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.

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.

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.

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.

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.

Lactobacillus plantarum isolated from kimchi regulates inflammation by increasing interleukin-10 secretion by antigen-presenting cells, leading to diminishing of STAT5 phosphorylation in Th2 cells.

The relationship between allergic inflammation and gut microbiota has been elucidated, and the effect of probiotics on immune disorders has been studied as well. Identifying the role of probiotics in individual diseases and immune responses and selecting and applying specific microorganisms based on these findings can be an effective strategy for using probiotics. Herein, lactobacilli isolated from kimchi were investigated in depth, focusing on their immune regulatory effects and the mechanisms involved. Lactic acid bacteria (LAB) effectively diminished the increased secretion of T helper 2 cytokines, such as IL-4, IL-5, and IL-13, from ovalbumin (OVA)-sensitized mouse splenocytes. The gene expression of GATA3, IL-4, IL-5, IL-9, and IL-13 was confirmed to be regulated by LAB. LAB also suppressed IL-2 production and STAT5 phosphorylation. An IL-10-neutralizing antibody attenuated these effects, indicating that LAB-induced upregulation of IL-10 in antigen-presenting cells was responsible at least partially for the increased IL-2 production and STAT5 phosphorylation in CD4+ T cells. In conclusion, the current study identified one immunomodulatory mechanism that allows LAB to regulate allergic immune reactions and the potential of LAB from kimchi to modulate various immune reactions.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

Eosinophil peroxidase promotes bronchial epithelial cells to secrete asthma-related factors and induces the early stage of airway remodeling.

Asthma is a heterogeneous disease characterized by chronic airway inflammation, reversible airflow limitation, and airway remodeling. Eosinophil peroxidase (EPX) is the most abundant secondary granule protein unique to activated eosinophils. In this study, we aimed to illustrate the effect of EPX on the epithelial-mesenchymal transition (EMT) in BEAS-2B cells. Our research found that both EPX and ADAM33 were negatively correlated with FEV1/FVC and FEV1%pred, and positively correlated with IL-5 levels. Asthma patients had relatively higher levels of ADAM33 and EPX compared to the healthy control group. The expression of TSLP, TGF-ß1 and ADAM33 in the EPX intervention group was significantly higher. Moreover, EPX could promote the proliferation, migration and EMT of BEAS-2B cells, and the effect of EPX on various factors was significantly improved by the PI3K inhibitor LY294002. The findings from this study could potentially offer a novel therapeutic target for addressing airway remodeling in bronchial asthma, particularly focusing on EMT.

Exploring novel perspectives on eosinophilic inflammation in severe asthma.

Two recent articles by the same research group documented that patients with severe eosinophilic asthma exhibit an increased proportion of a subtype of eosinophils, namely CD62Llow inflammatory eosinophils (iEos) and identified an intriguing correlation between such iEos and asthma control scores. Moreover, CD62Llow iEos were reduced after treatment with the anti-IL-5 monoclonal antibody mepolizumab. In the future, we believe that eosinophil subtypes could represent a useful biomarker in severe eosinophilic asthma, helping clinicians characterize patient endotypes and monitoring the response to biological drugs.

Patients with severe eosinophilic asthma (SEA) have an increased proportion of a subtype of eosinophils, CD62L^low inflammatory eosinophils (iEos), which are reduced after mepolizumab treatment. iEos might represent a novel useful biomarker in SEA.

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.

Obesity correlates with the immunosuppressive ILC2s-MDSCs axis in advanced breast cancer.

AIM: We investigated the relationship between the group 2 innate lymphoid cells (ILC2s)-myeloid-derived suppressor cells (MDSCs) axis and obesity-related breast cancer. METHODS: Fifty-eight patients with breast cancer who had first relapse and metastasis between January 2019 and August 2021 were enrolled. The proportions of ILC2s and MDSCs in blood and the levels of cytokines in serum were detected with flow cytometry. Correlation analysis among clinical characteristics (such as body mass index [BMI]), cytokines, ILC2s, and MDSCs was conducted. RESULTS: There was a significant difference in the proportions of ILC2s and MDSCs between the high BMI group and the normal BMI group (p < .05). In the triple-negative breast cancer (TNBC) patients, the proportions of ILC2s and MDSCs in the obese group were significantly higher than those in the nonobese group (p < .05). In all breast cancer patients, there was a positive correlation between BMI and the ILC2s-MDSCs axis (p < .05). However, there was no correlation observed between the number of metastases, progression-free survival, and the ILC2s-MDSCs axis (p > .05). Additionally, ILC2s showed positive correlations with MDSCs, interleukin-5 (IL-5), IL-10, IL-17A, (PD-L1), programmed cell death 2 ligand 2 (PD-L2), and molecular typing (p < .05). Similarly, MDSCs exhibited positive correlations with IL-5, IL-8, IL-9, IL-17A, PD-L1, and PD-L2 (p < .05). In patients with TNBC, there was a positive correlation between BMI and IL-5 (p < .05). CONCLUSION: Conclusively, obesity may enhance the immunosuppressive effect of the ILC2-MDSC axis in advanced breast cancer. IL-5 may play a vital role in the ILC2-MDSC axis and obesity in TNBC.

Eosinophil extracellular traps in eosinophilic chronic rhinosinusitis induce Charcot-Leyden crystal formation and eosinophil recruitment.

Eosinophil extracellular traps (EETs) are implicated in various eosinophil-associated diseases; however, their role in chronic rhinosinusitis (CRS) remains unclear. In the present study, 57 CRS patients were enrolled, and immunofluorescence was used to analyze EETs in eosinophilic (eCRS) and non-eosinophilic (Non-eCRS) tissues. MSD was used to examine IL-4, IL-5, and IL-13 concentrations in tissue homogenates. Charcot-Leyden crystals (CLCs) protein expression was detected in PMA, PMA+DNase I, and blank control eosinophils using ELISA. Eotaxin-3 mRNA and protein levels were measured in human nasal epithelial cells (HNECs) cultured with EETs, EETs+DNase I, DNase I, and unstimulated eosinophils using PCR and ELISA. EETs were significantly increased in eCRS tissues compared with Non-eCRS (P<0.001), and correlated with VAS and Lund-Mackay CT scores. IL-5 expression was related to EETs formation (r = 0.738, P<0.001). PMA-stimulated eosinophils exhibited higher CLCs protein levels (P<0.01). Co-culturing HNECs with EETs significantly increased eotaxin-3 mRNA and protein levels (P<0.0001, P<0.001) compared with other groups. The study suggests EETs formation is elevated in eCRS patients and is involved in CLCs formation and chemokine secretion, promoting eosinophilic inflammation.

Radical S-adenosyl methionine domain-containing 2, a potential target of D-tryptophan in asthma treatment, regulates T helper cell type 1/2 balance.

Asthma is a common chronic respiratory disease. D-tryptophan (D-TRP) can inhibit allergic airway inflammation and T helper cell type 2 (Th2) immune response. RNA-sequencing results have indicated that radical S-adenosyl methionine domain-containing 2 (RSAD2) might be a potential molecular target of D-TRP in asthma treatment. Herein, we established a mouse model of asthma using ovalbumin (OVA) via intraperitoneal injection and inhalational challenge. Gain- and loss-of-function studies of RSAD2 were performed in mice following the intratracheal delivery of lentiviral vectors (3 × 106 TU/mL). Naïve CD-4+ T cells were isolated from the spleen and used to explore the effects of RSAD2 on Th2 cell differentiation. RSAD2 expression was higher in the asthma group than in the control group. RSAD2 knockdown alleviated inflammatory cell infiltration and reduced the number of goblet cells. Low RSAD2 expression decreased the levels of IgE, IL-25, IL-33, and TSLP, and it reduced the number of inflammatory cells in the bronchoalveolar lavage fluid. RSAD2 silencing suppressed Th2-related cytokine levels (such as IL-4, IL-5, and IL-13) and increased Th1-related cytokine levels (such as IFN-γ). Additionally, RSAD2 knockdown inhibited the phosphorylation of JAK1, JAK3, and STAT6, and downregulated GATA-3 expression. RSAD2 overexpression increased inflammatory cell infiltration and mucus secretion in the lung tissues of mice pretreated with D-TRP. D-TRP pretreatment reduced OVA-specific IgE content and IL-4 and IL-5 levels, and it increased the IFN-γ levels; however, RSAD2 overexpression reversed these effects. In conclusion, RSAD2 knockdown can mitigate OVA-induced asthma by regulating the Th2 immune response via JAK/STAT6 pathway inhibition.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

OBJECTIVE: The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. METHOD: A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin (OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200 µg/ml Artemisia annua pollen extract and OVA for 24 h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. RESULT: On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). CONCLUSION: To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

Correlation between inflammatory biomarkers and disease control in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) may have a heterogeneous response to medical/surgical treatments based on endotypes. Data correlating biomarkers and severity of the disease are lacking. We aimed to determine if IL-5 and calprotectin may be useful in defining severity of disease and identifying uncontrolled patients. METHODS: This was a case-control study including 81 patients with diffuse CRSwNP who underwent at least one previous surgery and treated with intranasal steroids. We enrolled 39 uncontrolled patients (SNOT-22 ≥ 40 and two or more cycles of systemic corticosteroids in last year) (Group A) and 42 controlled one (SNOT-22 < 40 and less than two cycles of systemic corticosteroids in last year) (Group B). We analyzed IL-5 and calprotectin in both nasal secretions and nasal polyp tissue. RESULTS: Calprotectin and IL-5 were significantly higher in Group A in both secretions and tissue, and the higher the number of previous surgeries, the higher the levels detected in nasal secretions. At univariate analyses, smoking, asthma, non-steroidal anti-inflammatory drugs-exacerbated respiratory disease (NSAID-ERD), blood eosinophilia, neutrophils, and eosinophils at nasal cytology were significantly associated with uncontrolled disease. Multivariate analyses showed that asthma, NSAID-ERD, and IL-5 in nasal secretion/polyp tissue were significantly related to the risk of uncontrolled disease. CONCLUSIONS: Our data suggest that asthma, NSAID-ERD, and IL-5 in nasal secretions/tissue may be helpful to identify more severe patients, as they are related to the risk of uncontrolled disease. Nonetheless, high levels of calprotectin and neutrophilia were also observed in uncontrolled patients, especially after multiple surgeries.

Epithelial Tight Junction Anomalies in Nasal Inverted Papilloma.

OBJECTIVES: As a critical component of the epithelial barrier, tight junctions (TJs) are essential in nasal mucosa against pathogen invasion. However, the function of TJs has rarely been reported in nasal inverted papilloma (NIP). This study aims to investigate the potential factors of TJs' abnormality in NIP. METHODS: We assessed the expression of ZO-1, occludin, claudin-1, claudin-3, and claudin-7 in healthy controls and NIP by real-time quantitative polymerase chain reaction and immunofluorescent staining. The correlation between TJs expression and neutrophil count, TH 1/TH 2/TH 17 and regulatory T cell biomarkers, and the proportion of nasal epithelial cells was investigated. RESULTS: Upregulation of ZO-1, occludin, claudin-1, and claudin-7, along with downregulation of claudin-3, was found in NIP compared to control (all p < 0.05). An abnormal proportion with a lower number of ciliated cells (control vs. NIP: 37.60 vs. 8.67) and goblet cells (12.52 vs. 0.33) together with a higher number of basal cells (45.58 vs. 124.00) in NIP. Meanwhile, claudin-3 was positively correlated with ciliated and goblet cells (all p < 0.01). Additionally, neutrophils were excessively infiltrated in NIP, negatively correlated with ZO-1, but positively with claudin-3 (all p < 0.05). Furthermore, FOXP3, IL-10, TGF-ß1, IL-5, IL-13, and IL-22 levels were induced in NIP (all p < 0.01). Occludin level was negatively correlated with IL-10, IL-5, IL-13, and IL-22, whereas ZO-1 was positively with TGF-ß1 (all p < 0.05). CONCLUSION: Nasal epithelial barrier dysfunction with TJs anomalies is commonly associated with abnormal proliferation and differentiation of epithelial cells and imbalance of immune and inflammatory patterns in NIP. LEVEL OF EVIDENCE: NA Laryngoscope, 134:552-561, 2024.

Molecular design and systematic optimization of a halogen-bonding system between the asthma interleukin-5 receptor and its cyclic peptide ligand.

Human interleukin-5 (IL-5) functions as an important pro-inflammatory factor by binding to its specific receptor, IL-5Rα, which has been implicated in the pathogenesis of asthma. Previously, a disulfide-bonded cyclic peptide AF17121 obtained from random library screening and sequence variation was found to competitively disrupt the cognate IL-5Rα/IL-5 interaction with moderate potency. In this study, the crystal complex of IL-5Rα with AF17121 was investigated at structural and energetic levels. It is revealed that the side-chain indole moiety of the AF17121 Trp5 residue is a potential site for a stem putative halogen bond (X-bond) with IL-5Rα, which is just located within the key 3 EXXR6 motif region recognized specifically by IL-5Rα. We systematically examined four halogen substitution types at five positions of the indole moiety; QM/MM calculations theoretically unraveled that only halogenations at 5 and 6 positions can form effective X-bonds with the side-chain hydroxyl oxygen of the IL-5Rα Thr21 residue and the backbone carbonyl oxygen of Ala66 residue, respectively. Binding assays observed that I-substitution at the 5 position and Br-substitution at the 6 position can result in two potent halogenated peptides, [5I]AF17121 and [6Br]AF17121, which are improved by 1.6-fold and 3.5-fold relative to the native AF17121, respectively. 5I/6Br-double substitution, resulting in [5I/6Br]AF17121, can further enhance the peptide affinity by 7.5-fold. Structural analysis revealed that the X-bond stemming from 6Br-substitution is also involved in an orthogonal interaction system with a H-bond; they share a common backbone carbonyl oxygen acceptor of IL-5Rα Ala66 residue and exhibit a significant synergistic effect between them.