Journal: Scientific Reports

Article Title: Tuning IL-2 signaling by ADP-ribosylation of CD25

doi: 10.1038/srep08959

Figure Lengend Snippet: Splenocytes from DEREG mice were incubated in the absence or presence of the indicated concentrations of NAD + before staining with fluorochrome-conjugated mAbs directed against CD4 and CD25 (mAb PC61 or mAb 7D4). Gating was performed on CD4 + cells. (a) Representative dot plots of cells incubated in the absence or presence of 12 μM NAD + . (b) Percentages of CD4 + GFP + cells staining with anti-CD25 mAbs plotted as a function of the concentration of added NAD + . Results are representative of two independent experiments.

Article Snippet: Alternatively, after erythrocyte lysis with Ack lysis buffer (Bio Whittaker) and depletion of B-cells with Dynabead-conjugated sheep anti-mouse IgG (Invitrogen), Tregs were positively selected by magnetic cell sorting using PE conjugated anti-CD25 (7D4) and magnetic bead-conjugated anti-PE antibodies according to the manufacturer's instructions (Miltenyi Biotec).

Techniques: Incubation, Staining, Concentration Assay

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: International journal of molecular sciences

Article Title: Inhibitory Effects of Extracellular Vesicles from iPS-Cell-Derived Mesenchymal Stem Cells on the Onset of Sialadenitis in Sjögren's Syndrome Are Mediated by Immunomodulatory Splenocytes and Improved by Inhibiting miR-125b.

doi: 10.3390/ijms24065258

Figure Lengend Snippet: Figure 3. Effects of iEVs on splenic Th17 cells. Four-month-old female NOD.B10.H2b mice were IV injected with PBS, PD15 iEVs, or PD45 iEVs twice a week for two weeks. Splenocytes were collected at two weeks after last injection. (A,B) Percentages of IL17+ cells in splenic CD4+ T cells were examined with flow cytometry. (C) The mRNA levels of Th17 markers (IL17a, IL21, and Rorc) and negative regulator IL1rn in spleen were examined with qRT-PCR. N = 5, ns: not significant, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: These cells were stained with fluorescent-labeled antibodies against F4/80 (BD Pharmingen 123116, 123107, San Diego, CA, USA), CD3 (BioLegend 100236, San Diego, CA, USA), CD19 (BioLegend 152409), CD38 (BioLegend 102707), CD206 (BioLegend 141706), CD4 (BioLegend 100408), IL17a (130-112-009, Miltenyi Biotec, San Diego, CA, USA), or the corresponding isotype controls (BioLegend 400608, 400511, 400612, 400207, 1:100).

Techniques: Injection, Cytometry, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Inhibitory Effects of Extracellular Vesicles from iPS-Cell-Derived Mesenchymal Stem Cells on the Onset of Sialadenitis in Sjögren's Syndrome Are Mediated by Immunomodulatory Splenocytes and Improved by Inhibiting miR-125b.

doi: 10.3390/ijms24065258

Figure Lengend Snippet: Figure 6. Effects of inhibiting miR-125b in aging iEVs on the polarization of splenic macrophages. Four-month-old female NOD.B10.H2b mice were IV injected with PBS, Ctrl EVs, or 125KD EVs twice a week for two weeks. Splenocytes were collected at two weeks after last injection. (A,B) Percentages of IL17+ cells in CD4+ splenocytes were examined with flow cytometry. (C) The mRNA levels of Th17 markers (IL17a, IL21, and Rorc) and negative regulator IL1rn in spleen were examined with qRT-PCR. N = 5, ns: not significant, *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

Article Snippet: These cells were stained with fluorescent-labeled antibodies against F4/80 (BD Pharmingen 123116, 123107, San Diego, CA, USA), CD3 (BioLegend 100236, San Diego, CA, USA), CD19 (BioLegend 152409), CD38 (BioLegend 102707), CD206 (BioLegend 141706), CD4 (BioLegend 100408), IL17a (130-112-009, Miltenyi Biotec, San Diego, CA, USA), or the corresponding isotype controls (BioLegend 400608, 400511, 400612, 400207, 1:100).

Techniques: Injection, Cytometry, Quantitative RT-PCR

Journal: Mucosal Immunology

Article Title: Butyrate regulates neutrophil homeostasis and impairs early antimicrobial activity in the lung

doi: 10.1016/j.mucimm.2023.05.005

Figure Lengend Snippet: Effect of butyrate on acute Pseudomonas aeruginosa immunity. (A) Quantification of the total number of cells in BALF 18 hours post-inoculation with 1 × 10 6 CFU of P. aeruginosa strain PAO1 in Ctrl and But mice. (B) Frequency and quantification of cell types in cytospins of BALF from control and butyrate-treated mice 18 hours after PAO1 infection; (C) Quantification of P. aeruginosa CFU in BALF 18 hours post-challenge. (D, E) Cytokine and chemokine production in BALF 18 hours following infection. (F) MFI of CXCR2 on lung AM and IM from Ctrl and But mice. (G) MFI of CXCR2 on Ctrl and But lung macrophages US or stimulated for 2 hours with LPS in vitro . (H) transmigration of Ctrl and But neutrophils toward a CXCL2 or CCL2 chemokine gradient after 3 hours. Results are a mean of two independent experiments. Values are expressed as mean ± standard error of mean; n = 6–14. Statistical significance was determined with one-way analysis of variance in (A–E, G, H) and Student’s t test (unpaired, two-tailed) in (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. AM = alveolar macrophages; BALF = bronchoalveolar lavage fluid; But = butyrate-treated; CFU = colony-forming units; Ctrl = control; IFN = interferon; IL = interleukin; IM = interstitial macrophages; LPS = lipopolysaccharide; MFI = mean fluorescence intensity; PAO1 = P. aeruginosa strain 1; TNF = tumor necrosis factor; US = unstimulated.

Article Snippet: To differentiate progenitor and neutrophil populations in the BM, cells were stained with antibodies to lineage cocktail, CD115 (BD Biosciences, clone 2B8), SiglecF (BD Biosciences, clone 2B8), CD117 BUV117 (BD Biosciences, clone 2B8), Sca1 APCFire (Biolegend, clone D7), FcgR biot (Miltenyi, clone 93), CD34 BV421 (Biolegend, clone SA376A4), CD11b BV510 (BD Biosciences, clone M1/70), Ly6G Pecy5 (eBioscience, clone 1A8-Ly6g), CXCR2 Pecy7 (Miltenyi, clone REA942) and streptavidin BUV395 (BD Biosciences).

Techniques: Control, Infection, In Vitro, Transmigration Assay, Two Tailed Test, Fluorescence

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: (A, E) Pretreatment with IL-35 neutralizing antibodies (anti-P35 or anti-Ebi3) aggravated LPS- and CLP-induced ARDS. Lungs from each experimental group were processed for histological examination after H&E staining. Compared with the LPS + IgG group, thickened alveolar wall, alveolar hemorrhage and collapse, inflammatory cells in filtration were more severe and pretreated with IL-35 neutralizing antibodies prior to LPS challenge. (B, D) Lung injury scores were estimated by the method of Mikawa, which is from the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. 0 marks: no or very slight damage, 1 marks: mild injury, 2 marks: moderate injury, 3 marks: severe injury, 4 marks: very severe damage, the cumulative increase in the number of lesions of the total score is the pathological score of the ARDS. Pretreatment with IL-35 neutralizing antibodies lungs injury were more severe than LPS + IgG group. **p < 0.01, ***p < 0.001, by the two-way ANOVA followed by LSD multiple comparisons test, compared with the LPS + IgG group.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Staining, Filtration, Membrane

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: Interleukin 35 (IL-35) blockade upregulated the production of proinflammatory cytokines and downregulated anti-inflammatory cytokines during ARDS models. Cytokine and chemokine concentrations in BALF and blood specimens from five mice treated with or without anti–Ebi3 or anti–P35 blocking antibodies were determined by mice cytokine/chemokine magnetic bead panel kit assays 24 h after onset of ARDS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by the one-way ANOVA followed by LSD multiple comparisons test, compared with mice treated with isotypical IgG control. ARDS featured increased CXCL-1, TNF-α, IL-6, and IL-17A and reduced IFN-γ, IL-10, IL-2, and IL-13 after neutralizing antibody treatment (anti-IL-35 Ebi3 or anti-IL-35 P35) in BALF and serum.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Blocking Assay, Control