Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: A Illustration of experimental timeline and outcome measures. B Lung lesion scores of vaccinated-then-challenged animals. Bronchoalveolar lavage fluid (BALF) concentrations of C TNF-α, D IL-1β, E IL-6, F IL-17A, and G KC in vaccinated-then-challenged animals. Positive correlations between disease severity (lung lesion scores) and BALF H IL-17A, and I KC concentrations. * p < 0.5, ** p < 0.1, *** p < 0.01, **** p < 0.001. Error bars for B indicate median and interquartile range and mean and SEM for C – G . Dotted lines for linear regression graphs indicate 95% confidence intervals. Each point represents data from an individual animal. Nonparametric lesion score data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cytokine concentration data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons. Linear regression was utilized to establish correlations.

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Concentration Assay

Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: Representative H&E stained lung sections ( A , C , E , G , I , K ) and RNAScope in situ hybridization processed slides staining IL-17A transcript (blue) and CD4 transcript (red) ( B , D , F , H , J , L ) from Sham-vaccinated/Mp-challenged animals (top), LAMPs-vaccinated/Mp-challenged animals (middle) and dLAMPs-vaccinated/Mp-challenged animals (bottom). Scale bars indicate 500 um (4x) or 100 um (10x).

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Staining, RNAscope, In Situ Hybridization

Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: Percentage ( A , E , I ) and number ( B , F , J ) of IL-17A positive cells when analyzing all live-single-cells (top), lymphocyte-like live-single cells (middle) and other-cells (bottom). Overlaid histograms ( C , G , K ) of cell count vs IL-17A signal on all live-single-cells (top), lymphocyte-like live-single cells (middle) and other-cells (bottom). Dot-plots ( D , H , L ) of Forward Scatter area vs IL-17A signal on all live-single-cells (top), lymphocyte-like live-single cells (middle) and other-cells (bottom). * p < 0.5, ** p < 0.1, *** p < 0.01, **** p < 0.001. Error bars indicate mean and SEM. Each point represents data from an individual animal. Data from single representative animals from each vaccination group are shown on histograms and dot-plots. Nonparametric percent frequency data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cell count data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons.

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Cell Counting

Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: H&E stained lung sections (left) and RNAScope in situ hybridization processed slides staining IL-17A transcript (blue) and CD4 transcript (red) (right) displaying vessels and airways to show that CD4 mRNA and IL-17A mRNA co-localization was more frequent in the areas of perivascular cuffing.

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Staining, RNAscope, In Situ Hybridization

Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: IL-17A producing CD3 + CD4 + cells as a percent of IL-17A positive lymphocyte-like cells ( A ) and raw counts per 50k analyzed events ( E ). IL-17A producing CD3 + CD4- cells as a percent of IL-17A positive lymphocyte-like cells ( B ) and raw counts per 50k analyzed events ( F ). IL-17A producing CD3-CD4 + cells as a percent of IL-17A positive lymphocyte-like cells ( C ) and raw counts per 50k analyzed events ( G ). IL-17A producing CD3-CD4- cells as a percent of IL-17A positive lymphocyte-like cells ( D ) and raw counts per 50k analyzed events ( E ). * p < 0.5, ** p < 0.1, *** p < 0.01, **** p < 0.001. Error bars indicate mean and SEM. Each point represents data from an individual animal. Nonparametric percent frequency data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cell count data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons.

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Cell Counting

Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: A Illustration of experimental timeline and outcome measures. B Numbers of lung-infiltrating leukocytes, proportion C and numbers D of lung-infiltrating neutrophils in vaccinated-then-challenged animals. E Positive correlations between lung-infiltrating neutrophil proportions and Lung Lesion Scores. F – I Correlations between proportions and numbers of lung-infiltrating neutrophils and IL-17A and KC concentrations. * p < 0.5, ** p < 0.1, *** p < 0.01, **** p < 0.001. Error bars for B – D indicate mean and SEM. Dotted lines for linear regression graphs indicate 95% confidence intervals. Each point represents data from an individual animal. Nonparametric percent frequency/proportion data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cell count data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons. Linear regression was utilized to establish correlations.

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Cell Counting

Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: A Illustration of experimental timeline and outcome measures. BALF concentrations of B IL-17A, C TNF-α, D IL-1β, E IL-6, and F KC in LAMPs-vaccinated/ Mp -challenged animals receiving an anti-IL-17A neutralizing monoclonal antibody (17F3) or isotype control (MOPC-21). BALF numbers of lung-infiltrating leukocytes ( G ), and proportion of H and number of ( I ) lung-infiltrating neutrophils. J Lung Lesion Scores and K bacterial loads of vaccinated-then-challenged animals treated with anti-IL-17A antibody or isotype control. * p < 0.5, ** p < 0.1, *** p < 0.01, **** p < 0.001. Error bars for J and K indicate median and interquartile range and mean and SEM for B – I . Each point represents data from an individual animal. Nonparametric lesion score, bacterial burden and percent frequency/proportion data were analyzed via an unpaired, two-tailed Mann–Whitney U -test. Parametric cytokine concentration and cell count data were analyzed via an unpaired, two-tailed t -test.

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Control, Two Tailed Test, MANN-WHITNEY, Concentration Assay, Cell Counting

Journal: NPJ Vaccines

Article Title: Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

doi: 10.1038/s41541-022-00513-w

Figure Lengend Snippet: Anamnestic reactivation of IL-17A recall responses in LAMPs-vaccinated/ Mp -challenged animals results in the further production of TNF-α, IL-1β, IL-6, and KC. TNF-α and IL-1β can further induce the expression of the neutrophil chemotactic factor KC (Supplementary References , ), and in the presence of IL-6, further potentiate IL-17A production by helper T-cells (Supplementary Reference ), establishing a positive feedback loop of neutrophil recruitment and inflammation. Neutrophils also contribute to TNF-α production which can further potentiate KC production, contributing to the positive neutrophil recruitment loop that is associated with the more severe disease observed in Mp VED. ( Created in biorender.com by ABM ).

Article Snippet: Starting 1 day prior to challenge (day −1) and continuing daily until the end of the study period (day 4), mice were intraperitoneally injected with 150 μg/250 μl/dose of either murine monoclonal anti-IL-17A antibody (BioXcell; clone 17F3, InVivoMAb anti-mouse IL-17A Cat#. BE0173) or the IgG1 isotype control antibody (BioXCell; clone MOPC-21, InVivoMAb IgG1 isotype control, Cat#. BE0083; San Antonio, TX).

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: Astragaloside IV alleviates septic myocardial injury through DUSP1-Prohibitin 2 mediated mitochondrial quality control and ER-autophagy

doi: 10.1016/j.jare.2024.10.030

Figure Lengend Snippet: Astragaloside IV improves SCM myocardial inflammatory injury via DUSP1-Prohibitin 2(PHB2). (A) Immunofluorescence staining showed the expression of Tnt and Gr-1 in cardiac tissue. The images showed the expression changes in the control group (Ctrl), LPS-treated group, and after treatment with different concentrations of AS (AS-L, AS-M, and AS-H). (B) The levels of Gr-1 in mice were detected. Scale bar, 150 μm. (C–E)Protein expression and transcriptional level detection of DUSP1/PHB2 (F–H) Echocardiography was used to analyze the cardiac function of mice. The parameters analyzed included left ventricular ejection fraction (LVEF%), FS%, and LVDd. (I-K) The levels of serum myocardial injury markers, including creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), and troponin (TnT), were detected. (L-N) Analysis of inflammatory factor levels, detecting changes in MMP-9, interleukin-10 (IL-10), and interleukin-17 (IL-17).(O-Q) Transcription level detection of mitochondrial pathway necroptosis mediated genes (Caspase-3/-9/-12). Statistical conditions: Data are shown as mean ± SEM. Ten animals were used in each group and the dotes in each panel represent the average data of three replicates in each animal. *p < 0.05.

Article Snippet: NO: Mouse MMP-9(Matrix Metalloproteinase 9) ELISA Kit E-EL-M3052, Elabscience, China);Mouse IL-10(Interleukin 10) ELISA Kit(E-EL-M0046, Elabscience, China);Mouse IL-17A(Interleukin 17A) ELISA Kit(E-EL-M0047, Elabscience, China);MDA(Malondialdehyde) ELISA Kit(E-EL-0060, Elabscience, China);A001-2-1 Superoxide Dismutase (SOD) Typing Test Kit(E-BC-K020-M, Elabscience, China);H545-1-1 Glutathione Peroxidase 4 (gpx4) Kit(E-BC-K096-S, Elabscience, China);Cell Mitochondrial Complex I (NADH-CoQ Reductase) Activity Assay Kit(E-BC-K834-M, Elabscience, China);Cell Mitochondrial Complex V (F0F1-ATPase/ATP Synthase) Activity Assay Kit (E-BC-K838-M, Elabscience, China);Cell Mitochondrial Complex III (Coenzyme Q-Cytochrome C Reductase) Activity Assay Kit(E-BC-K836-M, Elabscience, China);.

Techniques: Immunofluorescence, Staining, Expressing, Control

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 + CD4 + T cells show an increased Th1-like Th17 phenotype during inflammatory colitis (A and B) Acute colitis was induced in wild-type C57BL/6 mice by administering 2% DSS in their drinking water, and body weight loss was monitored. On day 10, spleen (SP), mesenteric lymph nodes (mLN), Peyer’s patches (PPs), and lamina propria (LP) cells were harvested, and cells were analyzed using flow cytometry. (A) Body weight was monitored and plotted (upper panel). CCL20 expression in colon tissue sections was analyzed by immunofluorescence staining (lower panel). Original magnification 400x, scale bars 100 μm. (B) The dot plot represents the expression of RORγt on CD4 + CCR6 - and CD4 + CCR6 + T cells (upper left), and IL-17A + and IFN-γ+ expression (upper right). Mean percentages of RORγt + cells (lower left), mean percentages of IL-17A + cells (lower middle), and IL-17 and IFN-γ expression (lower right) on CD4 + CCR6 - and CD4 + CCR6 + T cells. (C) Representative immunofluorescence images of PP of wild-type mice treated with or without DSS are shown (upper right). Original magnification 400x, scale bars 100 μm. (D) CCR6 −/− or CCR6 +/+ mice were given 2% DSS in the drinking water. The body weight changes in mice were monitored (left), and RORγt + T-bet + cells were analyzed in the indicated organs by flow cytometry (right). (E) CCR6 −/− splenocytes (CD45.2 congenic; 10 × 10 6 cells/mouse) were adoptively transferred into CD45.1 congenic mice (CCR6 +/+ genotype), and the mice were administered 2% DSS in their drinking water. On day 12, the mice were sacrificed. (F) The immunofluorescence staining of CD45.2 and CD4 markers was performed on the colon tissue. Original magnification 400x, scale bars 100 μm. (G and H) RORγt + and RORγt + T-bet + cells were analyzed after gating on CD45.2 + CD4 + or CD45.2 − CD4 + T cells. (I) IL-17A + IFN-γ + cells were analyzed after gating on CD45.2 + CD4 + or CD45.2 − CD4 + T cells. (J) Naive CD4 + T cells (CD4 + CD25 − CD44 lo CD45RB hi CD62L hi Foxp3rfp − T cells; 0.5 × 10 6 cells/mouse) from CCR6 gfp/+ Foxp3 rfp/rfp mice were adoptively transferred into RAG1 −/− mice. The mean percentage in body weight change from the initial weight was plotted. ( n = 4 mice/group). The error bar represents ±SD. (K) On day 21, RORγt + T-bet + cells were analyzed after gating on CCR6gfp + and CCR6gfp − CD4 + T cells in the SP, mLN, and lamina propria (LP) (left panel). The mean percentage and MFI of RORγt + cells were analyzed after gating on CD4 + CCR6gfp − T cells or CD4 + CCR6gfp + cells (right panel). The bar represents the mean ± SEM; each symbol represents data from an individual mouse, with n = 5–6 mice/group (A–K). The p -value for the comparison between the two groups is indicated in the graphs. The data shown are representative of three independent experiments (A–K). Student’s t test was used, not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: pGL4 plasmid containing mouse 2 Kbp IL-17 promoter , Adgene , Cat# 20127.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Staining, Comparison

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 intrinsic signaling induces the phosphorylation of the PI3K/Akt/mTORC1/STAT3 pathway and promotes RORγt binding on IL-17A regulatory elements (A) CCR6 + and CCR6 - CD4 T cells were sorted based on the expression of eGFP and stimulated with 100 ng/mL CCL20 at different time points (0, 5, 10, 15, 30, and 60 min). They were then immunoblotted with antibodies against various kinases in the downstream signaling pathways of Akt/mTOR/STAT3. (B) Sorted CCR6 + cells were rested overnight with or without rapamycin (50 ng/mL) and, the next day, stimulated with recombinant CCL20 (100 ng/mL) at two time points, 0 and 30 min. The phosphorylation status of STAT3 was analyzed by immunoblotting. Cyclophilin B and total STAT3 were used as loading controls. Blots are from 2 to 3 independent experiments. (C) The schematic presentation of IL-17A promoter (IL-17AP), IL-17F promoter (IL-17FP), conserved non-coding sequences (CNSs), and binding of RORγt. (D) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid vector containing the mouse IL-17 promoter (2 Kbp) and CNS5 enhancer elements. Transfected cells were stimulated with purified recombinant CCL20 (100 ng/mL) or TGF-β1 plus IL-6 at 37°C for 72 h. Then, cells were lysed, and luciferase activity was measured. The data shown are normalized to the Renilla luciferase activity. Each dot represents an independent experiment. Student’s t test. p -values are shown. (E) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid containing the IL-17 promoter (2 Kb) or IL-17 promoter (2 Kb) having a mutation in the RORγt binding site. Cells were cultured as given above, and luciferase activity was measured and plotted. The data shown are representative of one of the two independent experiments. The error bar represents ±SD. (F) Naive CD4 T cells were differentiated into the Th17 lineage condition (TGF-β + IL-6) in the presence or absence of CCL20 and with or without rapamycin (25 ng/mL) for 4 days. Expression of RORγt was analyzed after gating on CD4 + T cells. n = 3 experiments. (G) The schematic CCR6-CCL20 signaling pathways are shown. The p -value for the comparison between the two groups is indicated in the graphs. not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: pGL4 plasmid containing mouse 2 Kbp IL-17 promoter , Adgene , Cat# 20127.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Protein-Protein interactions, Recombinant, Western Blot, Transfection, Plasmid Preparation, Purification, Luciferase, Activity Assay, Mutagenesis, Cell Culture, Comparison