutic target to treat diseases involving these cell types. However, preclinical studies of Siglec-8 targeting in vivo are lacking because this protein is only found in humans, apes, and some monkeys. Therefore, we have developed a mouse strain in which SIGLEC8 transcription is activated by Cre recombinase and have crossed this mouse with the eoCre mouse to achieve eosinophil-specific expression. We confirmed that Siglec-8 is expressed exclusively on the surface of mature eosinophils in multiple tissues at levels comparable to those on human blood eosinophils. Following ovalbumin sensitization and airway challenge, Siglec-8 knock-in mice generated a pattern of allergic lung inflammation indistinguishable from that of littermate controls, suggesting that Siglec-8 expression within the eosinophil compartment does not alter allergic eosinophilic inflammation. Using bone marrow from these mice, we demonstrated that, during maturation, Siglec-8 expression occurs well before the late eosinophil developmental marker C-C motif chemokine receptor 3, consistent with eoCre expression. Antibody ligation of the receptor induces Siglec-8 endocytosis and alters the phosphotyrosine profile of these cells, indicative of productive signaling. Finally, we demonstrated that mouse eosinophils expressing Siglec-8 undergo cell death when the receptor is engaged, further evidence that Siglec-8 is functional on these cells. These mice should prove useful to investigate Siglec-8 biology and targeting in vivo in a variety of eosinophilic disease models.

FACS isolation of live mouse eosinophils at high purity via a protocol that does not target Siglec F

Flow cytometry protocols designed to identify mouse eosinophils typically target Siglec F, an α-2,3-sialic acid binding transmembrane protein expressed universally on cells of this lineage. While a convenient target, antibody-mediated ligation of Siglec F induces eosinophil apoptosis, which limits its usefulness for isolations that are to be followed by functional and/or gene expression studies. We present here a method for FACS isolation which does not target Siglec F and likewise utilizes no antibodies targeting IL5Rα (CD125) or CCR3. Single cell suspensions are prepared from lungs of mice that were sensitized and challenged with Aspergillus fumigatus antigens; eosinophils were identified and isolated by FACS as live SSChi/FSChi CD11c−Gr1−/loMHCII− cells. This strategy was also effective for eosinophil isolation from the lungs of IL5tg mice. Purity by visual inspection of stained cytospin preparations and by Siglec F-diagnostic flow cytometry was 98–99% and 97–99%, respectively. Eosinophils isolated by this method (yield, ~4×106/mouse) generated high-quality RNA suitable for gene expression analysis.

Hypoxia causes IL-8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid-induced apoptosis in human eosinophils.

Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.

Mechanisms and consequences of Siglec-8 internalization and toxic payload delivery in human eosinophils

Abstract Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell surface protein expressed selectively on human eosinophils, mast cells, and basophils that induces eosinophil apoptosis and inhibits mast cell mediator release. Hence, Siglec-8 is an ideal target for diseases involving these cells. However, the effective delivery of therapeutic agents to these cells through Siglec-8 requires an understanding of the dynamics of Siglec-8 surface expression, which have not been elucidated. We observed by flow cytometry that Siglec-8 endocytosis in eosinophils proceeds slowly and plateaus at 80% loss in response to antibody or polyvalent synthetic ligand. Surprisingly, based on sensitivity to pharmacological inhibition, Siglec-8 appears to be internalized in both a clathrin- and lipid raft-dependent manner. Actin cytoskeletal rearrangement and tyrosine kinase and PKC activity are necessary for endocytosis. Interestingly, Siglec-8 is shuttled to the cell surface in a manner dependent on actin rearrangement, microtubule polymerization, and the activities of tyrosine kinases, dynamin, and PI3K. Neither process is significantly affected by inhibitors of Src family kinases or PTPs. Internalized Siglec-8 localizes to the lysosome, and sialidase treatment of eosinophils enhances ligand binding, indicating the presence of masking sialylated cis ligands on the cell surface. Finally, conjugating the ribosome-inactivating protein saporin to an anti-Siglec-8 mAb enhanced cell death induction in unstimulated eosinophils. The dynamics of Siglec-8 surface expression therefore appear to be suitable for sustained targeting and may deliver drugs to effectively treat diseases involving these cell types, including malignancies.

Mucins and Their Sugars. Critical Mediators of Hyperreactivity and Inflammation.

Excessive mucus causes severe airflow obstruction in fatal asthma. It is also present in mild to moderate disease, but is poorly understood and treated. Mucus overproduction is associated with dysregulated expression of the mucins MUC5AC and MUC5B. Whereas increased MUC5AC is a consistent finding, MUC5B varies-remaining stably produced in some patients but strongly repressed in others (>90%). Patients with lower MUC5B display worsened asthma phenotypes including airway hyperreactivity (AHR) to methacholine (MCh) and eosinophilic inflammation. To better understand the roles of mucins in asthma, we generated Muc5ac and Muc5b knockout ((-/-)) mice. AHR to MCh was abolished in antigen-challenged Muc5ac(-/-) mice, due to prevention of heterogeneous mucous plugging that occurred in allergic wild-type mice during MCh-induced bronchoconstriction. Thus, in addition to the established role of smooth muscle-mediated airway narrowing, Muc5ac is an essential noncontractile AHR component. We also found that, unlike Muc5ac(-/-) mice, Muc5b-deficient mice were not protected from asthma phenotypes. Furthermore, whereas inflammation was unaffected by Muc5ac deficiency, it was exaggerated in the absence of Muc5b. On the basis of these differential effects, we are now determining how asthma phenotypes are regulated by mucin isoform specificity. Glycosylation is dramatically different: Muc5ac is heavily fucosylated whereas Muc5b is mainly sialylated. Fucosylation increases mucus viscoelasticity, and FUT2, the enzyme that catalyzes mucin α1,2-fucosylation, is associated with severe asthma exacerbation risk. Sialylation is required for binding to siglec (sialic acid-binding immunoglobulin-like lectin) receptors on leukocytes. Eosinophils express Siglec-F (mouse) or Siglec-8 (human). Engagement by sialoside ligands induces eosinophil apoptosis, and Muc5b via sialylated termini that require the α2,3-sialyltransferase ST3Gal3 for synthesis binds Siglec-F and induces apoptosis in mouse eosinophils. Because Muc5b is required for host defense in mouse lungs, inhibiting MUC5AC while preserving or enhancing MUC5B functions may be effective for treating asthma.

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3.

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.

Fasciola hepatica induces eosinophil apoptosis in the migratory and biliary stages of infection in sheep

The aim of the present work was to evaluate the number of apoptotic eosinophils in the livers of sheep experimentally infected with Fasciola hepatica during the migratory and biliary stages of infection. Four groups (n=5) of sheep were used; groups 1–3 were orally infected with 200 metacercariae (mc) and sacrificed at 8 and 28 days post-infection (dpi), and 17 weeks post-infection (wpi), respectively. Group 4 was used as an uninfected control. Apoptosis was detected using immunohistochemistry with a polyclonal antibody against anti-active caspase-3, and transmission electron microscopy (TEM). Eosinophils were identified using the Hansel stain in serial sections for caspase-3, and by ultrastructural features using TEM. At 8 and 28 dpi, numerous caspase-3+ eosinophils were mainly found at the periphery of acute hepatic necrotic foci. The percentage of caspase -3+ apoptotic eosinophils in the periphery of necrotic foci was high (46.1–53.9) at 8 and 28 dpi, respectively, and decreased in granulomas found at 28 dpi (6%). Transmission electron microscopy confirmed the presence of apoptotic eosinophils in hepatic lesions at 8 and 28 dpi. At 17 wpi, apoptotic eosinophils were detected in the infiltrate surrounding some enlarged bile ducts containing adult flukes. This is the first report of apoptosis induced by F. hepatica in sheep and the first study reporting apoptosis in eosinophils in hepatic inflammatory infiltrates in vivo. The high number of apoptotic eosinophils in acute necrotic tracts during the migratory and biliary stages of infection suggests that eosinophil apoptosis may play a role in F. hepatica survival during different stages of infection.

Natural killer cells protect against cardiac inflammation and fibrosis by directly limiting eosinophilic accumulation (BA7P.144)

Abstract Myocarditis is the inflammation of the cardiac muscle and is among the leading causes of sudden cardiac failure in young adults. We demonstrate here that natural killer (NK) cells, a subset of the Type I innate lymphoid cell (ILC) group, suppress experimental autoimmune myocarditis (EAM). EAM was initiated in BALB/c mice by immunization with myocarditogenic peptide emulsified in complete Freund's adjuvant. During disease, activated cardiac NK cells accumulated in the heart, secreted interferonγ, perforin, and granzyme-B, and expressed activation markers CD69, TRAIL, and CD27. The depletion of NK cells during EAM with anti-asialo GM1 antibody resulted in a 10-fold increase from 0.12 - 12.0% in the percentage of eosinophils found in the cardiac tissue at the peak of disease. This was accompanied by significantly increased myocarditis severity and elevated fibrosis. Eosinophils were required for the change in disease severity in the absence of NK cells, as treatment with anti-asialo GM1 did not increase myocarditis severity in eosinophil-deficient ΔdoubleGATA1 mice. We show that NK cells limited eosinophilic infiltration during EAM indirectly through altering eosinophil-related chemokine production by resident cardiac fibroblasts both in vitro and in vivo. Additionally, the co-culture of NK cells and eosinophils directly induced eosinophil apoptosis in vitro. Altogether, we define a new pathway of eosinophilic regulation through interactions with NK cells.

Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation.

Eosinophils are key effector cells in allergic diseases, including allergic rhinitis, eczema, and asthma. Their tissue presence is regulated by both recruitment and increased longevity at inflamed sites. To investigate the ability of the flavone wogonin to induce eosinophil apoptosis in vitro and attenuate eosinophil-dominant allergic inflammation in vivo in mice. Human and mouse eosinophil apoptosis in response to wogonin was investigated by cellular morphology, flow cytometry, mitochondrial membrane permeability, and pharmacological caspase inhibition. Allergic lung inflammation was modeled in mice sensitized and challenged with ovalbumin. Bronchoalveolar lavage (BAL) and lung tissue were examined for inflammation, mucus production, and inflammatory mediator production. Airway hyperresponsiveness to aerosolized methacholine was measured. Wogonin induced time- and concentration-dependent human and mouse eosinophil apoptosis in vitro. Wogonin-induced eosinophil apoptosis occurred with activation of caspase-3 and was inhibited by pharmacological caspase inhibition. Wogonin administration attenuated allergic airway inflammation in vivo with reductions in BAL and interstitial eosinophil numbers, increased eosinophil apoptosis, reduced airway mucus production, and attenuated airway hyperresponsiveness. This wogonin-induced reduction in allergic airway inflammation was prevented by concurrent caspase inhibition in vivo. Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation, suggesting that it has therapeutic potential for the treatment of allergic inflammation in humans.

Induction of eosinophil apoptosis by hydrogen peroxide promotes the resolution of allergic inflammation.

Eosinophils are effector cells that have an important role in the pathogenesis of allergic disease. Defective removal of these cells likely leads to chronic inflammatory diseases such as asthma. Thus, there is great interest in understanding the mechanisms responsible for the elimination of eosinophils from inflammatory sites. Previous studies have demonstrated a role for certain mediators and molecular pathways responsible for the survival and death of leukocytes at sites of inflammation. Reactive oxygen species have been described as proinflammatory mediators but their role in the resolution phase of inflammation is poorly understood. The aim of this study was to investigate the effect of reactive oxygen species in the resolution of allergic inflammatory responses. An eosinophilic cell line (Eol-1) was treated with hydrogen peroxide and apoptosis was measured. Allergic inflammation was induced in ovalbumin sensitized and challenged mouse models and reactive oxygen species were administered at the peak of inflammatory cell infiltrate. Inflammatory cell numbers, cytokine and chemokine levels, mucus production, inflammatory cell apoptosis and peribronchiolar matrix deposition was quantified in the lungs. Resistance and elastance were measured at baseline and after aerosolized methacholine. Hydrogen peroxide accelerates resolution of airway inflammation by induction of caspase-dependent apoptosis of eosinophils and decrease remodeling, mucus deposition, inflammatory cytokine production and airway hyperreactivity. Moreover, the inhibition of reactive oxygen species production by apocynin or in gp91phox−/− mice prolonged the inflammatory response. Hydrogen peroxide induces Eol-1 apoptosis in vitro and enhances the resolution of inflammation and improves lung function in vivo by inducing caspase-dependent apoptosis of eosinophils.

Natural Killer Cells Limit Cardiac Inflammation and Fibrosis by Halting Eosinophil Infiltration

Myocarditis is a leading cause of sudden cardiac failure in young adults. Natural killer (NK) cells, a subset of the innate lymphoid cell compartment, are protective in viral myocarditis. Herein, we demonstrated that these protective qualities extend to suppressing autoimmune inflammation. Experimental autoimmune myocarditis (EAM) was initiated in BALB/c mice by immunization with myocarditogenic peptide. During EAM, activated cardiac NK cells secreted interferon γ, perforin, and granzyme B, and expressed CD69, tumor necrosis factor-related apoptosis-inducing ligand treatment, and CD27 on their cell surfaces. The depletion of NK cells during EAM with anti-asialo GM1 antibody significantly increased myocarditis severity, and was accompanied by elevated fibrosis and a 10-fold increase in the percentage of cardiac-infiltrating eosinophils. The resultant influx of eosinophils to the heart was directly responsible for the increased disease severity in the absence of NK cells, because treatment with polyclonal antibody asialogangloside GM-1 did not augment myocarditis severity in eosinophil-deficient ΔdoubleGATA1 mice. We demonstrate that NK cells limit eosinophilic infiltration both indirectly, through altering eosinophil-related chemokine production by cardiac fibroblasts, anddirectly, by inducing eosinophil apoptosis invitro. Altogether, we define a new pathway of eosinophilic regulation through interactions with NK cells.