Journal: Cellular and Molecular Immunology

Article Title: The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

doi: 10.1038/s41423-022-00873-2

Figure Lengend Snippet: p38α deletion in DCs promotes Th2 priming and allergic inflammation during the sensitization phase. a – c WT and p38α ΔDC mice were sensitized with HDM on Days 0–2 and challenged with HDM on Days 14–16. Mice were sacrificed for analysis on Day 17. ELISA analysis of IL-4, IL-5, IL-13, IL-17 and IFNγ production in the BALF ( a ) ( n = 3–4). The percentages of IL-4 + , IL-5 + , IL-13 + , IL-17 + and IFNγ + cells in CD4 + T cells were determined by intracellular staining ( b , c ) ( n = 13). Percentages of GATA3 + cells in CD4 + T cells ( d ) ( n = 5). e – h WT and p38α ΔDC mice were sensitized with HDM for 3 days and analyzed 7 days later. Total cell number in the BALF ( e ) ( n ≥ 14). The percentage and cell number of eosinophils were detected by flow cytometry ( f ) ( n ≥ 14). The percentages of IL-4 + , IL-5 + , IL-13 + , IL-17 + and IFNγ + cells in CD4 + T cells were determined by intracellular staining ( g ) ( n ≥ 19). ELISA analysis of ex vivo-isolated mLN cells restimulated with or without HDM for 72 h ( h ) ( n = 3–4). * P < 0.05; ** P < 0.01; ns, not significant. Data are representative of three ( a , d , h ) independent experiments or pooled from three ( b – c , e and f ) or four ( g ) experiments with consistent results. Student’s t test ( c – g ) or two-way ANOVA ( a and h ) was performed, and the data are presented as the mean ± SEM

Article Snippet: For intracellular staining (ICS), cells were stimulated with PMA (Sigma–Aldrich) and ionomycin (Sigma–Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 h and then stained according to the manufacturer’s instructions (BD Biosciences) with antibodies against ms IL-4 (11B11; eBioscience), ms IL-5 (TRFK5; BD Biosciences),ms IL-13 (eBio13A; eBioscience), ms IFNγ (XMG1.2; eBioscience), and ms IL-17 (eBio17B7; eBioscience).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Ex Vivo, Isolation

Journal: Cellular and Molecular Immunology

Article Title: The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

doi: 10.1038/s41423-022-00873-2

Figure Lengend Snippet: p38α signaling in cDC1s regulates Th2 responses upon HDM treatment. a Flow cytometry-sorted lung DCs from HDM-treated WT and p38α ΔDC mice were cocultured with naïve OT-II CD4 + T cells in the presence of OVA 323-339 for 72 h. ELISA analysis of IL-4, IL-5, IL-13, IL-17 and IFNγ production ( n = 3). b Analysis of Il4 , Il5 , Il13 , Gata3 , Il10 and Il9 mRNA expression in OT-II CD4 + T cells ( n = 4). c , d WT and p38α ΔcDC mice were sensitized and challenged with HDM to induce asthma ( n = 3). Percentages and cell numbers of eosinophils in the BALF and lung tissues ( c ). Percentages of IL-4, IL-5, IL-13 and IL-17 in lung CD4 + T cells ( d ). e mRNA expression of Il4 , Il13 , Gata3 and Tbx21 in OT-II CD4 + T cells activated by WT or p38α-deficient lung cDC1 or cDC2 subsets from HDM-treated mice in the presence of OVA 323-339 ( n = 2–3, 1 DC subset sample pooled from at least 3 mice). f and g WT and p38α ΔcDC1 mice were sensitized and challenged with HDM to induce asthma ( n = 3–7). Airway resistance was measured ( f ). The cell numbers of IL-4 + CD4 + T cells and IL-13 + CD4 + T cells in lung tissues were measured ( g ). h and i WT, p38α ΔDC , IRF8 ΔDC and IRF8/p38α ΔDC mice were sensitized and challenged with HDM to induce asthma ( n = 3–6). The numbers of eosinophils in the BALF and lung tissues ( h ) and the numbers of IL-4 + CD4 + T cells and IL-13 + CD4 + T cells in lung tissues were measured ( i ). * P < 0.05; ** P < 0.01; ns, not significant. Data are pooled from three ( a and b ) or two ( h and i ) experiments with consistent results or representative of three ( c – e ) or two ( f and g ) experiments with consistent results. Student’s t test ( a – d , g – i ) or two-way ANOVA ( e and f ) was performed, and the data are presented as the mean ± SEM

Article Snippet: For intracellular staining (ICS), cells were stimulated with PMA (Sigma–Aldrich) and ionomycin (Sigma–Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 h and then stained according to the manufacturer’s instructions (BD Biosciences) with antibodies against ms IL-4 (11B11; eBioscience), ms IL-5 (TRFK5; BD Biosciences),ms IL-13 (eBio13A; eBioscience), ms IFNγ (XMG1.2; eBioscience), and ms IL-17 (eBio17B7; eBioscience).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cellular and Molecular Immunology

Article Title: The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

doi: 10.1038/s41423-022-00873-2

Figure Lengend Snippet: p38α signaling in lung cDC1s regulates Th2-cell differentiation by modulating IL-12 expression. a GSEA of cDC1s. b IL-12p40 and ( c ) IL-12p35 expression in HDM-treated WT and p38α-deficient lung DC subsets analyzed by flow cytometry ( n = 4). d mRNA expression of Il4 and Il13 in CD4 + T cells activated with WT or p38α-deficient lung cDC1 or cDC2 subsets from HDM-treated mice with or without IL-12 for 3 days ( n = 2–3, 1 DC subset sample pooled from at least three mice). e – h WT and p38α ΔDC mice were sensitized with HDM on Days 0–2 and challenged with HDM on Days 14–16, and then IL-12p70 was i.n . administered during HDM treatment. Mice were sacrificed for analysis on Day 17 ( n = 3–4). Airway resistance was measured ( e ). Eosinophil infiltration in the BALF and lung tissues was detected by flow cytometry and quantified ( f and g ). The concentrations of IL-4, IL-5 and IL-13 in the BALF were detected by ELISA ( h ). ** P < 0.01; ns, not significant. Data are representative of three ( b – d and f – h ) or two ( e ) independent experiments. Student’s t test ( b , c , g and h ) or two-way ANOVA ( d and e ) was performed, and the data are presented as the mean ± SEM

Article Snippet: For intracellular staining (ICS), cells were stimulated with PMA (Sigma–Aldrich) and ionomycin (Sigma–Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 h and then stained according to the manufacturer’s instructions (BD Biosciences) with antibodies against ms IL-4 (11B11; eBioscience), ms IL-5 (TRFK5; BD Biosciences),ms IL-13 (eBio13A; eBioscience), ms IFNγ (XMG1.2; eBioscience), and ms IL-17 (eBio17B7; eBioscience).

Techniques: Cell Differentiation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Stimulation of a subset of natural killer T cells by CD103 + DC is required for GM-CSF and protection from pneumococcal infection

doi: 10.1016/j.celrep.2021.110209

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified Rat Anti-Mouse IL-2 , BD Biosciences , Cat# 554424, RRID:AB_395383.

Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Knock-In, Software

Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Genes differentially expressed in resident macrophages at 1 h postinfection with S. pyogenes

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Zinc-Fingers, Binding Assay

Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Confirmation of microarray data by RT-PCR and protein expression. (A) RT-PCR analysis of selected gene transcription in resident macrophages uninfected or infected with S. pyogenes. Uninfected samples were loaded in lanes 1, and infected samples were loaded in lanes 2. β-Actin expression served as a control. (B) IL-6 protein expression by S. pyogenes-infected macrophages. Resident macrophages were isolated from the peritoneal cavity of mice after 1 h of infection with S. pyogenes and cultured in vitro for 2 h. Noninfected macrophages were used as a control. The levels of IL-6 in the supernatants were determined by ELISA. Each column represents the mean ± standard deviation of triplicate samples obtained from three independent experiments. P < 0.0001 (ANOVA).

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Isolation, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation