Eosinophil Migration Research Articles

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin.

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMβ2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-β-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMβ2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 μg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 μg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.

MHTP, 2-Methoxy-4-(7-methoxy-1,2,3,4-tetrahydroisoquinolin-1-yl) phenol, a Synthetic Alkaloid, Induces IFN-γ Production in Murine Model of Ovalbumin-Induced Pulmonary Allergic Inflammation.

MHTP [2-methoxy-4-(7-methoxy-1,2,3,4-tetrahydroisoquinolin-1-yl) phenol], a synthetic isoquinolinic alkaloid, presented anti-inflammatory activity in several experimental models of acute inflammation as lipopolysaccharide (LPS)-induced acute lung injury and phlogistic agent-induced edema and presented low preclinical toxicity. The aim of this study was to determine the MHTP effect on ovalbumin (OVA)-induced pulmonary allergic inflammation. In other to realize this study, female BALFB/c mice were sensitized and challenged with OVA (OVA group) and treated with MHTP (MHTP group) by nasal instillation. Inflammatory, allergic, and immunomodulatory parameters such as migration of inflammatory cells to the lung tissue, pulmonary histological analysis, serum level of IgE-allergen specific, cytokine secretion, and lung T cell population characterization were analyzed and the data were considered statistically significant with p < 0.05. OVA-sensitized and OVA-challenged and MHTP (5.0mg/kg)-treated mice presented reduction on total leukocyte migration into the bronchoalveolar lavage (BALF) dependent of lymphocyte and eosinophil migration (p < 0.001 and p < 0.01) as compared with the OVA group. Flow cytometric analysis showed that MHTP treatment decreased the percentage of granulocytes (p < 0.001) into the BALF and lung tissue histological analyzes demonstrated that the MHTP treatment decreased leukocyte migration and mucus production. In addition, treatment with MHTP decreased the number of CD3+CD4+ T cells independently of CD8+ T cell reduction into the BALF. The treatment also reduced significantly (p < 0.05) the serum level of IgE-OVA specific followed by reduction of IL-4, IL-13, and IL-17 production. Surprisingly, the MHTP treatment increased significantly (p < 0.05) the IFN-γ production in the BALF of these animals. Therefore, the results presented here showed that MHTP treatment, by nasal instillation, in a mouse model of OVA-induced pulmonary allergy has anti-allergic and immunomodulatory effects dependent on a Th1-skewed cytokine production that ameliorate the pulmonary allergic inflammation.

Eosinophilic keratitis in horses

Eosinophilic keratitis is an immune-mediated condition of the cornea, in which eosinophils accumulate within the superficial stroma. It is often associated with corneal ulceration. The instigating trigger for the migration...

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.

Addition of Curcumin in Dairy Sheep Diet in the Control of Subclinical Mastitis

Background: Curcumin, a polyphenol derived from rhizome of Curcuma longa, has been successfully used in animals and humans due to its antibacterial and anti-inflammatory properties. Recently, a study demonstrated that curcumin ointment reduced pain, mammary tension and erythema in humans with mastitis 72 h after the treatment. Mastitis in sheep is an important problem due to restrictions associated to the use of antibiotics, affecting milk production and becoming a public health issue. Thus, the aim of this study was to verify whether the addition of curcumin in the diet of six female sheep with subclinical mastitis could have curative efficacy and benefits to animal health.Cases: Six Lacaune sheep with subclinical mastitis that persisted for more than thirty days after the parturition were used. The animals received feed containing the curcumin (60 mg/animal/day) during 10 days. On days 0 and 10 of the experiment, total blood was collected to perform the hemogram and serum biochemistry (triglycerides, cholesterol, glucose, total protein (TP), albumin, globulin, aspartate aminotransferase (AST) and urea), as well as individual milk samples for centesimal composition, somatic cell count (SCC) and total bacterial count (TBC). Total leukocytes and erythrocytes, as well as hemoglobin content, were obtained using the hematological counter CELM-CC-550. Blood smears were stained by theRomanowsky method and used for leukocytes differentiation. After ten days of treatment, five sheep were negative in the California Mastitis Test (CMT). Total erythrocytes increased, while total leukocytes decreased after the treatment. Seric levels of AST, triglycerides, cholesterol and globulins decreased, while seric levels of albumin and urea increased after tendays of treatment. No differences were observed regarding milk production and composition, as well as for SCC and TBC on days 0 and 10 post treatment.Discussion: The use curcumin to prevent or to delay the occurrence of subclinical mastitis in ewes was first tested. In an experiment of lipopolysaccharide-induced mastitis in rats, researchers identified receptors type Toll 4 which are related with the induction of the synthesis of pro-inflammatory cytokines, as tumor necrosis factor alpha, interleukin-6 and 1β, molecules involved in the migration of leukocytes to the site of inflammation. According to literature, curcumin was able to reduce theactivity of myeloperoxidase in the mammary gland, an enzyme used as indicator of neutrophil infiltration, demonstrating the potent anti-inflammatory effect of curcumin. This effect can be associated to the release of interleukin-8, a cytokine that promotes the migration of neutrophils, lymphocytes and eosinophils into the site of infection, i.e., the mammary gland. It is important to highlight that neutrophils, due to its capacity to release inflammatory mediators and to delay apoptosis, has the potential to maintain a permanent inflammatory process, effects that were prevented or reduced by curcumin, specially the apoptotic and cytotoxic effects. It is important to emphasize the direct effect of curcumin on bacteria present in the mammary gland, since it exerts potent bactericidal effect. In summary, the use of curcumin in sheep with subclinical mastitis exerted beneficial effects in the dose of 60 mg/sheep/day, since it showed 83.3% of curative efficacy in the test CMT. This effect of curcumin can be direct or indirect, since the curcumin provided in the diet showed anti-inflammatory properties byreducing oxidation reactions and cell damage. Overall, the addition of curcumin in sheep diet improved the hematological and biochemical variables after only ten days of treatment. Future studies should be performed to clarify and determine whether the curcumin provided in diet can be an alternative to treat clinical or subclinical mastitis.Keywords: curcumin, mammary infection, immunity, biochemistry parameters, ovine.

Treatment with galectin-1 eye drops regulates mast cell degranulation and attenuates the severity of conjunctivitis

Galectin-1 (Gal-1) is a β-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation, however the mechanisms by which Gal-1 modulates cellular responses in allergic inflammatory processes have not been fully determined. In this study, we evaluated the therapeutic potential of Gal-1 eye drops in an experimental model of conjunctivitis. Wistar rats received a topical application of compound (C)48/80 (100 mg/ml) into right eyes and a drop of vehicle into the contralateral eye. Another group of rats received Gal-1 (0.3 or 3 μg/eye) or sodium cromoglycate (SCG; 40 mg/ml) in both eyes and, after 15 min, right eye was challenged with C48/80. Conjunctivitis-induced by C48/80 was characterized by severe eyelid oedema and tearing, but clinical signs were ameliorated by eye drop doses of both Gal-1 (0.3/3 μg) and SCG. As expected, an increased proportion of degranulated mast cells (62%, P < 0.01) and lower histamine levels were observed after 6 h of C48/80 challenge, compared to control (32%). This effect was abrogated by Gal-1 and SCG, which reduced mast cell degranulation (31–36%), eosinophil migration and eosinophil peroxidase levels in the eyes. Gal-1 (3 μg) and SCG treatments also decreased IL-4 levels, as well as activation of mitogen activated protein kinases compared to untreated C48/80 eyes. Our findings suggest that Gal-1 eye drops represent a new therapeutic strategy for ocular allergic inflammation.

A potential role for adipose tissue eosinophils in allergy and asthma.

Abstract Allergy can take many forms, but a mutual trait is the hypersensitive immune response to what the body detects as foreign. A common form of airway allergy is asthma, which affects &amp;gt;235 million people worldwide and is the most common chronic disease among children. Interestingly, obese individuals have a staggering 92% increased risk of asthma, displaying enhanced asthmatic symptoms, airway hyper-responsiveness, &amp; atopy. Due to the clinical correlation between obesity &amp; asthma, scientific endeavors have sought to explain the causality. Studies have shown adipokines secreted from adipose tissue (AT) can have either pro- or anti-inflammatory effects on the lungs. Furthermore, diet-induced obesity increases migration of eosinophils from bone marrow to the lung, as well as delays exit from the airway epithelium to the lumen. Curiously, investigators have tried to understand the correlative link between asthma &amp; obesity without closely examining the AT itself. Our data shows for the first time that AT mounts a hypereosinophilic state upon repeated exposure to a foreign substance (i.e. a hallmark of allergy &amp; asthma in lung); increasing eosinophils from ~5% to 60% of AT leukocytes in obese mice. A single exposure does not elicit an eosinophilic response, potentially suggesting a role for the adaptive immune system. We found that obesity increased sensitivity of this immune response. Furthermore, the surge in AT eosinophils was dependent on IL33, a required cytokine for eosinophilic accumulation in lung during allergy. We are determining if other classic indicators of allergy are present in AT of our model. Ultimately, we hope to elucidate whether AT &amp; lung act within a feedback loop that contributes to the incidence/severity of allergy &amp; asthma.

FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation.

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced β2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

Activation of IL5R and CRTH2 on Human Eosinophils Elicit a Similar Molecular Response and Reveal a Synergistic Effect

Eosinophils represent a key cell driving inflammatory processes in diseases such as asthma and eosinophilic esophagitis. The entire process of migration and activation of eosinophils is orchestrated by a myriad of molecules, among which interleukin 5 (IL5) plays a crucial role. Another receptor expressed on eosinophils is chemo-attractant receptor like expressed on Th2 cells (CRTH2). CRTH2 activation has a prominent role in stimulating Th2 and type 2 innate lymphoid cells to produce IL5 in disease tissue, but also induces eosinophils migration and adhesion. Prostaglandin D2 is the endogenous ligand of CRTH2, predominately released by activated mast cells, another key cell involved in eosinophilic diseases. Consequently, the IL5 and CRTH2 pathway are likely triggered simultaneously in eosinophils in disease tissue. Understanding the molecular effects of CRTH2 and IL5 receptor (IL5R) activated eosinophils might provide new insights for efficient eosinophils blocking. We performed gene expression profiling with isolated human eosinophils to compare the molecular signatures of CRTH2 and IL5R activation. The identified genes were confirmed using an orthogonal technology, and simultaneous IL5R and CRTH2 activation was investigated on the protein level. This study reveals that IL5R and CRTH2 activation trigger a similar molecular response in eosinophils with stronger IL5R effects. C-C chemokine ligand 2 was produced synergistically upon IL5R and CRTH2 stimulation and might contribute to monocyte and macrophage recruitment to disease tissue. The effect on C-C chemokine receptor 1 suggests that eosinophils maturation in the tissue might even occur in absence of IL5 via the CRTH2 pathway.

DP1 receptor signaling prevents the onset of intrinsic apoptosis in eosinophils and functions as a transcriptional modulator

Prostaglandin (PG) D2 is the ligand for the G‐protein coupled receptors DP1 (D‐type prostanoid receptor 1) and DP2 (also known as chemoattractant receptor homologous molecule, expressed on Th2 cells; CRTH2). Both, DP1 and DP2 are expressed on the cellular surface of eosinophils; although it has become quite clear that PGD2 induces eosinophil migration mainly via DP2 receptors, the role of DP1 in eosinophil responses has remained elusive. In this study, we addressed how DP1 receptor signaling complements the pro‐inflammatory effects of DP2. We found that PGD2 prolongs the survival of eosinophils via a DP1 receptor‐mediated mechanism that inhibits the onset of the intrinsic apoptotic cascade. The DP1 agonist BW245c prevented the activation of effector caspases in eosinophils and protected mitochondrial membranes from depolarization which—as a consequence—sustained viability of eosinophils. DP1 activation in eosinophils enhanced the expression of the anti‐apoptotic gene BCL‐XL, but also induced pro‐inflammatory genes, such as VLA‐4 and CCR3. In HEK293 cells that overexpress recombinant DP1 and/or DP2 receptors, activation of DP1, but not DP2, delayed cell death and stimulated proliferation, along with induction of serum response element (SRE), a regulator of anti‐apoptotic, early‐response genes. We conclude that DP1 receptors promote the survival via SRE induction and induction of pro‐inflammatory genes. Therefore, targeting DP1 receptors, along with DP2, may contribute to anti‐inflammatory therapy in eosinophilic diseases.

Inhibition of Soluble Epoxide Hydrolase Attenuates Food Allergen‐induced Eosinophil Recruitment and Gastrointestinal Inflammation

The prevalence of food allergies is on the rise, affecting nearly 4–6% of the population in the US and impacting their daily life in various ways. Although present in the normal gastrointestinal (GI) mucosa under homeostatic conditions, eosinophils are recruited to the intestinal mucosa in increased numbers in food allergy and contribute to inflammation. Soluble epoxide hydrolase (sEH) is known to play a pro‐inflammatory role during inflammation by metabolizing anti‐inflammatory epoxyeicosatrienoic acids (EETs) to pro‐inflammatory diols. In the current study, the role of sEH in a murine model of food allergy was investigated and the potential therapeutic effect of a highly selective sEH inhibitor (t‐TUCB) was evaluated. Oral exposure of mice on a soy‐free diet to soy protein isolate (SPI) induced expression of intestinal sEH, increased circulating total and antigen‐specific IgE levels and caused significant weight loss. Additionally, SPI induced GI inflammation characterized by increased recruitment of eosinophils and mast cells, elevated eotaxin‐1 levels, mucus hypersecretion and decreased epithelial junction protein expression. Administration of t‐TUCB to SPI‐challenged mice not only inhibited IgE levels and prevented SPI‐induced weight loss but also significantly decreased eosinophilia, mast cell recruitment and mucus secretion relative to untreated mice while SPI‐induced loss of junction protein expression was prevented to variable levels. Inflammatory mediators such as TNF‐α and eotaxin‐1 were found to induce sEH expression in eosinophils. Treatment of eosinophils with t‐TUCB significantly inhibited eosinophil migration, an effect which was mirrored by treatment with 11,12‐EET, by inhibiting intracellular signaling events such as ERK (1/2) activation and eotaxin‐1‐induced calcium flux. These studies suggest that sEH induced by soy proteins promotes allergic responses including eosinophilic GI inflammation and that inhibition of sEH can attenuate these allergen‐induced responses.Support or Funding InformationSupport was provided in part by National Institute of Environmental Health Sciences and National Institute of Diabetes and Digestive and Kidney Diseases to B.D.H. and by the University of Minnesota College of Veterinary Medicine Research Office, Minnesota Agricultural Experiment Station, and General Ag Research Funds to R.J.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Integrin αM activation and upregulation on esophageal eosinophils and periostin-mediated eosinophil survival in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an increasingly recognized allergic disease associated with dysphagia and esophageal fibrosis. We aimed to determine expression patterns of specific eosinophil integrins that promote eosinophilic infiltration of the esophageal epithelium, and to determine how key EoE-related cytokines influence eosinophil activation and survival. Esophageal and peripheral eosinophils were isolated from 20 adult subjects with EoE for immunophenotyping and integrin profiling using multicolor flow cytometry and immunohistochemistry. Expression signatures of eosinophil integrins were further assessed by immunohistochemistry using serial sections of esophageal biopsy specimens. Purified eosinophils were used to assess the effect of EoE-relevant cytokines and recombinant periostin on expression of known eosinophil integrins and eosinophil survival and activation. We found that resting eosinophils express high levels of the β2-pairing integrins αL and αM, and lower levels of α4, α6 and α4β7. The migration of peripheral eosinophils to the esophagus is characterized by the specific induction of αM, and a significant increase in the proportion of αM in high-activity conformation. Periostin, a secreted extracellular matrix protein that is significantly overexpressed in EoE, enhances eosinophil survival, and this effect is mediated by αM interaction. Integrin αM is a specific marker of activated tissue eosinophils in EoE, and promotes eosinophil survival through interactions with periostin. The ability of αMβ2 to mediate eosinophil tissue residency via periostin represents a key mechanism for disease development and a potential therapeutic target in EoE.

The endocannabinoid system in canine Steroid-Responsive Meningitis-Arteritis and Intraspinal Spirocercosis.

Endocannabinoids (ECs) are involved in immunomodulation, neuroprotection and control of inflammation in the central nervous system (CNS). Activation of cannabinoid type 2 receptors (CB2) is known to diminish the release of pro-inflammatory factors and enhance the secretion of anti-inflammatory cytokines. Furthermore, the endocannabinoid 2-arachidonoyl glycerol (2-AG) has been proved to induce the migration of eosinophils in a CB2 receptor-dependent manner in peripheral blood and activate neutrophils independent of CB activation in humans. The aim of the current study was to investigate the influence of the endocannabinoid system in two different CNS inflammatory diseases of the dog, i.e. Steroid-Responsive Meningitis-Arteritis (SRMA) and Intraspinal Spirocercosis (IS). The two main endocannabinoids, anandamide (AEA) and 2-AG, were quantified by mass spectrometry in CSF and serum samples of dogs affected with Steroid- Responsive Meningitis-Arteritis in the acute phase (SRMA A), SRMA under treatment with prednisolone (SRMA Tr), intraspinal Spirocercosis and healthy dogs. Moreover, expression of the CB2 receptor was evaluated in inflammatory lesions of SRMA and IS and compared to healthy controls using immunohistochemistry (IHC). Dogs with SRMA A showed significantly higher concentrations of total AG and AEA in serum in comparison to healthy controls and in CSF compared to SRMA Tr (p<0.05). Furthermore, dogs with IS displayed the highest ECs concentrations in CSF, being significantly higher than in CSF samples of dogs with SRMA A (p<0.05). CSF samples that demonstrated an eosinophilic pleocytosis had the highest levels of ECs, exceeding those with neutrophilic pleocytosis, suggesting that ECs have a major effect on migration of eosinophils in the CSF. Furthermore, CB2 receptor expression was found in glial cells in the spinal cord of healthy dogs, whereas in dogs with SRMA and IS, CB2 was strongly expressed not only in glial cells but also on the cellular surface of infiltrating leukocytes (i.e. neutrophils, eosinophils, lymphocytes, plasma cells, and macrophages) at lesion sites. The present study revealed an upregulated endocannabinoid system in dogs with inflammatory CNS diseases, highlighting the endocannabinoid system as a potential target for treatment of inflammatory CNS diseases.

Inhibition of soluble epoxide hydrolase attenuates eosinophil recruitment and food allergen-induced gastrointestinal inflammation.

Prevalence of food allergies in the United States is on the rise. Eosinophils are recruited to the intestinal mucosa in substantial numbers in food allergen-driven gastrointestinal (GI) inflammation. Soluble epoxide hydrolase (sEH) is known to play a pro-inflammatory role during inflammation by metabolizing anti-inflammatory epoxyeicosatrienoic acids (EETs) to pro-inflammatory diols. We investigated the role of sEH in a murine model of food allergy and evaluated the potential therapeutic effect of a highly selective sEH inhibitor (trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]-cyclohexyloxy}-benzoic acid [t-TUCB]). Oral exposure of mice on a soy-free diet to soy protein isolate (SPI) induced expression of intestinal sEH, increased circulating total and antigen-specific IgE levels, and caused significant weight loss. Administration of t-TUCB to SPI-challenged mice inhibited IgE levels and prevented SPI-induced weight loss. Additionally, SPI-induced GI inflammation characterized by increased recruitment of eosinophils and mast cells, elevated eotaxin 1 levels, mucus hypersecretion, and decreased epithelial junction protein expression. In t-TUCB-treated mice, eosinophilia, mast cell recruitment, and mucus secretion were significantly lower than in untreated mice and SPI-induced loss of junction protein expression was prevented to variable levels. sEH expression in eosinophils was induced by inflammatory mediators TNF-α and eotaxin-1. Treatment of eosinophils with t-TUCB significantly inhibited eosinophil migration, an effect that was mirrored by treatment with 11,12-EET, by inhibiting intracellular signaling events such as ERK (1/2) activation and eotaxin-1-induced calcium flux. These studies suggest that sEH induced by soy proteins promotes allergic responses and GI inflammation including eosinophilia and that inhibition of sEH can attenuate these responses.

Activation state of circulating eosinophils in nasal polyposis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common disease with an uncertain pathophysiology. It is characterized by polyps rich in eosinophils, with an activation status already investigated at the tissue level. In a group of CRSwNP patients, we assessed the activation status of circulating eosinophils in the blood before migration into tissues. Thirteen patients with CRSwNP and 16 healthy volunteers were enrolled. Several biologic parameters were studied: blood count of eosinophils; plasma eosinophil cationic protein; oxidative metabolism by chemiluminescence at baseline or when activated by phorbol 12-myristate 13-acetate or platelet-activating factor, with or without interleukin-5 (IL-5); percent of granulosar cells; and mean fluorescence intensity (MFI) by flow cytometry. The mean number of eosinophils was significantly higher in patients with CRSwNP, whose eosinophils showed increased oxidative metabolism in the basal or activated state significantly decreasing in the presence of IL-5. There was also a higher percentage of CD49d+ , CD25+ , and CCR3+ cells in patients, and a nonsignificant decrease in descending order in MFI between the control group, patients with normal eosinophil levels, patients with hypereosinophilia, and patients with aspirin-exacerbated respiratory disease. This study demonstrates a priming state of circulating eosinophils in CRSwNP patients when compared with healthy controls, as evidenced by the extent of oxidative metabolism, with increased sensitivity to IL-5 and by the observed variations of percent and MFI of CD49d, CCR3, and CD25. This priming is thus found at the peripheral level and occurs before the migration of eosinophils to polyps, reflecting the systemic and not just local nature of abnormalities in CRSwNP.

Role of Natural Killer Cells in Airway Inflammation.

Natural killer (NK) cells have an immune regulatory function as well as cytotoxicity against tumor or infected cells. In the airway, although NK cells constitute a small proportion of the resident lymphocytes, they play an important role in the pathogenesis of chronic inflammatory airway diseases by modulating immune responses. NK cells can promote allergic airway inflammation by increasing the production of type 2 cytokines and inducing eosinophil migration. The increased activity of NK cells can develop or aggravate the destruction of lung parenchymal cells. On the other hand, decreased apoptotic activity of NK cells in eosinophils can serve as an aggravating factor for allergic airway inflammation. The increase in interferon-γ-producing NK cells and the inhibition of type 2 immune response by NK cells can alleviate allergic airway inflammation. This review aims to define the roles of NK cells in chronic inflammatory diseases of lower and upper airways.

Galectin-1: A Jack-of-All-Trades in the Resolution of Acute and Chronic Inflammation.

Regulatory signals provide negative input to immunological networks promoting resolution of acute and chronic inflammation. Galectin-1 (Gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, displays broad anti-inflammatory and proresolving activities by targeting multiple immune cell types. Within the innate immune compartment, Gal-1 acts as a resolution-associated molecular pattern by counteracting the synthesis of proinflammatory cytokines, inhibiting neutrophil trafficking, targeting eosinophil migration and survival, and suppressing mast cell degranulation. Likewise, this lectin controls T cell and B cell compartments by modulating receptor clustering and signaling, thus serving as a negative-regulatory checkpoint that reprograms cellular activation, differentiation, and survival. In this review, we discuss the central role of Gal-1 in regulatory programs operating during acute inflammation, autoimmune diseases, allergic inflammation, pregnancy, cancer, and infection. Therapeutic strategies aimed at targeting Gal-1-glycan interactions will contribute to overcome cancer immunosuppression and reinforce antimicrobial immunity, whereas stimulation of Gal-1-driven immunoregulatory circuits will help to mitigate exuberant inflammation.

Epigenome-wide association study of asthma and wheeze in childhood and adolescence

BackgroundAsthma heritability has only been partially explained by genetic variants and is known to be sensitive to environmental factors, implicating epigenetic modifications such as DNA methylation in its pathogenesis.MethodsUsing data collected in the Avon Longitudinal Study of Parents and Children (ALSPAC), we assessed associations of asthma and wheeze with DNA methylation at 7.5 and 16.5 years, at over 450,000 CpG sites in DNA from the peripheral blood of approx. 1000 participants. We used Mendelian randomization (MR), a method of causal inference that uses genetic variants as instrumental variables, to infer the direction of association between DNA methylation and asthma.ResultsWe identified 302 CpGs associated with current asthma status (FDR-adjusted P value < 0.05) and 445 with current wheeze status at 7.5 years, with substantial overlap between the two. Genes annotated to the 302 associated CpGs were enriched for pathways related to movement of cellular/subcellular components, locomotion, interleukin-4 production and eosinophil migration. All associations attenuated when adjusted for eosinophil and neutrophil cell count estimates. At 16.5 years, two sites were associated with current asthma after adjustment for cell counts. The CpGs mapped to the AP2A2 and IL5RA genes, with a − 2.32 [95% CI − 1.47, − 3.18] and − 2.49 [95% CI − 1.56, − 3.43] difference in percentage methylation in asthma cases respectively.Two-sample bi-directional MR indicated a causal effect of asthma on DNA methylation at several CpG sites at 7.5 years. However, associations did not persist after adjustment for multiple testing. There was no evidence of a causal effect of asthma on DNA methylation at either of the two CpG sites at 16.5 years.ConclusionThe majority of observed associations are driven by higher eosinophil cell counts in asthma cases, acting as an intermediate phenotype, with important implications for future studies of DNA methylation in atopic diseases.

Development of a robust reporter gene based assay for the bioactivity determination of IL-5-targeted therapeutic antibodies

Eosinophilic asthma is characterized by the eosinophilic inflammation with the allergen independent activation of Th2 lymphocytes. Since IL-5 plays an important role in the maturation, survival and migration of eosinophils, hence the pathogenesis of eosinophilic asthma, biotherapeutics targeting IL-5/IL-5Rα have been developed and/or marketed, including Mepolizumab, Reslizumab, and Benralizumab. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current mode of action (MOA) based method used in the quality control and stability tests for anti-IL-5 mAbs is anti-proliferation assay, which is tedious with long duration and high variation. We describe here the development and validation of a reporter gene assay (RGA), based on an IL-5-dependent TF-1 cell line variant we established that stably expresses the luciferase reporter under the control of STAT5 response elements. After careful optimization, we demonstrate the excellent specificity, precision, accuracy and linearity of the established RGA. Our study also proves that the assay is superior on precision, sensitivity and assay simplicity to the anti-proliferation assay. The established RGA is also applicable to another anti-IL-5Rα mAb. These results show for the first time that this novel RGA, based on the IL-5-IL-5R-STAT5 pathway, can be a valuable supplement to the anti-proliferation assay and employed in the bioactivity determination of anti-IL-5/anti-IL-5Rα biotherapeutics.

Structure-activity relationship study of β-oxidation resistant indole-based 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) receptor antagonists

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is formed from 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by the 5-lipoxygenase (5-LO) pathway under conditions associated with oxidative stress. 5-Oxo-ETE is an important pro-inflammatory mediator, which stimulates the migration of eosinophils via a selective G-protein coupled receptor, known as the OXE receptor (OXE-R). Previously, we designed and synthesized structural mimics of 5-oxo-ETE such as 1 using an indole scaffold. In the present work, we added various substituents at C-3 of this moiety to block potential β-oxidation of the 5-oxo-valerate side chain, and investigated the structure-activity relationships of the resulting novel β-oxidation-resistant antagonists. Cyclopropyl and cyclobutyl substituents were well tolerated in this position, but were less potent as the highly active 3S-methyl compound. It seems likely that 3-alkyl substituents can affect the conformation of the 5-oxovalerate side chain containing the critical keto and carboxyl groups, thereby affecting interaction with the OXE-receptor.