Expression of Epithelial Alarmin Receptor on Innate Lymphoid Cells Type 2 in Eosinophilic Chronic Obstructive Pulmonary Disease.

Studies have shown that eosinophilic COPD (eCOPD) is a distinct phenotype of the disease. It is well established that innate lymphoid cells are involved in the development of eosinophilic inflammation. Interleukin(IL)-25, thymic stromal lymphopoietin (TSLP) and IL-33 are a group of cytokines produced by epithelium in response to danger signals, e.g., cigarette smoke, and potent activators of ILC2s. In the present study, we examined circulating and sputum ILC2 numbers and expression of intracellular IL-5 as well as receptors for TSLP, IL-33 and IL-25 by ILC2s in non-atopic COPD patients with and without (neCOPD) airway eosinophilic inflammation and healthy smokers. In addition, we examined the association between ILC2s and clinical indicators of COPD burden (i.e., symptom intensity and risk of exacerbations). ILC2s were enumerated in peripheral blood and induced sputum by means of flow cytometry. We noted significantly greater numbers of airway IL-5+ILC2s and TSLPR+ILC2s in eCOPD compared with neCOPD (p < 0.05 and p < 0.01, respectively) and HSs (p < 0.001 for both). In addition, we showed that IL-5+ILC2s, IL-17RB+ILC2s and ST2+ILC2s are significantly increased in the sputum of eCOPD patients compared with HSs. In all COPD patients, sputum ILC2s positively correlated with sputum eosinophil percentage (r = 0.48, p = 0.002). We did not find any significant correlations between sputum ILC2s and dyspnea intensity as measured by the modified Medical Research Council scale (mMRC) and symptom intensity measured by the COPD Assessment Test (CAT). These results suggest the involvement of epithelial alarmin-activated ILC2s in the pathobiology of eosinophilic COPD.

Group 2 innate lymphocytes protect the balance between autophagy and apoptosis in cardiomyocytes during sepsis-induced cardiac injury.

Group 2 innate lymphocytes (ILC2) have an important role in orchestrating sepsis-induced immune response. However, the impact of LC2 on sepsis-induced cardiac injury is still not fully understood. This study investigated the mechanisms governing ILC2 activation within the cardiac tissue after sepsis. In vivo experiments using wild-type and IL-33 deficient mice indicated that the presence of interleukin (IL)-33, which participates in expanding and activating ILC2 cells, was correlated with higher ILC2 levels (246 ± 34 vs. 66 ± 18, p < 0.01), reduced cardiac dysfunction, and lower markers of cardiac injury. Conversely, IL-33 deficiency led to exacerbated cardiac damage. Additionally, heart ILC2 significantly increased the expression and secretion of IL-5 (2.18 ± 0.34 ng/ml vs. 1.18 ± 0.24 ng/ml, p < 0.05) and IL-13 (10.55 ± 1.13 ng/ml vs. 7.59 ± 1.13 ng/ml, p < 0.05) following sepsis, with this response being mediated by IL-33. Moreover, IL-5 deficient mice exhibited increased cardiac dysfunction and myocardial apoptosis post-sepsis (20.7 ± 4.28% vs. 29.61 ± 4.28%, p < 0.05). Furthermore, in vitro experiments involving co-cultures of ILC2 with mice cardiomyocytes after lipopolysaccharide (LPS) administration suggested that IL-5 derived from ILC2 protects cardiomyocytes from autophagy and apoptosis. These findings imply that IL-33, released in response to sepsis, induces ILC2 activation and IL-5 secretion, orchestrating the equilibrium between autophagy and apoptosis in cardiomyocytes and offering potential therapeutic avenues for mitigating sepsis-induced cardiac injury.

Interleukin-5 as a pleiotropic cytokine orchestrating airway type 2 inflammation: Effects on and beyond eosinophils.

Interleukin (IL)-5 is the key cytokine in the maturation, activation, proliferation, migration and survival of eosinophils, which are key effector cells in many upper and lower airway diseases. Through its effects on eosinophils, IL-5 indirectly contributes to various pathophysiological processes including tissue damage, repair and remodelling. Understanding the importance of IL-5 in eosinophil-associated diseases led to the development of anti-IL-5 therapies, which provide clinical benefits across a range of conditions. However, recent evidence suggests that eosinophil-depletion alone may not account for all of the therapeutic effects of anti-IL-5 therapy and that IL-5 may also contribute to disease independently of its effects on eosinophils. Indeed, evidence from ex vivo studies and targeted therapy in vivo demonstrates that IL-5 and its inhibition affects a much broader range of cells beyond eosinophils, including epithelial cells, plasma cells, mast cells, basophils, neutrophils, type 2 innate lymphoid cells, T regulatory cells and fibroblasts. This review will provide an update on the evidence supporting the breadth of IL-5 biology relevant to disease pathogenesis beyond eosinophil-associated inflammation, where there is a need for additional insight, and the clinical implications of a more central role of IL-5 in type 2 inflammation.

Asthma Biologics: Lung Function, Steroid-Dependence, and Exacerbations.

The development of multiple targeted biologic therapies over the past two decades has revolutionized the management of asthma. Currently, there are 6 monoclonal antibodies that target specific inflammatory mediators involved in the pathophysiology of asthma, and together, they provide the opportunity for personalized treatment options beyond bronchodilators and inhaled or systemic glucocorticoids in severe and difficult-to-control cases of asthma. These agents are the anti-IgE antibody omalizumab, the anti-IL-5 antibodies mepolizumab and reslizumab, the IL-5 receptor alpha antagonist benralizumab, the IL-4 receptor alpha antagonist dupilumab, and the anti-thymic stromal lymphopoietin antibody tezepelumab.

[Expression of prostacyclin receptor in chronic rhinosinusitis and its relationship with type 2 inflammation].

Objective:The purpose of this study is to explore the expression of prostacyclin receptor（IP） in patients with chronic rhinosinusitis（CRS） and its possible association with type 2 inflammation. Methods:HE staining was used to observe the morphological changes of nasal mucosa, qRT-PCR was used to detect the expression of IP in polyps and nasal mucosa, and IHC was used to detect the expression of IP, IL-4, IL-5 and IL-13 in polyps and nasal mucosa. Results:Compared with the control group, the nasal mucosa of patients with various types of CRS was obviously thickened, accompanied by inflammatory cell infiltration and gland hyperplasia. The statistical results of IHC showed that the expression levels of IL-4, IL-5 and IL-13 in CRS group were significantly higher than those in control group（P<0.05）, and the IP expression in control group was significantly higher than that in ECRS group and non-ECRS group（P<0.05）. The IP expression in ECRS group was negatively correlated with IL-4, IL-5 and IL-13. The results of qRT-PCR showed that the expression of IP mRNA in control group was significantly higher than that in ECRS group and non-ECRS group（P<0.05）. Conclusion:IL-4, IL-5 and IL-13 are highly expressed in the nasal mucosa of CRS patients, while IP is poorly expressed in the nasal mucosa of CRS patients, and IP is negatively correlated with IL-4, IL-5 and IL-13, suggesting that IP is related to the occurrence and development of type 2 inflammation and may be a potential therapeutic target for CRS patients.

[Screening and validation of therapeutic targets for chronic sinusitis with nasal polyps based on proteomics].

Objective: To identify potential therapeutic targets of chronic sinusitis with nasal polyps (CRSwNP) through proteomics screening of and verify its effectiveness experimentally. Methods: The nasal tissue samples were collected from patients undergoing surgical treatment in the Department of Otorhinolaryngology, Head and Neck Surgery in Yuhuangding Hospital of Yantai from June 2010 to December 2021, including 69 patients with CRSwNP and 39 patients in the control group. Tissue samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-independent acquisition (DIA) mode to find differentially expressed proteins. Bioinformatics tools were employed to analyze the functions of differentially expressed proteins. The expression of hematopoietic cell kinase (HCK) in nasal tissues of patients with CRSwNP was further confirmed by qPCR and western blot. The mouse model of CRSwNP was established and treated with HCK inhibitor. The levels of inflammatory factors IgE, IL-4 and IL-5 in serum of CRSwNP mice, both treated and untreated with HCK inhibitors, were detected by enzyme-linked immunosorbent assay (ELISA) across different experimental groups. The experimental data were analyzed by Graphpad Prism 9 software. Results: DIA analysis identified 1 850 differential proteins, including 760 up-regulated proteins and 1 090 down-regulated proteins. Weighted correlation network analysis (WGCNA) correlation analysis of phenotypic data such as cell count and CT score with the results of genomics indemnified 575 proteins of MEBrown module which intersected with 35 kinases further screened from 1 850 differential proteins, yielding eight protein kinases: HCK, SYK, PDK2, FGR, PRKCB, ROR1, CAMK1 and GRK6. qPCR showed that the expression of HCK in CRSwNP was significantly higher than that in the control group (P<0.05). Further experiments in mice confirmed that the secretion of IgE, IL-4 and IL-5 in the serum of CRSwNP group was significantly higher than the control group (all P<0.05), indicating successful model establishment. The intervention of HCK significantly decreased the secretion of IgE, IL-4 and IL-5 in serum of mice (all P<0.05). Conclusion: The HCK inhibitor can reduce the inflammatory index of mice with CRSwNP, and HCK is a potential therapeutic target of CRSwNP.

The influence of the oral microbiota in full-term pregnant women on immune regulation during pregnancy.

BACKGROUND: This study aims to conduct a preliminary exploration of the correlation between the oral microbiota of full-term pregnant women and both local placental immunity and the systemic immune system of the mother. METHODS: A total of 26 pregnant women participated in this study, with samples collected from oral swabs, placental tissue, and peripheral venous blood. High-throughput sequencing was used to examine the oral microbial community. Flow cytometry was employed to assess immune cells in placental tissue and peripheral venous blood. ELISA and Luminex liquid bead chip technology were utilized to detect cytokines in both placental tissue and peripheral venous blood. RESULTS: In placental tissue, The oral microbial community is primarily negatively correlated with placental CD3+CD4+CD8+T cells and positively correlated with placental IL-5. In the peripheral blood, The oral microbial community is primarily positively correlated with maternal systemic immune parameters, including CD3+CD4+ T cells and the CD4+/CD8+ ratio, as well as positively correlated with peripheral IL-18. CONCLUSIONS: The oral microbiota of full-term pregnant women participates in the regulatory function of the maternal immune system. Meanwhile, the oral microbial community may also be an important factor mediating local immune regulation in the placenta.

Interleukin-5 alleviates cardiac remodelling via the STAT3 pathway in angiotensin II-infused mice.

Interleukin-5 (IL-5) has been reported to be involved in cardiovascular diseases, such as atherosclerosis and cardiac injury. This study aimed to investigate the effects of IL-5 on cardiac remodelling. Mice were infused with angiotensin II (Ang II), and the expression and source of cardiac IL-5 were analysed. The results showed that cardiac IL-5 expression was time- and dose-dependently decreased after Ang II infusion, and was mainly derived from cardiac macrophages. Additionally, IL-5-knockout (IL-5-/-) mice were used to observe the effects of IL-5 knockout on Ang II-induced cardiac remodelling. We found knockout of IL-5 significantly increased the expression of cardiac hypertrophy markers, elevated myocardial cell cross-sectional areas and worsened cardiac dysfunction in Ang II-infused mice. IL-5 deletion also promoted M2 macrophage differentiation and exacerbated cardiac fibrosis. Furthermore, the effects of IL-5 deletion on cardiac remodelling was detected after the STAT3 pathway was inhibited by S31-201. The effects of IL-5 on cardiac remodelling and M2 macrophage differentiation were reversed by S31-201. Finally, the effects of IL-5 on macrophage differentiation and macrophage-related cardiac hypertrophy and fibrosis were analysed in vitro. IL-5 knockout significantly increased the Ang II-induced mRNA expression of cardiac hypertrophy markers in myocardial cells that were co-cultured with macrophages, and this effect was reversed by S31-201. Similar trends in the mRNA levels of fibrosis markers were observed when cardiac fibroblasts and macrophages were co-cultured. In conclusions, IL-5 deficiency promote the differentiation of M2 macrophages by activating the STAT3 pathway, thereby exacerbating cardiac remodelling in Ang II-infused mice. IL-5 may be a potential target for the clinical prevention of cardiac remodelling.

Analysis of 12 kinds of cytokines in seminal plasma by flow cytometry and their correlations with routine semen parameters.

OBJECTIVE: To investigate the levels of 12 kinds of cytokines in seminal plasma and their correlations with routine semen parameters. METHODS: The remaining seminal plasma samples of 134 patients undergoing routine semen examination were collected for detecting cytokines. The parameters for sperm concentration, percentage of progressively motile sperm (PR), and motility were analyzed by a computer-assisted sperm analysis (CASA) system. According to the results of sperm concentration, PR and motility, 134 patients were divided into the normal routine semen parameters group, oligoasthenospermia group and azoospermia group. The levels of 12 kinds of cytokines in seminal plasma, including interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17, interferin (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α, were detected by flow cytometry. Two seminal plasma samples were detected for 10 times, respectively, to calculate the coefficients of variation (CV) of each cytokine. The linear range of each cytokine was measured using the standard, and the correlation coefficient (r) was calculated. RESULTS: The r2 of 12 kinds of cytokines detected by flow cytometry were all greater than 0.99. The reproducibility of 2 seminal plasma samples showed that the CVs of all cytokines were lower than 15 % except for TNF-α in sample 1 (15.15 %). Seminal plasma IL-6 levels were negatively correlated with semen volume (P < 0.01). Seminal plasma IL-5 levels were positively correlated with sperm concentration (P < 0.01). Seminal plasma IL-8 levels were negatively correlated with sperm motility (P < 0.01). Seminal plasma IL-8, IL-17 and IL-12P70 levels were negatively correlated with sperm PR (P < 0.05). In addition to the significant negative correlation between IL-5 and IL-17 (P < 0.05), there was a significant positive correlation between the majority of other cytokines. The levels of seminal plasma IL-17 and IL-12P70 in the oligoasthenospermia group and IL-1ß and IL-12P70 in the azoospermia group were significantly higher than those in the normal routine semen parameters group (P ≤ 0.05), while the levels of IL-10 in the azoospermia group were significantly lower than that in the normal routine semen parameters group (P < 0.05). CONCLUSION: There are certain correlations between seminal plasma cytokines and routine semen parameters and strong correlations between different seminal plasma cytokines, suggesting that the imbalance between seminal plasma cytokines may affect sperm quality. However, it still needs to be further confirmed by large samples and multi-center clinical studies and related basic researches.

Endotype and Phenotype Related Postoperative Effects of Bacterial Lysate in Chronic Rhinosinusitis.

OBJECTIVES: Chronic rhinosinusitis (CRS) endotypes have demonstrated clinical value in guiding treatment decisions. Bacterial lysates are immunomodulators that have shown beneficial effects in various respiratory inflammatory diseases. This study aimed to evaluate the effect of postoperative bacterial lysate therapy on different CRS endotypes. METHODS: Patients diagnosed with CRS who underwent endoscopic sinus surgery were recruited. Bacterial lysates were administered postoperatively for 10 days per month for 3 months to the experimental group comprising patients with a history of frequent upper respiratory infections without adverse reactions. The remaining participants were allocated to the control group. The results of the postoperative 3-, 6-, and 12-month assessments, including the modified Lund-Kennedy (mLK) endoscopic and Sinonasal Outcome Test (SNOT) 22 scores, for the groups were compared. The tissue samples obtained from the participants were evaluated to detect the presence of relevant inflammatory mediators. RESULTS: Among the 92 participants, 47 started bacterial lysate therapy 2 weeks after the surgery. The tissue cytokine profiles and clinical parameters, such as the disease severity and blood eosinophil percentage, of the bacterial lysate and control groups were comparable before treatment. The mLK endoscopic and SNOT-22 scores did not differ after 3, 6, and 12 months of follow-up. The subgroup analysis revealed that the bacterial lysate group had significantly lower mLK endoscopic scores than the control group for CRS without nasal polyps, while there was a tendency toward significance for the interleukin (IL)-5 negative group after 6 months. CONCLUSION: Postoperative bacterial lysate therapy has some beneficial effects on the endoscopic findings of patients with CRS without nasal polyps or those who are negative for IL-5.

Helminth infection induces a distinct subset of CD101hi lung tissue-infiltrating eosinophils that are differentially regulated by type 2 cytokines.

Eosinophils play divergent roles in health and disease, contributing to both immunoregulatory and proinflammatory responses. Helminth infection is strongly associated with eosinophilia and the induction of the type 2 cytokines interleukin (IL)-5, IL-4 and IL-13. This study aimed to elucidate the heterogeneity of pulmonary eosinophils in response to helminth infection and the roles of IL-5, IL-4 and IL-13 in driving pulmonary eosinophil responses. Using the murine helminth model Nippostrongylus brasiliensis (Nb), we characterize a subtype of eosinophils, defined by high expression of CD101, that is induced in the lungs of Nb-infected mice and are phenotypically distinct from lung eosinophils that express low levels of CD101. Strikingly, we show that the two eosinophil subtypes have distinct anatomical localization within the lung: CD101low eosinophils are predominantly localized in the lung vasculature, whereas Nb-induced CD101hi eosinophils are predominantly localized in the extravascular lung niche. We show that CD101hi eosinophils are also induced across other models of pulmonary infection and inflammation, including a nonlung-migrating helminth infection, house dust mite-induced allergic inflammation and influenza infection. Furthermore, we demonstrate that the induction of CD101hi tissue eosinophils is independent of IL-5 and IL-4 signaling, but is dependent on intact IL-13 signaling. These results suggest that IL-13 produced during helminth infection and other disease states promotes a pulmonary tissue-infiltrating program in eosinophils defined by high expression of CD101.

Role of IL-5 in asthma and airway remodelling.

Asthma is a common and burdensome chronic inflammatory airway disease that affects both children and adults. One of the main concerns with asthma is the manifestation of irreversible tissue remodelling of the airways due to the chronic inflammatory environment that eventually disrupts the whole structure of the airways. Most people with troublesome asthma are treated with inhaled corticosteroids. However, the development of steroid resistance is a commonly encountered issue, necessitating other treatment options for these patients. Biological therapies are a promising therapeutic approach for people with steroid-resistant asthma. Interleukin 5 is recently gaining a lot of attention as a biological target relevant to the tissue remodelling process. Since IL-5-neutralizing monoclonal antibodies (mepolizumab, reslizumab and benralizumab) are currently available for clinical use, this review aims to revisit the role of IL-5 in asthma pathogenesis at large and airway remodelling in particular, in addition to exploring its role as a target for biological treatments.

The stimulation of TH2 cells results in increased IL-5 and IL-13 production via the H4 receptor.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting in decreased quality of life. Histamine and specifically the H4 receptor play a key role in the inflammatory process in AD and serve as targets for novel therapeutic approaches. OBJECTIVE: In the present study we aimed to elucidate the immunopathological mechanisms with which the H4 receptor impacts TH2 cells and contributes to AD pathophysiology. METHODS: Total CD4+ T cells obtained from healthy or AD individuals and in vitro differentiated TH2 cells were cultured under different conditions and the mRNA expression or protein production of target molecules were determined using quantitative real-time PCR and ELISA. RESULTS: H4 receptor mRNA expression was upregulated concentration dependent upon IL-4 stimulation in in vitro differentiated TH2 cells progressively during the differentiation. Transcriptomic analysis of in vitro differentiated TH2 versus TH1 cells revealed that the H4 receptor among other genes represents one of the highly upregulated genes in TH2 cells. Most importantly, increased amounts of IL-5 and IL-13 mRNA expression were detected in in vitro differentiated TH2 cells as well as protein secretion in the presence of histamine or of the H4 receptor-selective-agonist when compared to the untreated control. CONCLUSION: We show for the first time an H4 receptor dependent upregulation of the pro-inflammatory cytokines IL-5 and IL-13 in human TH2 cells by histamine. This suggests that the blockade of the H4 receptor may lead to downregulation of these cytokines and amelioration of AD symptoms as reported in first clinical studies.

Increased allergic inflammation and decreased lung insulin sensitivity in offspring of obese allergic mothers.

Epidemiological studies demonstrate that maternal obesity and maternal allergy are major risk factors for asthma in offspring. However, the impact of maternal allergy and obesity on offspring lung insulin signaling and allergen responsiveness is not known. To evaluate this, allergic and nonallergic female mice were fed a high-fat diet or low-fat diet from 7 wk before pregnancy until weaning. Neonatal pups were allergen-sensitized and allergen-challenged and then were assessed for obesity, insulin signaling, and allergic inflammation. Compared with pups of nonobese nonallergic mothers, allergen-challenged pups of obese nonallergic mothers, nonobese allergic mothers, and obese allergic mothers had bronchoalveolar lavage (BAL) eosinophilia, with the pups of obese allergic mothers having the highest BAL eosinophilia. These pups also had lower insulin-induced lung AKT phosphorylation, indicating a decrease in lung parenchymal insulin sensitivity. In cross-fostering experiments, allergen-challenged pups exposed to both pre- and postnatal obese allergic mothers had the highest level of BAL eosinophilia. Maternal obesity or allergy increased offspring serum allergen-specific IgE and interleukin-5 that was highest when the mother was both obese and allergic. Also, allergen-challenged pups exposed to both pre- and postnatal obese allergic mothers had the highest level of interleukin-5. In summary, offspring born to obese allergic mothers have decreased lung insulin sensitivity and increased lung allergic inflammation. Interestingly, our data also demonstrate that there is both a pregnancy and postpregnancy aspect of maternal allergy and obesity that enhances allergen responsiveness in offspring.

3-carene supresses inflammatory cytokine interleukin-4, interleukin-5 and interleukin-13 in a murine model of asthma.

Asthma is a common airway disease associated with allergic inflammation. Environmental factors, such as pollens, pollution, insect-borne antigens, or commercial chemicals, cause this disease. The common symptoms of this airway allergic reaction are increasing mucus, narrowing of the airway wall, coughing, and chest tightness. Medications, such as steroids, alleviate the disease but with severe side effects. Several studies have reported the anti-inflammatory effects of tree-based essential oil components, particularly 3-carene. Therefore, this study used 3-carene to determine if it alleviates asthmatic symptoms in the murine model. First, BALB/c mice were sensitized to an ovalbumin and aluminium hydroxide mixture on day 7th and 14th. From days 21st to 23rd, the mice were challenged with 3-carene and budesonide. The lung trachea, plasma, and bronchiolar lavage fluid (BAL fluid) were collected on day 24. The 3-carene treatment suppressed the cytokine gene expression, such as interleukin-4 (IL-4), IL-5, and IL-13, reducing the lung epithelial cell thickness in the asthmatic model. These results suggest that essential oil 3-carene has an anti-asthmatic effect.

Elastin-derived peptides favor type 2 innate lymphoid cells in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airway inflammation and obstruction, primarily caused by tobacco smoking. Although the involvement of immune cells in COPD pathogenesis is well established, the contribution of innate lymphoid cells (ILCs) remains poorly understood. ILCs are a type of innate immune cells that participate in tissue remodeling processes, but their specific role in COPD has not been fully elucidated. During COPD, the breakdown of pulmonary elastin generates elastin peptides that elicit biological activities on immune cells. This study aimed to investigate the presence of ILC in patients with COPD and examine the impact of elastin peptides on their functionality. Our findings revealed an elevated proportion of ILC2 in the peripheral blood of patients with COPD, and a general activation of ILC as indicated by an increase in their cytokine secretion capacity. Notably, our study demonstrated that serum from patients with COPD promotes ILC2 phenotype, likely due to the elevated concentration of IL-5, a cytokine known to favor ILC2 activation. Furthermore, we uncovered that this increase in IL-5 secretion is partially attributed to its secretion by macrophages upon stimulation by elastin peptides, suggesting an indirect role of elastin peptides on ILC in COPD. These findings shed light on the involvement of ILC in COPD and provide insights into the potential interplay between elastin breakdown, immune cells, and disease progression. Further understanding of the mechanisms underlying ILC activation and their interaction with elastin peptides could contribute to the development of novel therapeutic strategies for COPD management.NEW & NOTEWORTHY Elastin-derived peptides, generated following alveolar degradation during emphysema in patients with COPD, are able to influence the response of type 2 innate lymphoid cells. We show that the orientation of innate lymphoid cells in patients with COPD is shifted toward a type 2 profile and that elastin peptides are indirectly participating in that shift through their influence of macrophages, which in turn impact innate lymphoid cells.

[The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma].

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 µg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

Single-cell proteomics and transcriptomics capture eosinophil development and identify the role of IL-5 in their lineage transit amplification.

The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides resources, methods, and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics, and the regulation of eosinophil development and numbers in health and disease.

scRNA-seq profiling of human granulocytes reveals expansion of developmentally flexible neutrophil precursors with mixed neutrophil and eosinophil properties in asthma.

Neutrophils and eosinophils share common hematopoietic precursors and usually diverge into distinct lineages with unique markers before being released from their hematopoietic site, which is the bone marrow (BM). However, previous studies identified an immature Ly6g(+) Il-5Rα(+) neutrophil population in mouse BM, expressing both neutrophil and eosinophil markers suggesting hematopoietic flexibility. Moreover, others have reported neutrophil populations expressing eosinophil-specific cell surface markers in tissues and altered disease states, confusing the field regarding eosinophil origins, function, and classification. Despite these reports, it is still unclear whether hematopoietic flexibility exists in human granulocytes. To answer this, we utilized single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing to profile human BM and circulating neutrophils and eosinophils at different stages of differentiation and determine whether neutrophil plasticity plays role in asthmatic inflammation. We show that immature metamyelocyte neutrophils in humans expand during severe asthmatic inflammation and express both neutrophil and eosinophil markers. We also show an increase in trilobed eosinophils with mixed neutrophil and eosinophil markers in allergic asthma and that interleukin-5 promotes differentiation of immature blood neutrophils into trilobed eosinophilic phenotypes, suggesting a mechanism of emergency granulopoiesis to promote myeloid inflammatory or remodeling response in patients with chronic asthma. By providing insights into unexpectedly flexible granulocyte biology and demonstrating emergency hematopoiesis in asthma, our results highlight the importance of granulocyte plasticity in eosinophil development and allergic diseases.

A single infusion of engineered long-lived and multifunctional T cells confers durable remission of asthma in mice.

Asthma, the most prevalent respiratory disease, affects more than 300 million people and causes more than 250,000 deaths annually. Type 2-high asthma is characterized by interleukin (IL)-5-driven eosinophilia, along with airway inflammation and remodeling caused by IL-4 and IL-13. Here we utilize IL-5 as the targeting domain and deplete BCOR and ZC3H12A to engineer long-lived chimeric antigen receptor (CAR) T cells that can eradicate eosinophils. We call these cells immortal-like and functional IL-5 CAR T cells (5TIF) cells. 5TIF cells were further modified to secrete an IL-4 mutein that blocks IL-4 and IL-13 signaling, designated as 5TIF4 cells. In asthma models, a single infusion of 5TIF4 cells in fully immunocompetent mice, without any conditioning regimen, led to sustained repression of lung inflammation and alleviation of asthmatic symptoms. These data show that asthma, a common chronic disease, can be pushed into long-term remission with a single dose of long-lived CAR T cells.