Journal: The Journal of Experimental Medicine

Article Title: GM-CSF primes cardiac inflammation in a mouse model of Kawasaki disease

doi: 10.1084/jem.20151853

Figure Lengend Snippet: Radio-resistant CFs produce GM-CSF after CAWS challenge. (a and b) Reciprocal B6.Ly5.1 (WT) and GM-CSF −/− (GM −/− ) BM chimeras were challenged with CAWS, and 1 d later, the hearts were analyzed for cardiac vasculitis. (a) Dot plots are gated on heart-resident (in vivo Gr-1 − ) myeloid cells (CD11b + ). (b) Graphs depict the mean ± SEM number of neutrophils (neuts.) and monocytes (mono.) and ICAM/VCAM expression on cardiac endothelium (endo.; data are pooled from four to nine mice from four experiments). (c and d) Hearts from naive or CAWS-challenged mice (6 h after injection) were sorted into CD45 + leukocytes (leuko.), CD45 − CD31 + endothelial cells, CD45 − gp38 + CFs, and CD45 − CD31 − gp38 − stromal cells (stroma), and the expression of cytokines and chemokines was analyzed by qPCR. (d) Bar graphs show mean ± SEM gene expression relative (rel.) to Gapdh pooled from three experiments. fibro., fibroblasts; FSC, forward side scatter. (e) Fibroblasts (CD45 − CD31 − gp38 + ) were sorted from the heart, lung, and LN of naive and CAWS (∼10 h after challenge) mice, and GM-CSF expression was measured by qPCR. Expression is normalized to naive tissue fibroblasts and shows the mean ± SEM from two experiments ( n = 5–6 mice). (f) The expression of GM-CSF by CFs sorted from naive, day 1, or day 28 CAWS-challenged mice was measured by qPCR. Data points show individual experiments ( n = 2–3 mice per experiment), with the mean ± SEM indicated. (g) The expression of ICAM and VCAM on CFs was analyzed at various stages after CAWS challenge. FACS plots are gated on CFs (CD45 − CD31 − gp38 + ), and graphs depict the mean ± SEM of four to six mice pooled from two experiments. Statistical analysis was performed with unpaired, two-tailed Student’s t tests. *, P < 0.05; ***, P < 0.001.

Article Snippet: Sections were stained with anti-gp38 (eBio8.1.1; eBioscience), CD31 (MEC13.3; BD), and biotinylated anti–MHC II (M5/114; eBioscience) for detection with goat anti–hamster IgG AF488, goat anti–rat IgG AF594, and streptavidin 647, respectively (Thermo Fisher Scientific).

Techniques: In Vivo, Expressing, Injection, Gene Expression, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: GM-CSF primes cardiac inflammation in a mouse model of Kawasaki disease

doi: 10.1084/jem.20151853

Figure Lengend Snippet: Identifying activating stimuli and location of CFs. (a) Primary human CFs were stimulated with LPS, TNF, or CAWS, and 4 h later, GM-CSF expression was measured by qPCR. Results show fold-induction relative to nil stimulation from two donors conducted over four experiments. (b) FACS analysis of B6 mouse hearts before and after in vitro culture. A representative contour plot of gp38 and CD31 expression is shown (inset value shows the mean from three individual CF lines). (c) Primary mouse CFs were stimulated with LPS, TNFα, or CAWS for 4 h, and GM-CSF , Ccl2 , and Ccl7 expression were measured by qPCR (values show gene expression relative to nil stimulation). The four data points represent individual experiments with different CF lines. (d–g) Hearts from naive and CAWS-injected (∼20 h after challenge) RAG-1 −/− mice were divided into upper (d and e) and lower (f and g) halves, stained for gp-38 (green), CD31 (red), and MHC-II (gray), and analyzed by confocal microscopy (isotype control [IC; green] of gp-38 antibody is shown). Yellow, dashed-lined boxes show image insets. Representative images of five to six mice from three experiments are shown. Bars, 100 µm.

Article Snippet: Sections were stained with anti-gp38 (eBio8.1.1; eBioscience), CD31 (MEC13.3; BD), and biotinylated anti–MHC II (M5/114; eBioscience) for detection with goat anti–hamster IgG AF488, goat anti–rat IgG AF594, and streptavidin 647, respectively (Thermo Fisher Scientific).

Techniques: Expressing, In Vitro, Gene Expression, Injection, Staining, Confocal Microscopy, Control

Journal: Nature Communications

Article Title: Desmoplastic stroma restricts T cell extravasation and mediates immune exclusion and immunosuppression in solid tumors

doi: 10.1038/s41467-023-40850-5

Figure Lengend Snippet: a Representative (top) and quantification of (bottom) multiplex immunofluorescence images in tumors 1 week (left) and 2 weeks (right) post-treatment with PBS, MigR control or FAP-CAR T cells. First row depicts staining for stromal cell profiles, including FAP (green), α-SMA (red) and PDGFR-α (magenta). Second row depicts staining for ECM components including collagen hybridizing peptide (CHP) that detects remodeled collagen (red) and FN (green). Third row depicts distribution of T cells (CD3, red) and their spatial relationships to tumor cells (PanCK, green) and stromal cells (PDPN, magenta). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. b t-SNE of multi-parametric flow cytometry demonstrating the immune cell profiles based on the gating strategy as depicted in Supplementary Fig. , including lymphocyte (left) and myeloid cells (right) in tumors 1 week (Top row) and 2 weeks (bottom row) post-treatment with PBS, MigR control or FAP-CAR T cells. The percentages of indicated immune cell subpopulations in total live cells or CD45.2 + cells from tumors for each group of mice quantified based on multi-parametric flow cytometry analysis. Data points are mean ± SD ( n = 5 per group) and groups were compared using two-way ANOVA with Tukey’s multiple comparisons tests. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. a . PDGFRα (post 1 week): PBS vs. FAP-CAR, p = 0.032. PDGFRα (post 2 weeks): PBS vs. FAP-CAR, p = 0.002. Fibronectin (post 1 week): MigR vs. FAP-CAR, p = 0.022. CD3 (post 1 week): MigR vs. FAP-CAR, p = 0.005. PanCK (post 2 weeks): MigR vs. FAP-CAR, p = 0.004. b post 1 week: CD3 PBS vs. FAP-CAR, p = 0.047. CD8 PBS vs. FAP-CAR, p = 0.004, MigR vs. FAP-CAR, p = 0.020. post 2 weeks: Ly6C low Ly6G + MigR vs. FAP-CAR, p = 0.006. The p values for remaining comparisons are all <0.001 or <0.0001. Source data are provided as a Source Data file.

Article Snippet: Samples were stained with the following antibodies: FAP (1:250, Abcam), PDGFRα (1:200, R&D System), PDPN (1:200, R&D System), FITC-αSMA (1:500, Sigma), AF647-EpCAM (1:100, BioLegend), GFP (1:500, Abcam), CD3 (1:100, Abcam), CD8 (1:500, Abcam), CD4 (1:500, Abcam), FoxP3 (1:100, Abcam), F4/80 (1:100, Abcam), CD103 (1:100, Abcam), Ly6C + Ly6G (1:100, Abcam), CK19 (1:100, Abcam), Ki-67 (1:100, Abcam), CD31 (1:200, R&D System), IFN-γ (1:100, R&D System), TGF-β1 (1:100, Bioss), AF488 Pan-CK (1:100, eBiosciences), CD206 (1:100, R&D System), PD-1 (1:100, R&D System), biotin CHP (1:10, 3Helix), FN (1:300, Sigma-Aldrich), and cleaved caspase-3 (1:100, Cell Signaling Technology).

Techniques: Multiplex Assay, Immunofluorescence, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A dynamic variation of pulmonary ACE2 is required to modulate neutrophilic inflammation in response to Pseudomonas Areuginosa lung infection in mice

doi: 10.4049/jimmunol.1900579

Figure Lengend Snippet: List and application of antibodies.

Article Snippet: I. caption a7 Antigen primary Ab Secondary Ab application Vendor ACE2 rat donkey IF R & D sys actin mouse(HRP) western blot GenScript Aqp3 rabbit donkey IF abcam CC3 rabbit donkey IF Biocare Medical CD11b rat flow cytometry Biolegend Foxj1 mouse donkey IF eBioscience IL-17A goat donkey neutralization R & D sys iNOS mouse donkey IF BD biosciences Ly6G rat flow cytometry Biolegend Ly6G rat depletion R & D sys MPO goat donkey IF Santa Cruz Muc5A mouse donkey IF Thermofisher p -STAT3 rabbit goat western blot Cell signaling PdPn rabbit donkey IF ABBiotec Pgp9.5 mouse donkey IF abcam Scgb1A1 rabbit donkey IF millipore STAT3 rabbit Goat Western blot Cell signaling Open in a separate window List and application of antibodies.

Techniques: Western Blot, Flow Cytometry, Neutralization

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A dynamic variation of pulmonary ACE2 is required to modulate neutrophilic inflammation in response to Pseudomonas Areuginosa lung infection in mice

doi: 10.4049/jimmunol.1900579

Figure Lengend Snippet: (A). IL-17A concentration in BALF from mice with or without P.a lung infection was determined by ELISA. (B). Neutrophil counts in BALF from wild-type mice with P.a lung infection with or without pre-existing reduced ACE2 and in the presence or absence of A IL-17A neutralizing antibody. The neutrophils were detected by flow cytometry gated as Ly6G+CD11b+ live cells. The counts were normalized with the volume of BALF in the individual experimental group of mice. (C). Recombinant IL-17A (100 ng/ml, 30 μl intra-nasal instillation) induces neutrophil infiltration in wild type mice lungs, and the induction is more potent in ACE2 deficient mice. Recombinant human ACE2 inhibits the IL-17A induced neutrophil infiltration in mice (D-E). rhACE2 alleviates rIL-17 mediated cytokine and chemokine production in BALF. (F). IL-17A production in BALF from mice that ACE2 activity was manipulated either by pharmacological reagents or genetically modified. IL-17A concentration was determined by ELISA and normalized with respective BALF volume. (G-H). rhACE2 inhibited IL-17A induced STAT3 phosphorylation in vitro (G, mouse lung organoids) and in vivo (H, mouse lung). (I). rhACE2 represses bacterial lung infection induced STAT3 activation in both wild-type and ACE2 deficient mice. (J). Blocking STAT3 signaling alleviates the lack of pulmonary ACE2 induced exuberant neutrophil infiltration in bacterially infected mouse lung as manifested by western blot for phosphorylated STAT3. WP1066, a STAT3 antagonist. Data were analyzed for statistical significance by two-tailed student’s T-test or analysis of variance (ordinary one way ANOVA multiple comparisons) using Prism software (GraphPad).In all experimental groups, n≥3. * p<0.05, ** p<0.01 and *** p<0.001.

Article Snippet: I. caption a7 Antigen primary Ab Secondary Ab application Vendor ACE2 rat donkey IF R & D sys actin mouse(HRP) western blot GenScript Aqp3 rabbit donkey IF abcam CC3 rabbit donkey IF Biocare Medical CD11b rat flow cytometry Biolegend Foxj1 mouse donkey IF eBioscience IL-17A goat donkey neutralization R & D sys iNOS mouse donkey IF BD biosciences Ly6G rat flow cytometry Biolegend Ly6G rat depletion R & D sys MPO goat donkey IF Santa Cruz Muc5A mouse donkey IF Thermofisher p -STAT3 rabbit goat western blot Cell signaling PdPn rabbit donkey IF ABBiotec Pgp9.5 mouse donkey IF abcam Scgb1A1 rabbit donkey IF millipore STAT3 rabbit Goat Western blot Cell signaling Open in a separate window List and application of antibodies.

Techniques: Concentration Assay, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Recombinant, Activity Assay, Genetically Modified, In Vitro, In Vivo, Activation Assay, Blocking Assay, Western Blot, Two Tailed Test, Software