Wogonoside Ameliorates Airway Inflammation and Mucus Hypersecretion via NF-κB/STAT6 Signaling in Ovalbumin-Induced Murine Acute Asthma.

Asthma is recognized as a chronic respiratory illness characterized by airway inflammation and airway hyperresponsiveness. Wogonoside, a flavonoid glycoside, is reported to significantly alleviate the inflammation response and oxidative stress. Herein, this study aimed to investigate the therapeutic effect and underlying mechanism of wogonoside on airway inflammation and mucus hypersecretion in a murine asthma model and in human bronchial epithelial cells (16HBE). BALB/c mice were sensitized and challenged with ovalbumin (OVA). Pulmonary function and the number of cells in the bronchoalveolar lavage fluid (BALF) were examined. Pathological changes in lung tissue in each group were evaluated via hematoxylin and eosin and periodic acid-Schiff staining, and changes in levels of cytokines in BALF and of immunoglobulin E in serum were determined via an enzyme-linked immunosorbent assay. The expression of relevant genes in lung tissue was analyzed via real-time PCR. Western blotting and immunofluorescence were employed to detect the expression of relevant proteins in lung tissue and 16HBE cells. Treatment with 10 and 20 mg/kg wogonoside significantly attenuated the OVA-induced increase of inflammatory cell infiltration, mucus secretion, and goblet cell percentage and improved pulmonary function. Wogonoside treatment reduced the level of T-helper 2 cytokines including interleukin (IL)-4, IL-5, and IL-13 in BALF and of IgE in serum and decreased the mRNA levels of cytokines (IL-4, IL-5, IL-6, IL-13, and IL-1ß and tumor necrosis factor-α), chemokines (CCL-2, CCL-11, and CCL-24), and mucoproteins (MUC5AC, MUC5B, and GOB5) in lung tissues. The expression of MUC5AC and the phosphorylation of STAT6 and NF-κB p65 in lung tissues and 16HBE cells were significantly downregulated after wogonoside treatment. Thus, wogonoside treatment may effectively decrease airway inflammation, airway remodeling, and mucus hypersecretion via blocking NF-κB/STAT6 activation.

Amygdalin Improves Allergic Asthma via the Thymic Stromal Lymphopoietin-dendritic Cell-OX40 Ligand Axis in a Mouse Model.

Asthma, characterized by persistent inflammation and increased sensitivity of the airway, is the most common chronic condition among children. Novel, safe, and reliable treatment strategies are the focus of current research on pediatric asthma. Amygdalin, mainly present in bitter almonds, has anti-inflammatory and immunoregulatory potential, but its effect on asthma remains uninvestigated. Here, the impact of amygdalin on the thymic stromal lymphopoietin (TSLP)-dendritic cell (DC)-OX40L axis was investigated. A BALB/c mouse model for allergic asthma was established using the ovalbumin-sensitization method. Amygdalin treatment was administered between days 21 and 27 of the protocol. Cell numbers and hematoxylin and eosin (H&E) staining in bronchoalveolar lavage fluid (BALF) were used to observe the impact of amygdalin on airway inflammation. TSLP, IL-4, IL-5, IL-13, and IFN-γ concentrations were determined via Enzyme-linked immunosorbent assay (ELISA). TSLP, GATA-3, and T-bet proteins were measured using western blotting. Cell-surface receptor expression on DCs (MHC II, CD80, and CD86) was assessed via flow cytometry. OX40L mRNA and protein levels were detected using western blotting and qRT-PCR, respectively. Amygdalin treatment attenuated airway inflammation decreased BALF TSLP levels, inhibited DC maturation, restrained TSLP-induced DC surface marker expression (MHCII, CD80, and CD86), and further decreased OX40L levels in activated DCs. This occurred together with decreased Th2 cytokine levels (IL-4, IL-5, and IL-13) and GATA3 expression, whereas Th1 cytokine (IFN-γ) levels and T-bet expression increased. Amygdalin thus regulates the Th1/Th2 balance through the TSLP-DC-OX40L axis to participate in inflammation development in the airways, providing a basis for potential allergic asthma treatments.

[CGRP inhibits proliferation, activation and cytokine secretion of group 2 innate lymphoid cells (ILC2) in peripheral blood from patients with allergic rhinitis].

Objective To investigate the effect of calcitonin gene-related peptide (CGRP) on the regulation of group 2 innate lymphoid cells (ILC2) in the peripheral blood of patients with allergic rhinitis (AR). Methods Peripheral blood mononuclear cells (PBMCs) were extracted from normal healthy individuals and AR patients, then stimulated with CGRP, interleukin 33 (IL-33) and CGRP combined with IL-33 for 3 days, with blank stimulus as control. The percentage of ILC2 in the four groups was measured by flow cytometry. After being sorted, ILC2 was given to CGRP, IL-33 and CGRP combined with IL-33 stimulation for 3 days, with blank stimulus as control. The percentage of IL-5 and IL-13 positive cells in ILC2 was detected by flow cytometry, and the levels of IL-5 and IL-13 in ILC2 supernatant were measured by ELISA. Results The percentage of ILC2 in the peripheral blood of AR patients was significantly higher than that of the control group. The levels of IL-5+ILC2 and IL-13+ILC2 were significantly increased by IL-33 single stimulation after culturing PBMCs. After adding IL-33 combined with CGRP stimulation, the levels of IL-5+ILC2 and IL-13+ILC2 in PBMCs were significantly reduced; after CGRP single stimulation, the levels of IL-5+ILC2 and IL-13+ILC2 in PBMCs were further decreased. After ILC2 was sorted and cultured, the levels of IL-5+ILC2 and IL-13+ILC2 showed significant increase after IL-33 single stimulation. The levels of IL-5+ILC2 and IL-13+ILC2 were decreased by IL-33 and CGRP co-stimulation, and they were further reduced after CGRP single stimulation. Compared to IL-33 single stimulation, IL-5 and IL-13 levels dropped significantly due to the IL-33 and CGRP co-stimulation. The levels of IL-5 and IL-13 were further reduced by CGRP single stimulation. Conclusion CGRP inhibits the proliferation and activation of peripheral blood ILC2 in AR and exert anti-inflammatory effects in AR.

Effects of Lidocaine-Derived Organic Compounds on Eosinophil Activation and Survival.

Lidocaine, a local anesthetic, is known to possess anti-inflammatory properties. However, its clinical use is limited by inconveniences, such as its local synesthetic effects. This study evaluated lidocaine analogs designed and synthesized to overcome the disadvantages of lidocaine, having anti-inflammatory properties. Interleukin 5 (IL-5)-induced eosinophil activation and survival were evaluated using 36 lidocaine analogs with modified lidocaine structure on the aromatic or the acyl moiety or both. Eosinophil survival was evaluated using a CellTiter 96® aqueous cell proliferation assay kit. Superoxide production was determined using the superoxide dismutase-inhibitable reduction of cytochrome C method. Eosinophil cationic protein (ECP), IL-8, and transcription factor expression were determined using enzyme-linked immunosorbent assay. The platelet-activating factor (PAF)-induced migration assay was performed using a Transwell insert system. Compounds EI137 and EI341 inhibited IL-5-induced eosinophil survival and superoxide and ECP production in a concentration-dependent manner. These compounds also significantly reduced IL-8 production. Although compounds EI137 and EI341 significantly reduced phosphorylated ERK 1/2 expression, they did not influence other total and phosphorylated transcription factors. Moreover, 1000 µM of compound EI341 only inhibited PAF-induced migration of eosinophils. Lidocaine analogs EI137 and EI341 inhibited IL-5-mediated activation and survival of eosinophils. These compounds could be new therapeutic agents to treat eosinophilic inflammatory diseases.

Aflatoxin B1 can aggravate BALB/c mice allergy to ovalbumin through changing their Th2 cells immune responses.

Foods contaminated by Aflatoxin B1 (AFB1) frequently happen in the world and can cause a lot healthy damages to human beings, meanwhile, some of these foods are easily irritate food allergy. To investigate the effect of AFB1 exposure on food allergy, three doses of AFB1 were set, including 0.3 µg/kg · bw (LDAF), 7.5 µg/kg · bw (MDAF), and 100.0 µg/kg · bw (HDAF), respectively; food allergy model was constructed by the BALB/c mice allergy to ovalbumin (OVA). The changes of titer in OVA-specific immunoglobulin E (IgE), IgG, IgG1, IgG2a, as well as level of the mMCP-1 in sera were determined by enzyme linked immunosorbent assay (ELISA), respectively; the levels of interleukin (IL-4, IL-5, IL-13) and interferon (IFN)-γ in spleen were separately assessed using ELISA kits, and their relative genes expression were verified by Real-time fluorescence quantitative PCR (Q-PCR); the population of Th1/Th2/Treg cells were analyzed by flow cytometry. Results showed that when OVA-allergic mice were exposed to AFB1, the production of OVA-specific IgE, IL-4, IL-5 and IL-13, and mMCP-1 were all increased, whereas the level of IFN-γ was decreased; the Th1/Th2 balance was disrupted and the development of Th cells tilted to the Th2 phenotype. The study would contribute to further understand the risk of fungal toxins in food allergy.

Considerations for Use of Pharmacodynamic Biomarkers to Support Biosimilar Development - (II) A Randomized Trial with IL-5 Antagonists.

The US Food and Drug Administration (FDA) guidance describes how pharmacodynamic (PD) biomarkers can be used to address residual uncertainty and demonstrate no clinically meaningful differences between a proposed biosimilar and its reference product without relying on clinical efficacy end point(s). Pilot studies and modeling can inform dosing for such PD studies. To that end, we conducted a randomized, double-blinded, placebo-controlled, single-dose, parallel-arm clinical study in healthy participants to evaluate approaches to address information gaps, inform best practices for analysis of biomarker samples and study results, and apply emerging technologies in biomarker characterization. Seventy-two healthy participants (n = 8 per arm) received either placebo or 1 of 4 doses of the interleukin-5 inhibitors mepolizumab (3-24 mg) or reslizumab (0.1-0.8 mg/kg). A clinical study using doses lower than approved therapeutic doses was combined with modeling and simulation to evaluate the dose-response relationship of the biomarker eosinophils. There was no dose-response relationship for eosinophil counts due to variability, although the mepolizumab 24 mg and reslizumab 0.8 mg/kg doses showed clear effects. Published indirect-response models were used to explore eosinophil data across doses from this study and the unstudied therapeutic doses. Simulations were used to calculate typical PD metrics, such as baseline-adjusted area under the effect curve and maximum change from baseline. The simulation results demonstrate sensitivity of eosinophils as a PD biomarker and indicate doses lower than the approved doses would have PD responses overlapping with variability in the placebo arm. The simulation results further highlight the utility of model-based approaches in supporting use of PD biomarkers in biosimilar development.

Th2 cells inhibit growth of colon and pancreas cancers by promoting anti-tumorigenic responses from macrophages and eosinophils.

BACKGROUND: Immunotherapy of gastrointestinal cancers is challenging; however, several lines of evidence suggest that adoptive transfer of stimulated or modified immune cells support not only protective role of immune cells in tumor microenvironment, but actively participate in the elimination of cancer cells. METHODS: In vivo studies employing cancer cell-derived allograft murine models of gastrointestinal cancers were performed. The effects of T helper (Th) 2 cells on gastrointestinal cancers growth and tumor microenvironment composition using adoptive transfer of Th2 cells, interleukin (IL)-5 treatment, and immunofluorescence, multiplex and real-time PCR were explored. RESULTS: Here, we show that Th2 cells play an essential role in the inhibition of colon and pancreas cancers progression. In murine models of gastrointestinal tumors using adoptive transfer of Th2 cells, we identify that Th2 cells are responsible for generation of apoptotic factors and affect macrophage as well as eosinophil recruitment into tumors where they produce cytotoxic factors. Moreover, we found that Th2 cells lead to IL-5 hypersecretion, which links the anti-tumorigenic function of Th2 cells and eosinophils. Importantly, we noted that recombinant IL-5 administration is also related with inhibition of gastrointestinal tumor growth. Finally, using an in vitro approach, we documented that both Th2 cells and eosinophils are directly responsible for gastrointestinal cancer cell killing. CONCLUSIONS: These data demonstrate the significance of Th2 cells, eosinophils and IL-5 in the inhibition of gastrointestinal tumor growth, and pointed toward tumor microenvironment reprogramming as a Th2 cell-mediated anti-tumorigenic mechanism of action.

Ameliorative Effect of Imperatorin on Dermatophagoides pteronyssinus-Induced Allergic Asthma by Suppressing the Th2 Response in Mice.

Imperatorin is a furanocoumarin derivative and an effective ingredient in several Chinese medicinal herbs. It has favorable expectorant, analgesic, and anti-inflammatory effects. In this study, we investigated whether imperatorin has protective effects against Dermatophagoides pteronyssinus (Der p)-induced asthma in mice. Lung and bronchial tissues were histopathologically examined through hematoxylin-eosin staining. The concentrations of immunoglobin E (IgE), IgG1, IgG2a in serum and those of T helper 1 (Th1) and two cytokines and eosinophil-activated chemokines in bronchoalveolar lavage fluid (BALF) were detected using an enzyme immunoassay. Histological examination revealed that imperatorin reduced inflammatory cell infiltration, mucus hypersecretion, and endothelial cell hyperplasia. The examination also indicated that imperatorin could reduce the inflammatory cell count in BALF as well as IgE and IgG1 expression in serum, but IgG2a expression was significantly increased. Imperatorin reduced the production of interleukin (IL)-4, IL-5, and IL-13 by Th2, promoted the production of interferon-γ and IL-12 by Th1, and increased the production of IL-10 in bronchoalveolar lavage fluid. These findings suggest that imperatorin has a considerable anti-inflammatory effect on Der p-induced allergic asthma in mice.

Pulmonary Delivery of Levamisole Nanoparticles as an Immunomodulator Affecting Th and a Potential ADAM10 Inhibitor to Ameliorate Severe Allergic Asthma.

Asthma is a common chronic lung disease without absolute treatment, and hypersensitivity reactions and type 2 immune responses are responsible for asthma pathophysiology. ADAM10 as a metalloproteinase transmembrane protein is critical for development of Th2 responses, and levamisole as an anthelmintic drug has immunomodulatory effects, which not only regulates ADAM10 activity but also can suppress the bone marrow and neutrophil production. Therefore, in the present study, nanoparticles were used as a levamisole delivery system to reduce bone marrow suppression, and the immunomodulatory and ADAM10 inhibitory effects of levamisole were studied in allergic asthma. Asthmatic mice were treated with PLGA-levamisole nanoparticles. Then, AHR, BALF, and blood cell counts, levels of the IgG1 subclass, total and OVA-specific IgE, IL2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-25, IL-33, INF-γ, and TNF-α, gene expression of FoxP3, T-bet, RORγt, PU.1, GATA3, FcεRII, CysLT1R, eotaxin, and ADAM10, and lung histopathology were evaluated. PLGA-LMHCl with considered characteristics could control airway hyper-responsiveness, eosinophils in the BALF, levels of immunoglobulins, Th2-, Th9-, and Th17-derived cytokines and pivotal genes, eosinophilic inflammation, hyperplasia of the goblet cell, and hyperproduction of mucus and could increase Th1- and Treg-derived cytokines and also pivotal genes. It could also modulate the ADAM10 activity and had no effect on the number of neutrophils in the bloodstream. The novel safe nanodrug had no side effect on the bone marrow to produce neutrophils and could control the allegro-immuno-inflammatory response of asthma.

Oxysophocarpine inhibits airway inflammation and mucus hypersecretion through JNK/AP-1 pathway in vivo and in vitro.

Asthma is a high-incidence disease in the world. Oxysophocarpine (OSC), a quinolizidine alkaloid displays various pharmacological functions including anti-inflammation, neuroprotective, anti-virus and antioxidant. Here, we established mice and cell asthmatic model to explore the effects of OSC for asthma treatment. Mice were sensitized and challenged with ovalbumin (OVA) and treated with OSC before challenge. Enzyme-linked immuno sorbent assay (ELISA), hematoxylin and eosin (H&E), periodic acid-schiff (PAS), tolonium chloride staining and immunohistochemical assay were performed. OSC treatment inhibited inflammatory cell infiltration and mucus secretion in the airway, reduced IgE level in mouse serum and decreased IL-4, IL-5 production in bronchoalveolar lavage fluid (BALF). OSC also reduced the spleen index to regulate immune function. Meanwhile, NCI-H292 cells were induced by lipopolysaccharide (LPS) to simulate airway epithelial injury. OSC pretreatment decreased the IL-6 and IL-8 cytokine levels, mucin 5 AC expression, and mucin 5 AC mRNA level in the cell model. Further, OSC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1, Fos and Jun). These findings revealed that OSC alleviated bronchial asthma associated with JNK/AP-1 signaling pathway.

The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target IL-5 receptor alpha to negatively regulate eosinophilic airway inflammation.

Interleukin 5 (IL-5) plays crucial roles in type 2-high asthma by mediating eosinophil maturation, activation, chemotaxis and survival. Inhibition of IL-5 signaling is considered a strategy for asthma treatment. Here, we identified MARCH2 and MARCH3 as critical negative regulators of IL-5-triggered signaling. MARCH2 and MARCH3 associate with the IL-5 receptor α chain (IL-5Rα) and mediate its K27-linked polyubiquitination at K379 and K383, respectively, and its subsequent lysosomal degradation. Deficiency of MARCH2 or MARCH3 modestly increases the level of IL-5Rα and enhances IL-5-induced signaling, whereas double knockout of MARCH2/3 has a more dramatic effect. March2/3 double knockout markedly increases the proportions of eosinophils in the bone marrow and peripheral blood in mice. Double knockout of March2/3 aggravates ovalbumin (OVA)-induced eosinophilia and causes increased inflammatory cell infiltration, peribronchial mucus secretion and production of Th2 cytokines. Neutralization of Il-5 attenuates OVA-induced airway inflammation and the enhanced effects of March2/3 double deficiency. These findings suggest that MARCH2 and MARCH3 play redundant roles in targeting IL-5Rα for degradation and negatively regulating allergic airway inflammation.

Time-dependent dual beneficial modulation of interferon-γ, interleukin 5, and Treg cytokines in asthma patient peripheral blood mononuclear cells by ganoderic acid B.

Th2 cytokines play a dominant role in the pathogenesis of allergic asthma. Interferon gamma (IFN-γ), a Th1 cytokine, links to therapeutic mechanisms of allergic asthma. Interleukin (IL)-10, a regulatory cytokine, is involved in the induction of immune tolerance. We previously demonstrated that Anti-Asthma Simplified Herbal Medicine Intervention (ASHMI) suppressed Th2 and increased IFN-γ in patients with asthma and in animal models, but its bioactive compound is unknown. Ganoderic acid beta (GAB) was isolated from Ganoderma lucidum (one herb in ASHMI). Human peripheral blood mononuclear cells (PBMCs) from adult patients with asthma were cultured with GAB or dexamethasone (Dex) in the presence of environmental allergens. The cytokine levels of IL-10, IFN-γ, IL-5, transcription factors T-bet, Foxp-3, and GATA3 were measured. Following 3-day culture, GAB, but not Dex, significantly increased IL-10 and IFN-γ levels by allergic patients' PBMCs. Following 6-day treatment, GAB inhibited IL-5 production, but IL-10 and IFN-γ remained high. Dex suppressed production of all three cytokines. GAB suppressed GATA3 and maintained Foxp-3 and T-bet gene expression, while Dex significantly suppressed GATA3 and T-bet expression. GAB simultaneously increased IL-10, IFN-γ associated with induction of T-bet and Foxp3, while suppressing IL-5, which was associated with suppression of GATA3, demonstrating unique beneficial cytokine modulatory effect, which distinguishes from Dex's overall suppression.

Anti-toxoplasma and Cytotoxic Activities of Holothuria leucospilota Extract and TiO2NPs In vitro and In vivo.

BACKGROUND: An impressive treatment for toxoplasmosis is the combinatory use of sulfadiazine and pyrimethamine. However, both the drugs involve significant side effects and toxicity for the host. Therefore, the discovery of new anti-toxoplasma medications with high efficacy and less to no side effects is urgently needed. OBJECTIVE: This study aimed to evaluate the anti-toxoplasmic effects of Holothuria leucospilota (H. leucospilota) extract and TiO2NPs on the cell death of Toxoplasma gondii (T. gondii) tachyzoites in vitro and serum liver enzymes (AST, ALT, and ALP), and also to evaluate the immune response and production of IL-5, IFN-γ, and TNF-α in a mouse model. MATERIALS AND METHODS: The cytotoxicity of TiO2NPs and H. leucospilota extract against the tachyzoite of T. gondii was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. The levels of serum TNF-α, IFN-γ, IL-5, and liver enzymes were measured, as well. All the groups were subjected to T. gondii, and the survival rate of experimental mice was evaluated. RESULTS: Our findings suggested in vivo and in vitro anti-toxoplasmic activity of TiO2NPs and H. leucospilota extract by inhibiting the proliferation and invasion of T. gondii tachyzoite. In addition, a significant increase in IFN-γ and TNF-α production was observed in mice treated with high doses of TiO2NPs and H. leucospilota extract. However, IL-5 levels decreased in TiO2NPs and H. leucospilota extract-treated mice. Our results also showed a highly significant increase (P < 0.05) in the levels of ALT, AST, and ALP in the groups injected with TiO2NPs and H. leucospilota extract, but not the control group. CONCLUSION: TiO2NPs and H. leucospilota extract have greater anti-toxoplasma effects in vitro and in vivo. These two compounds could be considered as a candidate for use against toxoplasmosis, both therapeutically and prophylactically.

Interleukin-5 (IL-5) Therapy Prevents Allograft Rejection by Promoting CD4+CD25+ Ts2 Regulatory Cells That Are Antigen-Specific and Express IL-5 Receptor.

CD4+CD25+Foxp3+T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4+CD25+T cells generated from CD4+CD25- T cells' activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4+CD25+T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo, we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4+CD25+T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts' CD4+CD25+ cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4+CD25+ cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg including Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg.

IL-3 in the development and function of basophils.

The ß common chain (ßc) cytokine family includes granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5, all of which use ßc as key signaling receptor subunit. GM-CSF, IL-3 and IL-5 have specific roles as hematopoietic growth factors. IL-3 binds with high affinity to the IL-3 receptor α (IL-3Rα/CD123) and then associates with the ßc subunit. IL-3 is mainly synthesized by different subsets of T cells, but is also produced by several other immune [basophils, dendritic cells (DCs), mast cells, etc.] and non-immune cells (microglia and astrocytes). The IL-3Rα is also expressed by immune (basophils, eosinophils, mast cells, DCs, monocytes, and megacaryocytes) and non-immune cells (endothelial cells and neuronal cells). IL-3 is the most important growth and activating factor for human and mouse basophils, primary effector cells of allergic disorders. IL-3-activated basophils and mast cells are also involved in different chronic inflammatory disorders, infections, and several types of cancer. IL-3 induces the release of cytokines (i.e., IL-4, IL-13, CXCL8) from human basophils and preincubation of basophils with IL-3 potentiates the release of proinflammatory mediators and cytokines from IgE- and C5a-activated basophils. IL-3 synergistically potentiates IL-33-induced mediator release from human basophils. IL-3 plays a pathogenic role in several hematologic cancers and may contribute to autoimmune and cardiac disorders. Several IL-3Rα/CD123 targeting molecules have shown some efficacy in the treatment of hematologic malignancies.

Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a glycan-binding receptor bearing immunoreceptor tyrosine-based inhibitory and switch motifs (ITIM and ITSM, respectively) that is selectively expressed on eosinophils, mast cells, and, to a lesser extent, basophils. Previous work has shown that engagement of Siglec-8 on IL-5-primed eosinophils causes cell death via CD11b/CD18 integrin-mediated adhesion and NADPH oxidase activity and identified signaling molecules linking adhesion, reactive oxygen species (ROS) production, and cell death. However, the proximal signaling cascade activated directly by Siglec-8 engagement has remained elusive. Most members of the Siglec family possess similar cytoplasmic signaling motifs and recruit the protein tyrosine phosphatases SHP-1/2, consistent with ITIM-mediated signaling, to dampen cellular activation. However, the dependence of Siglec-8 function in eosinophils on these phosphatases has not been studied. Using Siglec-8 antibody engagement and pharmacological inhibition in conjunction with assays to measure cell-surface upregulation and conformational activation of CD11b integrin, ROS production, and cell death, we sought to identify molecules involved in Siglec-8 signaling and determine the stage of the process in which each molecule plays a role. We demonstrate here that the enzymatic activities of Src family kinases (SFKs), Syk, SHIP1, PAK1, MEK1, ERK1/2, PLC, PKC, acid sphingomyelinase/ceramidase, and Btk are all necessary for Siglec-8-induced eosinophil cell death, with no apparent role for SHP-1/2, SHIP2, or c-Raf. While most of these signaling molecules are necessary for Siglec-8-induced upregulation of CD11b integrin at the eosinophil cell surface, Btk is phosphorylated and activated later in the signaling cascade and is instead necessary for CD11b activation. In contrast, SFKs and ERK1/2 are phosphorylated far earlier in the process, consistent with their role in augmenting cell-surface levels of CD11b. In addition, pretreatment of eosinophils with latrunculin B or jasplakinolide revealed that actin filament disassembly is necessary and sufficient for surface CD11b integrin upregulation and that actin polymerization is necessary for downstream ROS production. These results show that Siglec-8 signals through an unanticipated set of signaling molecules in IL-5-primed eosinophils to induce cell death and challenges the expectation that ITIM-bearing Siglecs signal through inhibitory pathways involving protein tyrosine phosphatases to achieve their downstream functions.

Interleukin IL-5 alleviates sepsis-induced acute lung injury by regulating the immune response in rats.

To study the effect of IL-5 on the immune response and lung injury in rats with sepsis. We constructed a rat model of sepsis by cecal ligation and puncture (CLP). The rats were randomly divided into the control group, the sham group, the CLP group and the IL-5 group, with 6 rats in each group. With the induction of CLP, the lung tissue of rats was severely injured, and the water content of lung tissue was significantly increased. Moreover, the ratio of CD4+/CD8+ was significantly decreased and Th1/Th2 was significantly increased in the peripheral blood. The content of IL-6, TNF-α, and HMGB1 was found to be increased in the CLP group. However, with the injection of IL-5, the degree of lung tissue injury in CLP rats was alleviated and the water content of lung tissue was significantly reduced. The ratio of CD4+/CD8+ was increased and Th1/Th2 was significantly down-regulated in the peripheral blood and the levels of IL-6, TNF-α, and HMGB1 in serum were significantly decreased. In conclusion, IL-5 can alleviate lung injury by regulating the immune response and inhibiting the systemic inflammatory response induced by sepsis.

Regulatory eosinophils induce the resolution of experimental arthritis and appear in remission state of human rheumatoid arthritis.

OBJECTIVES: Eosinophils possess pro-inflammatory functions in asthma. However, our recent studies have suggested that innate lymphoid cells type 2 (ILC2s) and eosinophils have proresolving properties in rheumatoid arthritis (RA). Nothing is known yet about the mechanisms determining the double-edged role of eosinophils. Therefore, we investigated whether asthma, a paradigm eosinophilic disease, can elicit resolution of chronic arthritis. METHODS: Ovalbumin-triggered eosinophilic asthma was combined with K/BxN serum-induced arthritis, where lung and synovial eosinophil subsets were compared by single-cell RNA sequencing (scRNA-seq). To investigate the involvement of the ILC2-interleukin-5 (IL-5) axis, hydrodynamic injection (HDI) of IL-25 and IL-33 plasmids, IL-5 reporter mice and anti-IL-5 antibody treatment were used. In patients with RA, the presence of distinct eosinophil subsets was examined in peripheral blood and synovial tissue. Disease activity of patients with RA with concomitant asthma was monitored before and after mepolizumab (anti-IL-5 antibody) therapy. RESULTS: The induction of eosinophilic asthma caused resolution of murine arthritis and joint tissue protection. ScRNA-seq revealed a specific subset of regulatory eosinophils (rEos) in the joints, distinct from inflammatory eosinophils in the lungs. Mechanistically, synovial rEos expanded on systemic upregulation of IL-5 released by lung ILC2s. Eosinophil depletion abolished the beneficial effect of asthma on arthritis. rEos were consistently present in the synovium of patients with RA in remission, but not in active stage. Remarkably, in patients with RA with concomitant asthma, mepolizumab treatment induced relapse of arthritis. CONCLUSION: These findings point to a hitherto undiscovered proresolving signature in an eosinophil subset that stimulates arthritis resolution.

IL-13 and IL-4, but not IL-5 nor IL-17A, induce hyperresponsiveness in isolated human small airways.

BACKGROUND: Specific inflammatory pathways are indicated to contribute to severe asthma, but their individual involvement in the development of airway hyperresponsiveness remains unexplored. OBJECTIVE: This experimental study in human small bronchi aimed to provide insight into which of the type 2 and type 17 cytokines cause hyperresponsiveness of airway smooth muscle. METHODS: Explanted small bronchi isolated from human lung tissue and human airway smooth muscle cells were treated for 2 and 1 day(s), respectively, with 100 ng/mL of IL-4, IL-5, IL-13, or IL-17A, and contractile responses, Ca2+ mobilization, and receptor expression were assessed. RESULTS: Treatment with IL-13 increased the potency of histamine, carbachol, and leukotriene D4 as contractile agonists. IL-4, but not IL-5 or IL-17A, also increased the potency of histamine. In human airway smooth muscle cells, IL-13 and IL-4, but not IL-5 and IL-17A, enhanced the histamine-induced Ca2+ mobilization that was accompanied with increased mRNA expression of histamine H1 and cysteinyl leukotriene CysLT1 receptors. RNA sequencing of isolated bronchi confirmed the IL-13-mediated upregulation of H1 and CysLT1 receptors, without showing an alteration of muscarinic M3 receptors. Dexamethasone had no effects on IL-13-induced hyperresponsiveness in human bronchi, the increased Ca2+ mobilization, or the enhanced receptor expression. In contrast, antagonism of the common receptor for IL-13 and IL-4 by the biologic dupilumab prevented the effects of both IL-13 and IL-4 in human bronchi and human airway smooth muscle cells. CONCLUSIONS: The glucocorticoid-insensitive hyperrresponsiveness in isolated human airways induced by IL-13 and IL-4 provides further evidence that the IL-4Rα pathway should be targeted as a new strategy for the treatment of airway hyperresponsiveness in asthma.

Comparison of IL-33 and IL-5 family mediated activation of human eosinophils.

Eosinophils are the prominent inflammatory cell involved in allergic asthma, atopic dermatitis, eosinophilic esophagitis, and hypereosinophilic syndrome and are found in high numbers in local tissue and/or circulating blood of affected patients. There is recent interest in a family of alarmins, including TSLP, IL-25 and IL-33, that are epithelial-derived and released upon stimulation of epithelial cells. Several genome wide association studies have found SNPs in genes encoding IL-33 to be risk factors for asthma. In two studies examining the direct role of IL-33 in eosinophils, there were differences in eosinophil responses. We sought to further characterize activation of eosinophils with IL-33 compared to activation by other cytokines and chemokines. We assessed IL-33 stimulated adhesion, degranulation, chemotaxis and cell surface protein expression in comparison to IL-3, IL-5, and eotaxin-1 on human eosinophils. Our results demonstrate that IL-33 can produce as potent eosinophil activation as IL-3, IL-5 and eotaxin-1. Thus, when considering specific cytokine targeting strategies, IL-33 will be important to consider for modulating eosinophil function.