Epithelial Damage Research Articles

Estrogen prevented gingival barrier injury from Porphyromonas gingivalis lipopolysaccharide.

The postmenopausal population usually suffers from more severe periodontal disease than non-menopausal women due to the decrease and low levels of estrogen, especially β-estradiol (E2). While additional estrogen therapy can effectively relieve alveolar bone resorption, this suggests that estrogen has played an important role in the development of periodontitis. The integrity of the gingival epithelial barrier plays a key role in protecting gingival tissue from inflammatory injury caused by pathogens. However, it remains unclear whether estrogen can maintain the integrity of the gingival epithelial barrier to reduce inflammatory injury. Here, using an infection model established with Porphyromonas gingivalis lipopolysaccharide (LPS) in human gingival epithelial cells (hGECs) and ovariectomized or Sham mice, we assessed the protective effect of estrogen on the gingival barrier using qPCR, western blotting, immunohistochemistry, and transcriptome analysis. The results showed that estrogen restored epithelial barrier function to inhibit P. gingivalis-LPS invasion and further downregulate the inflammatory reaction (P < 0.05) by upregulating expressions of tight junction proteins (such as JAM1 and OCLN) at mRNA and protein levels in both hGECs and ovariectomized or Sham mice (P < 0.05). Estrogen also protected against alveolar bone resorption and preserved barrier integrity in both ovariectomized and Sham mice (P < 0.05). In conclusion, E2 prevented the progression of gingival epithelial barrier damage and inflammation induced by P. gingivalis-LPS by increasing the expression of tight junction proteins. The protective effect of estrogen on gingival epithelial barrier injury highlighted its potential application in treating periodontitis and inflammatory diseases involving epithelial barrier dysfunction.

Efficacy and the underlying mechanisms of berberine in treatment of recurrent Clostridioides difficile infection.

Efficacy and the underlying mechanisms of berberine in treatment of recurrent Clostridioides difficile infection.

Escherichia coli HPI-induced duodenitis through ubiquitin regulation of the TLR4/NF-κB pathway

BackgroundThe Highly Pathogenic Island (HPI) found in Yersinia pestis can be horizontally transferred to E. coli, enhancing its virulence and pathogenicity. Ubiquitin (Ub) acts as an activator of the NF-κB pathway and plays a critical role in the inflammatory response. However, the precise mechanism by which Ub and the regulated TLR4/NF-κB pathway contribute to HPI-induced intestinal inflammation in E. coli remains unclear.ResultsIn this study, we established Ub overexpression models of small intestinal epithelial cells (in vitro) and BALB/c mice (in vivo) and infected these models with HPI-rich E. coli. We investigated the role of the Ub-regulated TLR4/NF-κB pathway in E. coli HPI-induced intestinal inflammation through qPCR, ELISA, immunofluorescence, immunohistochemistry, and H&E staining. Our findings confirmed that E. coli HPI promoted the expression of Ub, TLR4, and NF-κB in IPEC-J2 cells and induced the translocation of NF-κB p65 protein to the nucleus. Further investigations revealed that Ub overexpression enhanced epithelial cell damage induced by E. coli HPI. This was accompanied by up-regulation of mRNA levels of TLR4, MyD88, NF-κB, IL-1β, and TNF-α, as well as increased release of the inflammatory factors IL-1β and TNF-α. In a mouse model with Ub overexpression infected with E. coli HPI, we observed that Ub overexpression promoted E. coli HPI-induced intestinal inflammation. Mechanistically, E. coli HPI induced intestinal epithelial cell damage by inducing Ub overexpression and modulating the TLR4/NF-κB pathway.ConclusionsIn conclusion, this study sheds light on the significant role of the Ub-regulated TLR4/NF-κB pathway in E. coli HPI-induced duodenitis, offering novel insights into the pathogenesis of E. coli infections.

Preparation and characterization of multilayered microcapsules of Lactobacillus rhamnosus encapsulated with sodium alginate, hyaluronic acid and carrageenan and their protective effects on the retina.

Preparation and characterization of multilayered microcapsules of Lactobacillus rhamnosus encapsulated with sodium alginate, hyaluronic acid and carrageenan and their protective effects on the retina.

Gut commensal Alistipes shahii improves experimental colitis in mice with reduced intestinal epithelial damage and cytokine secretion.

The commensal bacterium Alistipes shahii is a core microbe of the human gut microbiome and its abundance is negatively correlated with inflammatory bowel diseases (IBDs). However, its fundamental role in regulating inflammatory response remains unknown. Using a dextran sulfate sodium (DSS)-induced colitis mouse model, we examined the effect of A. shahii strain As360 intervention on host inflammatory response and found that A. shahii As360 alleviated disease activity index, colon shortening, and colonic histopathological lesion. The levels of tight junction proteins (mainly ZO1 and claudin-1) were decreased in DSS-induced colitis mice, whereas the levels of these proteins were elevated in colitis mice with A. shahii As360 treatment. In addition, A. shahii As360 treatment led to alterations in cytokine release, especially an increase of IL10. It also led to reduced expressions of mtor and Nlrp3 and increased expression of mTOR inhibitor Ddit4 at the transcriptional level. 16S rRNA amplicon sequencing found that Bacteroides, a producer of short-chain fatty acids (SCFAs), was enriched in the fecal samples of mice with A. shahii treatment. Metabolic analyses found that, following A. shahii As360 treatment, the SCFAs in the fecal content was increased whereas lactic acid was decreased in the cecal content. These findings suggest that supplementation with A. shahii As360 is a promising strategy to prevent colitis.IMPORTANCEAs one of the core microbes and keystone species in the human gut, Alistipes shahii has the potential to inhibit inflammation and improve inflammatory bowel diseases (IBDs) conditions. In this study, we experimentally demonstrated that oral administration of A. shahii As360 alleviated symptoms of colitis, altered the release of cellular inflammatory factors, reduced the intestinal epithelial barrier damage, and changed gut microbiota and fecal metabolites. These findings provide a deeper understanding of the beneficial effects of A. shahii and its perspective for better strategies to prevent IBD.

A25 INVESTIGATING THE ROLE OF TOLL-INTERACTING PROTEIN DURING CITROBACTER RODENTIUM INFECTION

Abstract Background Diarrheal diseases are a threat to human health, being the second leading cause of death in children. Diarrheic enterocolitis can be caused by attaching/effacing (A/E) pathogens like enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). Citrobacter rodentium (CR), a mouse A/E pathogen, mimics EPEC and EHEC infections, causing goblet cell depletion, crypt hyperplasia, and leukocyte infiltration. Autophagy, a critical immune pathway linked to goblet cell mucus secretion, is involved in the defence against CR. Toll-interacting protein (Tollip) is an intracellular mediator of autophagy and immunity, but its role in A/E infections remains elusive. This study aims to test the hypothesis that Tollip plays a protective role in CR infection. Aims - Assess CR infection kinetics in WT and Tollip-/- mice. - Assess colitis in CR-infected WT and Tollip-/- mice. Methods Bacterial load was assessed by fecal colony forming unit (CFU) count in CR-infected WT and Tollip-/- mice. Histological damage was evaluated by scoring epithelial damage, leukocyte infiltration and crypt hyperplasia in H&amp;E-stained distal colon sections. Apoptotic cells were identified by TUNEL. Acid and neutral mucin production was evaluated by Alcian-PAS staining. Glycosylation patterns were determined by immunofluorescence with lectins specific to sialic acid- and fucose-bound mucins. Results Tollip -/- and WT mice showed similar infection kinetics during the establishment and expansion phases (2-6 days post-infection (dpi)) and steady-state (7-14 dpi), with a peak of bacterial counts at 10 dpi. WT mice cleared CR by day 15, but Tollip-/- still shed bacteria by 17 dpi. The colitis grade was similar between WT and Tollip-/- mice, with marked epithelial damage at 8 dpi and increased leukocyte infiltration at 17 dpi. Scarce apoptotic cells (&amp;lt;4 per field) were detected in WT and Tollip-/- mice during infection. Tollip-/- showed a thinner colonic mucin barrier and less mucin-filled goblet cells at 17 dpi compared to WT mice. CR-infected Tollip-/- mice showed altered mucin glycosylation patterns, with lower fucosylated mucins in the upper crypt and lumen and increased sialic acid-bound mucins in the luminal barrier and lower crypt. The mucin barrier, goblet cells, and mucin glycosylation were similar in uninfected Tollip-/- and WT mice. Conclusions This study demonstrates a new protective role for Tollip, particularly during the resolution phase, as a lack of Tollip delayed the clearance of CR. This phenotype could be explained by deficiencies in the mucin layer observed in Tollip-/- mice, evidenced by lower mucin production and anomalies in mucin glycosylation. By enhancing our understanding of host-pathogen interactions during enteric infections, this research serves as a step in the development of new treatments and prevention strategies. Funding Agencies None

A195 MALNUTRITION IMPAIRS WOUND HEALING DURING DSS COLITIS AND AN IN-VITRO EPITHELIAL MIGRATION ASSAY

Abstract Background Malnutrition is a major public health burden. Caused by insufficient intake of food/nutrients, malnutrition often stems from poverty and lack of access to food. Malnutrition is also a common complication of many gastrointestinal diseases, including inflammatory bowel disease (IBD). Reportedly, more than half of all IBD patients suffer from micronutrient or macronutrient malnutrition, resulting from malabsorption, low nutrient intake, and excessive bleeding or diarrhea. Nutritional deficiencies can alter the course of IBD, prolong hospitalizations, alter treatment response, and delay wound healing. Notably, epithelial wound healing is critical in IBD, but efficient re-epithelization rely on nutrients such as zinc and vitamin A. I thus hypothesize that malnutrition will impair intestinal epithelial wound repair, in vivo and in vitro. Aims We tested how malnutrition affects wound healing following epithelial damage caused by dextran sulphate sodium (DSS)-induced colitis and in a 2D monolayer gap closure assay. Methods We used our previously published mouse model of multiple micronutrient deficiencies (MND) and induced colitis using dextran sodium sulfate (DSS). Weanling three-week-old male C57BL/6N mice were fed a diet deficient in (zinc, vitamin A, B12, folate and iron) or a control diet for 4 weeks. At 3 weeks, we induced colitis by administering 2.5% DSS in drinking water for 4 days, plus 3 days of water recovery. We investigated epithelial damage, wound repair and molecular responses through histological and gene expression profiling. We also generated 3D colonoids from non-colitic malnourished mice, converted them to a 2D monolayer and subjected them to a wound migration assay cultured using an established malnourished media containing standard nutrients, and 50% of normal levels. Results We found that while DSS induced tissue damage both in our control and MND mice, those on the MND diet exhibited impaired tissue repair with decreased re-epithelization over the wounded area compared to controls. Organoids cultured in the 50% malnourished media showed diminished growth, being smaller and less dense compared to standard media. Notably, epithelial migration towards gap closure in the 2D monolayer was delayed by 75% compared to standard media. Conclusions Our work demonstrates that malnutrition impairs intestinal wound repair mechanisms. We also demonstrate an effective organoid model for examining the effects of malnutrition in vitro. We propose that efforts to address underlying malnutrition may improve wound healing leading to better outcomes for patients with IBD. Funding Agencies CIHRTRIANGLE/Crohn’s and Colitis Canada, BC Children’s Hospital Research Foundation

Hollow cerium nanoparticles synthesized by one-step method for multienzyme activity to reduce colitis in mice

BACKGROUND Inflammatory bowel disease (IBD) is a common chronic intestinal inflammatory disease. High oxidative stress is a treatment target for IBD. Cerium oxide (CeO2) nanomaterials as nanozymes with antioxidant activity are potential drugs for the treatment of colitis. AIM To synthesize hollow cerium (H-CeO2) nanoparticles by one-step method and to validate the therapeutic efficacy of H-CeO2 in IBD. METHODS H-CeO2 was synthesized by one-step method and examined its characterization and nanoenzymatic activity. Subsequently, we constructed dextran sulfate sodium (DSS)-induced colitis in mice to observe the effects of H-CeO2 on colonic inflammation. The effects of H-CeO2 on colon inflammation and reactive oxygen species (ROS) levels in IBD mice were detected by hematoxylin and eosin staining and dichlorofluorescein diacetate staining, respectively. Finally, the biological safety of H-CeO2 on mice was evaluated by hematoxylin and eosin staining, blood routine, and blood biochemistry. RESULTS H-CeO2 nanoparticles prepared by the one-step method were uniform, monodisperse and hollow. H-CeO2 had a good ability to scavenge ROS, ∙OH and ∙OOH. H-CeO2 reduced DSS-induced decreases in body weight and colon length, colonic epithelial damage, inflammatory infiltration, and ROS accumulation. H-CeO2 administration reduced the disease activity index of DSS-induced animals from about 8 to 5. H-CeO2 had no significant effect on body weight, total platelet count, hemoglobin, white blood cell, and red blood cell counts in healthy mice. No significant damage to major organs was observed in healthy mice following H-CeO2 administration. CONCLUSION The one-step synthesis of H-CeO2 nanomaterials had good antioxidant activity, biosafety, and inhibited development of DSS-induced IBD in mice by scavenging ROS.

Akkermansia muciniphila identified as key strain to alleviate gut barrier injury through Wnt signaling pathway.

As the largest mucosal surface, the gut has built a physical, chemical, microbial, and immune barrier to protect the body against pathogen invasion. The disturbance of gut microbiota aggravates pathogenic bacteria invasion and gut barrier injury. Fecal microbiota transplantation (FMT) is a promising treatment for microbiome-related disorders, where beneficial strain engraftment is a significant factor influencing FMT outcomes. The aim of this research was to explore the effect of FMT on antibiotic-induced microbiome-disordered (AIMD) models infected with enterotoxigenic Escherichia coli (ETEC). We used piglet, mouse, and intestinal organoid models to explore the protective effects and mechanisms of FMT on ETEC infection. The results showed that FMT regulated gut microbiota and enhanced the protection of AIMD piglets against ETEC K88 challenge, as demonstrated by reduced intestinal pathogen colonization and alleviated gut barrier injury. Akkermansia muciniphila (A. muciniphila) and Bacteroides fragilis (B. fragilis) were identified as two strains that may play key roles in FMT. We further investigated the alleviatory effects of these two strains on ETEC infection in the AIMD mice model, which revealed that A. muciniphila and B. fragilis relieved ETEC-induced intestinal inflammation by maintaining the proportion of Treg/Th17 cells and epithelial damage by moderately activating the Wnt/β-catenin signaling pathway, while the effect of A. muciniphila was better than B. fragilis. We, therefore, identified whether A. muciniphila protected against ETEC infection using basal-out and apical-out intestinal organoid models. A. muciniphila did protect the intestinal stem cells and stimulate the proliferation and differentiation of intestinal epithelium, and the protective effects of A. muciniphila were reversed by Wnt inhibitor. FMT alleviated ETEC-induced gut barrier injury and intestinal inflammation in the AIMD model. A. muciniphila was identified as a key strain in FMT to promote the proliferation and differentiation of intestinal stem cells by mediating the Wnt/β-catenin signaling pathway.

Akkermansia muciniphila identified as key strain to alleviate gut barrier injury through Wnt signaling pathway

As the largest mucosal surface, the gut has built a physical, chemical, microbial, and immune barrier to protect the body against pathogen invasion. The disturbance of gut microbiota aggravates pathogenic bacteria invasion and gut barrier injury. Fecal microbiota transplantation (FMT) is a promising treatment for microbiome-related disorders, where beneficial strain engraftment is a significant factor influencing FMT outcomes. The aim of this research was to explore the effect of FMT on antibiotic-induced microbiome-disordered (AIMD) models infected with enterotoxigenic Escherichia coli (ETEC). We used piglet, mouse, and intestinal organoid models to explore the protective effects and mechanisms of FMT on ETEC infection. The results showed that FMT regulated gut microbiota and enhanced the protection of AIMD piglets against ETEC K88 challenge, as demonstrated by reduced intestinal pathogen colonization and alleviated gut barrier injury. Akkermansia muciniphila (A. muciniphila) and Bacteroides fragilis (B. fragilis) were identified as two strains that may play key roles in FMT. We further investigated the alleviatory effects of these two strains on ETEC infection in the AIMD mice model, which revealed that A. muciniphila and B. fragilis relieved ETEC-induced intestinal inflammation by maintaining the proportion of Treg/Th17 cells and epithelial damage by moderately activating the Wnt/β-catenin signaling pathway, while the effect of A. muciniphila was better than B. fragilis. We, therefore, identified whether A. muciniphila protected against ETEC infection using basal-out and apical-out intestinal organoid models. A. muciniphila did protect the intestinal stem cells and stimulate the proliferation and differentiation of intestinal epithelium, and the protective effects of A. muciniphila were reversed by Wnt inhibitor. FMT alleviated ETEC-induced gut barrier injury and intestinal inflammation in the AIMD model. A. muciniphila was identified as a key strain in FMT to promote the proliferation and differentiation of intestinal stem cells by mediating the Wnt/β-catenin signaling pathway.

Neutralizing IL-15 Inhibits Tissue-Damaging Immune Response in Ex Vivo Cultured Untreated Celiac Intestinal Mucosa.

In celiac disease (CeD), interleukin 15 (IL-15) affects the epithelial barrier by acting on intraepithelial lymphocytes, promoting interferon γ (IFN-γ) production and inducing strong cytotoxic activity as well as eliciting apoptotic death of enterocytes by the Fas/Fas ligand system. This study investigates the effects of a monoclonal antibody neutralizing the effects of IL-15 (aIL-15) on tissue-damaging immune response in untreated CeD patients by using an organ culture system. Jejunal biopsies from 10 untreated CeD patients were cultured ex vivo with or without aIL-15. Epithelial expressions of CD95/Fas, HLA-E and perforin were analyzed by immunohistochemistry. Apoptosis was detected in the epithelium by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Additionally, the surface epithelium compartment of ex vivo cultured biopsy samples was isolated by laser capture microdissection (LCM). RNA from each LCM sample was extracted and the relative expression of IFN-γ was evaluated by quantitative reverse transcriptase-PCR (qRT-PCR). Biopsies cultured with the aIL-15 antibody showed a reduction in Fas, HLA-E and perforin epithelial expression, as well as a decrease in epithelial TUNEL+ cells compared to biopsies cultured without the aIL-15 antibody. Moreover, downregulation of epithelial IFN-γ expression was recorded in biopsies incubated with aIL-15, compared to those cultured without aIL-15. Our findings suggest that neutralizing the effects of IL-15 in ex vivo cultured untreated CeD intestinal mucosa could block apoptosis by downregulating Fas and HLA-E expression and the release of cytotoxic proteins, such as perforin. Furthermore, it can dampen the hyperactive immune response by reducing IFN-γ expression. More generally, our study provides new evidence for the effects of anti-IL-15 neutralizing monoclonal antibodies in preventing or repairing epithelial damage and further supports the concept that IL-15 is a meaningful therapeutic target in CeD, or inflammatory diseases associated with the upregulation of IL-15.

Cloned airway basal progenitor cells to repair fibrotic lung through re-epithelialization

Irreversible damage of the lung epithelium in idiopathic pulmonary fibrosis (IPF) patients causes high mortality worldwide, with no lung repair approaches available currently. Here we show that in murine and monkey models, the KRT5+ P63+ progenitor cells in airway basal layer can enter the alveolar area post fibrotic injury. Aided with an automated culture system, we clone and characterize airway basal progenitor cells from 44 donors with various lung conditions. Transplantation of human progenitor cells into the mouse lung efficiently re-epithelializes the injured alveolar area, forms new respiratory tract and saccule-like structures, which ameliorates fibrotic lesions and improves survival of mice. Mechanistically, the engrafted human progenitor cells do not function by differentiating into mature alveolar cells in mouse lung; instead, they differentiate into saccular cells expressing multiple tight junction proteins such as CLDN4, which help the lung to re-establish epithelial barriers. Furthermore, by cloning P63+ airway basal progenitors from larger mammals and birds, we construct multiple lung-chimerism animals and uncover the evolutionarily conserved roles of these progenitor cells in lung repair. Overall, our data highlight the fate of airway basal progenitor cells in fibrotic lung and provide a potential therapeutic strategy for pulmonary diseases that lack inherent recovery mechanisms.

Cytolethal distending toxin-producing Escherichia coli clinical isolates from Mexican children harbor different cdt types causing CDT-induced epithelial pathological phenotypes

Cytolethal distending toxins (CDTs), encoded by cdtABC genes, have DNase activity leading to cellular and nuclear distention, resulting in actin remodeling, irreversible cell cycle arrest and apoptosis of target cells. PCR cdt-positive Escherichia coli strains have been isolated from children with diarrhea worldwide. However, toxin production and biological activity of cdt+ strains are rarely confirmed. Here, we characterized the biological activity of cdt+E. coli of clinical isolates from Mexican children with severe diarrhea and its relationship with the harbored cdt type. Ten isolates from seven patients containing cdt+E. coli, one isolate from a patient containing cdt− E. coli, and a prototype CDT-producing E. coli were used to determine the harbored cdt-type, cell distention, actin remodeling and cell cycle arrest on epithelial cells. Three isolates harbored cdt type I, one type II, two type III, two type IV and two simultaneously type II and III. Lysates from eight cdt+E. coli isolates caused cell distention, actin cytoskeletal remodeling and cell cycle arrest but two isolates from the same patient harboring simultaneously cdt type II/III did not. The cdt genes were necessary and enough to cause the cytolethal distending pathology. Mutants in cdtABIC (O86:H34 strain; cdt-I) and cdtABIIC (isolate; cdt-II) were complemented by cdtABIIC genes and both recovered the CDT-induced phenotypes. Transformation of E. coli BL21 by cdtABIIC genes caused this cytolethal distending pathology. These data indicate that cdt + E. coli isolates are potentially dangerous bacteria to cause serious epithelial cell damage and cell death to aggravate childhood diarrhea.

Anti-ulcerogenic activity of the marine-pearl derived medicine mukta Pishti in Rat model of pylorus ligation-induced peptic ulcer.

Anti-ulcerogenic activity of the marine-pearl derived medicine mukta Pishti in Rat model of pylorus ligation-induced peptic ulcer.

Active components unveiling and pharmacodynamic research on Valeriana jatamansi jones for ameliorating ulcerative colitis based on pharmacokinetics and network pharmacology.

Active components unveiling and pharmacodynamic research on Valeriana jatamansi jones for ameliorating ulcerative colitis based on pharmacokinetics and network pharmacology.

Exploring the Mechanism of Action and Potential Targets of Saorilao-4 Decoction in the Treatment of Pulmonary Fibrosis in Rats by Metabolomics.

Pulmonary fibrosis (PF) is a chronic progressive disease marked by alveolar epithelial cell damage. Saorilao-4 decoction (SRL), a traditional Mongolian prescription, has demonstrated therapeutic effects on PF, though its mechanism of action remains elusive. This study used a bleomycin-induced fibrosis rat model to evaluate SRL's effects by measuring inflammatory factors, assessing fibrosis-related indices, and performing histopathological lung examinations. Serum metabolite levels in the experimental groups were measured using high-performance liquid chromatography coupled with mass spectrometry. Data analysis involved principal component and partial least-squares discriminant analyses, followed by functional enrichment analysis of differential metabolites. SRL significantly ameliorated alveolar interstitial injury, fibrosis, and metabolic disorders induced by bleomycin. Additionally, we identified 71 metabolic components related to PF progression, including sphingolipids and fatty acids. Administration of SRL affected 59 metabolic components involved in purine, cysteine and methionine, and arginine and proline metabolisms. Specifically, SRL regulated the levels of hexadecanoic acid, S-adenosylmethionine, 3-oxopalmitoyl coenzyme A, and dodecanoic acid metabolites, thereby improving the metabolic course of PF. In conclusion, this study offers insights into the potential mechanisms of SRL in treating PF from a metabolomics perspective. It provides valuable information for its clinical application.

Epigallocatechin gallate ameliorates retinal pigment epithelial cell damage via the CYFIP2 /AKT pathway.

Epigallocatechin gallate ameliorates retinal pigment epithelial cell damage via the CYFIP2 /AKT pathway.

Markers of Environmental Enteric Dysfunction Are Associated with Poor Growth and Developmental Outcomes among Young Children in Lusaka, Zambia

Markers of Environmental Enteric Dysfunction Are Associated with Poor Growth and Developmental Outcomes among Young Children in Lusaka, Zambia

The inflammatory bowel disease and gut microbiome are restored by employing metformin-loaded alginate-shelled microcapsules.

The inflammatory bowel disease and gut microbiome are restored by employing metformin-loaded alginate-shelled microcapsules.

Effect of Mepolizumab in airway’s remodelling in patients with late-onset severe asthma with an eosinophilic phenotype

Effect of Mepolizumab in airway’s remodelling in patients with late-onset severe asthma with an eosinophilic phenotype