Journal: Frontiers in Allergy

Article Title: IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol

doi: 10.3389/falgy.2022.1062412

Figure Lengend Snippet: E2 treatment reduces IL-33 mediated IL-5 and IL-13 expression in ILC2. ( A ) Flow cytometry gating strategy of ILC2 defined as live, singlet cells lacking lineage markers (CD11b, CD11c) and expressing KLRG-1, CD127 and GATA3. Cytokine expression by ILC2 in Male-sham, female-sham, and estrogen-treated OVX mice is shown. ( B,C ) Concentration of IL-5-producing ILC2 and the mean fluorescence intensity of IL-5 measured by flow cytometry. ( D,E ) il5 and il13 mRNA expression by ILC2 measured by qRT-PCR. ( F,G ) IL-5 and IL-13 protein expression measured by ELISA. Error bars denote means ± SEM ( n = 6–8 mice/group). Data from two experiments are shown. Groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lung tissue was dissociated and ILC2 enrichment was performed with the EasySep Mouse ILC2 Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Expressing, Flow Cytometry, Concentration Assay, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Allergy

Article Title: IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol

doi: 10.3389/falgy.2022.1062412

Figure Lengend Snippet: Broad screening of mRNA levels of mediators of the NF-κB pathway in E2-treated ILC2. ILC2 were sorted from the lungs of Male, female, and estrogen-treated OVX mice that had been sensitized and challenged with OVA (OVX-E2). Purified ILC2 were cultured with IL-2, IL-7 and IL-33 for 5 days and NF-κB pathway components gene expression was measured using qRT-PCR. ( A ) Heatmap shows fold-change gene expression of relA , relB , NFκB1 , NFκB2 , cRel , IkBkb1 and chuk . ( B ) Counts of ILC2 with phosphorylated p65 and the mean fluorescence intensity of phosphorylated p65 measured by flow cytometry. Groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lung tissue was dissociated and ILC2 enrichment was performed with the EasySep Mouse ILC2 Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Purification, Cell Culture, Expressing, Quantitative RT-PCR, Fluorescence, Flow Cytometry

Journal: Frontiers in Allergy

Article Title: IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol

doi: 10.3389/falgy.2022.1062412

Figure Lengend Snippet: Single cell-RNA seq reveals differentially expressed genes in Male and female lung ILC2. ( A ) UMAP visualization shows 11 clusters of ILC2, ( B ) heat map shows top two DEG in each cluster ( C ) UMAP visualization shows sex differences in lung ILC2, ( D ) Violin plot of myd88 , il13 , il5 , nfkb2 , gata3 and nfkb1 I genes, and ( E ) heat map of top 20 highly significantly differentially expressed genes in Male and female lung ILC2.

Article Snippet: Lung tissue was dissociated and ILC2 enrichment was performed with the EasySep Mouse ILC2 Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: RNA Sequencing Assay

Journal: Frontiers in Allergy

Article Title: IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol

doi: 10.3389/falgy.2022.1062412

Figure Lengend Snippet: Differentially expressed genes in male and female ILC2.

Article Snippet: Lung tissue was dissociated and ILC2 enrichment was performed with the EasySep Mouse ILC2 Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Expressing

Journal: Frontiers in Allergy

Article Title: IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol

doi: 10.3389/falgy.2022.1062412

Figure Lengend Snippet: Gene ontology, pathway and TRRUST analysis reveals differences in male and female ILC2 ( A ) gene ontology analysis of top 20 DEG in male and female ILC2 using enrichr GO biological process 2021, ( B ) bioplanet pathway analysis of top 100 DEG using enrichr ( C ) TRRUST analysis of top 100 DEG in male and female ILC2 isolated following challenge with alternaria using metascape, bar graph are sorted by p -value ranking.

Article Snippet: Lung tissue was dissociated and ILC2 enrichment was performed with the EasySep Mouse ILC2 Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Isolation

Journal: Frontiers in Allergy

Article Title: IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol

doi: 10.3389/falgy.2022.1062412

Figure Lengend Snippet: Genes associated with ILC2 phenotype are reduced with estrogen treatment in males and females. Male (black circles) and female (maroon circles) ILC2 were sorted from the lungs and cultured with titrated amounts of E2 and IL-2 and IL-7 with IL-33 for 5 days. ( A ) il-5 , ( B ) il-13 , ( C ) areg and ( D ) gata3 mRNA expression was quantified via qRT-PCR. Error bars denote means ± SEM ( n = 8–10 mice/group). Data from two experiments are shown. Groups were compared using two-way ANOVA and Tukey's multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (“#” symbolizes comparison of male vs. female group using Mann-Whitney test, # p = 0.028).

Article Snippet: Lung tissue was dissociated and ILC2 enrichment was performed with the EasySep Mouse ILC2 Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Frontiers in Allergy

Article Title: IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol

doi: 10.3389/falgy.2022.1062412

Figure Lengend Snippet: NF-κβ pathway components are reduced in ILC2 by E2 treatment. Male (black circle) and female (maroon circle) ILC2 were sorted from the lungs and cultured with titrated amounts of E2 and IL-2 and IL-7 with IL-33 for 5 days. ( A–G ) Components of the NF-κB pathway ( A ) NFκβ1 (p50) ( B ) NFκβ2 (p52) , ( C ) MyD88 , ( D ) relA , ( E ) relB , ( F ) cRel and ( G ) IκBa mRNA were evaluated for responsiveness to low 2.5 pg/ml and moderate dose (25 pg/ml) estrogen in combination with IL-33 treatment. Groups were compared using one-way ANOVA and Tukey's multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lung tissue was dissociated and ILC2 enrichment was performed with the EasySep Mouse ILC2 Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Cell Culture