Journal: Infection and Immunity

Article Title: Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii

doi: 10.1128/IAI.00376-19

Figure Lengend Snippet: SSChigh granulocytes express CD11b and the interleukin-5 receptor. BALB/c mice were vaccinated s.c. with 10 μg of PIV or PIIV, and spleens were processed for flow cytometry as previously described. Mice receiving Alhydrogel adjuvant alone served as unvaccinated controls. Naïve mice were used to establish baseline values. (A) Gating strategy. FSC and SSC were used to remove cell debris from the analysis. CD45+ leukocytes were then identified and further subgated based on SSC and expression of CD11b. CD11b+ SSChigh granulocytes were then further subgated based on expression of the interleukin-5 receptor alpha subunit (IL-5Rα, CD125). (B and C) Population frequencies and absolute cell counts were determined at 7 and 14 dpv. Each experimental group includes 4 to 6 mice, with error bars representing the standard deviation from the mean. *, P < 0.05; **, P < 0.01, as determined by one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Recombinant anti-mouse CD125 (IL-5Ra, clone REA343; conjugated to phycoerythrin [PE]), Siglec-F (clone REA798; PE-Vio770), and CD45R (B220, clone REA755; fluorescein isothiocyanate [FITC]) as well as rat anti-mouse CD19 (clone 6D5; FITC) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Adjuvant, Expressing, Standard Deviation, Comparison

Journal: Infection and Immunity

Article Title: Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii

doi: 10.1128/IAI.00376-19

Figure Lengend Snippet: SSChigh CD11b+ CD125+ cells are Siglec-F+ eosinophils. BALB/c mice were vaccinated s.c. with 10 μg of PIV or PIIV, and spleens were processed for flow cytometry. Mice receiving Alhydrogel adjuvant alone served as unvaccinated controls. Naïve mice were used to establish baseline values. (A) Gating strategy. SSChigh CD11b+ CD125+ cells were examined for expression of Siglec-F (eosinophils) and CD19/B220 (B cells). (B and C) Population frequencies and absolute cell counts were determined at 7 and 14 dpv. Each experimental group includes 4 to 6 mice, with error bars representing the standard deviations from the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as determined by one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Recombinant anti-mouse CD125 (IL-5Ra, clone REA343; conjugated to phycoerythrin [PE]), Siglec-F (clone REA798; PE-Vio770), and CD45R (B220, clone REA755; fluorescein isothiocyanate [FITC]) as well as rat anti-mouse CD19 (clone 6D5; FITC) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Adjuvant, Expressing, Comparison

Journal: Infection and Immunity

Article Title: Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii

doi: 10.1128/IAI.00376-19

Figure Lengend Snippet: Eosinophil accumulation in PIV-vaccinated mice is CD4+ T cell dependent. WT C57BL/6 and CD4-deficient mice were vaccinated s.c. with 10 μg of PIV, and 7 days later spleens were harvested for flow cytometry or ex vivo restimulation studies. Mice receiving Alhydrogel adjuvant alone served as unvaccinated controls. Naïve mice were used to establish baseline values. (A) SSChigh CD11b+ CD125+ Siglec-F+ eosinophils were enumerated using the gating strategy described in the legends for Fig. 4 and ​and5.5. (B) Total splenocytes were isolated and restimulated with medium or with 1 × 107 C. burnetii NMI bacteria for 3 and 6 days, and cell culture supernatants were evaluated for IFN-γ production by cytokine-specific ELISA. Each experimental group includes 5 mice, with error bars representing the standard deviations from the means. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as determined by one-way ANOVA with Tukey’s multiple-comparison test. Cb, C. burnetii; KO, knockout.

Article Snippet: Recombinant anti-mouse CD125 (IL-5Ra, clone REA343; conjugated to phycoerythrin [PE]), Siglec-F (clone REA798; PE-Vio770), and CD45R (B220, clone REA755; fluorescein isothiocyanate [FITC]) as well as rat anti-mouse CD19 (clone 6D5; FITC) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Ex Vivo, Adjuvant, Isolation, Bacteria, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison, Knock-Out

Journal: Respiratory Research

Article Title: IL-5 signaling in asthmatic derived fibroblasts exacerbates airway remodeling through ECM dysregulation and apoptosis resistance

doi: 10.1186/s12931-025-03371-x

Figure Lengend Snippet: IL-5 inducing the expression of IL-5Ra in lung derived fibroblasts. A mRNA levels of IL5RA in both normal and asthmatic derived lung fibroblasts. 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2. −ΔΔct . Data shown are the mean ± SD of four separate experiment; two-tailed paired student’s t-Test statistical analysis was done * p < 0.05, ** p < 0.01, *** p < 0.001 . B Cultured normal and asthmatic fibroblasts, captured at 40X magnification, display immunofluorescence staining for IL-5Rα (green), vimentin (red), and DAPI (blue) in both cell types, along with mouse and rabbit IgG isotype controls. C Evaluation of the mean fluorescence intensity (MFI) of IL-5Rα expression in lung fibroblasts, quantified following 24 h of IL-5 stimulation. Statistical significance was determined using two-tailed paired Student's t-test based on data from three independent experiments, * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: Subsequently, the cells were incubated overnight at 4 °C with primary antibodies against IL-5Rα (Cat: MAB253, R&D Systems) Vimentin (Cat: 5741, Cell Signalling Technology), Rabbit mAb IgG XP® Isotype Control (Cat: 3900, CST), Mouse IgG isotype control (Cat: MAB002, R&D systems) at a 1:100 dilution.

Techniques: Expressing, Derivative Assay, Control, Two Tailed Test, Cell Culture, Immunofluorescence, Staining, Fluorescence