Journal: iScience

Article Title: Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

doi: 10.1016/j.isci.2024.109950

Figure Lengend Snippet: Heterogeneous clearance dynamics in a panel of high-grade serous ovarian cancer models in vitro and tumor implant formation efficiency in orthotopic xenografts in vivo (A) Schematic representation of cancer-mesothelial interactions during the process of ovarian cancer spheroid (red) transmigration across a mesothelial monolayer (green). We term this process of transmesothelial migration as clearance of the mesothelial barrier (see Figure S1 A for alternative colors in schematic representation). (B) Representative images of ovarian cancer cells (RFP+) clearing the ZTGFP mesothelial cell monolayer (GFP+) as a function of time. The white line represents the GFP- mesothelial-free area and the yellow line represents the RFP+ cancer spheroid area. Scale bar = 100μm. (C) Normalized clearance area calculated as the ratio of GFP- area (white line in panel B) to the RFP+ cancer area (yellow line in panel B) (see Equation 1 in ) in a panel of four ovarian cancer models (OVCAR8, OVCAR3, OVCA432 and OV90) transmigrating across the ZTGFP mesothelial model. Data is mean ± SEM in N = 3 biological replicates. One-way ANOVA ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001. (D) Quantification of OVCAR8 and OVCAR3 spheroid adhesion to ZTGFP mesothelial monolayers after T = 3h. Data is mean ± SEM in N = 3 biological replicates. T-test ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001. (E) Quantification of OVCAR8 and OVCAR3 spheroid adhesion to a murine peritoneal wall after T = 3h. Data is mean ± SEM in N = 3 biological replicates. T-test ∗: p < 0.05. (F) Representative images of the invasive tumor implants on murine omentum stained for cell nuclei (DAPI) and pan-cytokeratin (yellow) in an orthotopic xenograft. Scale bar = 80μm. (G) Quantification of tumor size in xenografts with OVCAR8 and OVCAR3. Black line = median. N = 2 mice with at least n = 10 fields analyzed. T-test ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001. (H) Representative H&E images of the metastatic tumor implants on omentum. Yellow lines outline tumor cells. Scale bar = 100μm.

Article Snippet: Human Mesothelial Cells , ZenBio , N/A.

Techniques: In Vitro, In Vivo, Transmigration Assay, Migration, Staining

Journal: iScience

Article Title: Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

doi: 10.1016/j.isci.2024.109950

Figure Lengend Snippet: Forskolin reduces clearance efficiency across multiple ovarian cancer spheroids with different baseline clearance rates (A) Schematic of clearance assay and pre-treatment schedule with forskolin. (B) Images of mesothelial clearance for control and forskolin pre-treated mesothelial barriers (ZTGFP cells). Green: mesothelial cells; red: ovarian cancer cells. Scale bar = 300 μm. (C) Analysis of clearance dynamics for control (red) and forskolin-pretreated (20μM – yellow) ZTGFP mesothelial barriers. Data is mean ± SEM in N = 3 biological replicates. T-test ∗: p < 0.05, ∗∗: p < 0.01.

Article Snippet: Human Mesothelial Cells , ZenBio , N/A.

Techniques: Control

Journal: iScience

Article Title: Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

doi: 10.1016/j.isci.2024.109950

Figure Lengend Snippet: Inhibition of protein kinase A in forskolin-treated mesothelial barriers reverts the protective effects of forskolin across multiple ovarian cancer models (A) Quantification of phospho-PKA staining of ZTGFP cells in panel B. Data is mean ± SEM for N = 3 biological replicates. One-way ANOVA: ∗: p < 0.05. (B) Representative images of staining of ZTGFP cells for phospho-PKA (white) and cell nuclei (blue) under the conditions described in panel A. Scale bar = 20 μm. (C) Representative images at 24 h of ovarian cancer spheroid (red) and ZTGFP mesothelial barrier (green) for control and combination treatment with forskolin + PKAi (forskolin: 20μM; PKA-14-22: 10μM). Scale bar = 500μm. (D) Clearance dynamics following treatment with forskolin as a single agent (yellow), PKAi as a single agent (gray), PKAi and forskolin combination (cyan), and control (red) in ZTGFP mesothelial cells. Data is mean ± SEM in N = 3 biological replicates. T-test: ∗ p < 0.05, ∗∗: p < 0.01.

Article Snippet: Human Mesothelial Cells , ZenBio , N/A.

Techniques: Inhibition, Staining, Control

Journal: iScience

Article Title: Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

doi: 10.1016/j.isci.2024.109950

Figure Lengend Snippet: Forskolin enhances mesothelial cell-cell junction integrity and impairs actomyosin fiber formation in a PKA-dependent manner (A) Staining of cell-cell junctions for β-Catenin (red) and ZO-1 (magenta), cell nuclei (blue) in ZTGFP cells. Scale bar = 20μm. (B) Quantification of coverage index for ZO-1 stained ZTGFP cells. See for Equation 2 . Black line = median. N = 3 biological replicates. Control n = 93, Forskolin n = 93, PKAi n = 80, and Forskolin+PKAi n = 77 junctions. Nonparametric Kruskal-Wallis one-way ANOVA: ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001. (C) Quantification of intensity per interface area for ZO-1 stained ZTGFP cells. See for Equation 3 . Black line = median. N = 3 biological replicates. Control n = 82, Forskolin n = 79, PKAi n = 67, and Forskolin+PKAi n = 69 junctions. Nonparametric Kruskal-Wallis one-way ANOVA: ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001. (D) Quantification of maximum intensity of β-catenin junctions of ZTGFP mesothelial cells. Black line = median. N = 3 biological replicates. n = 30 junctions analyzed for all conditions. Nonparametric Kruskal-Wallis one-way ANOVA: ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001. (E) Quantification of actin staining intensity of ZTGFP mesothelial cells. Data is mean ± SEM for N = 3 biological replicates; One-way ANOVA: ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001. (F) Representative images of actin cytoskeleton staining (white), ZO-1 tight junctions (magenta) and phospho-myosin light chain 2 (green) in ZTGFP mesothelial barriers. Scale bar = 10 μm. (G) Schematic of analysis methodology for angle between stress fiber and cell-cell interface. ZO-1 (magenta), actin (white) and pMLC2 (green). (H) Quantification of angle between stress fibers and cell-cell interface. N = 3 biological replicates, n = 5 junctions per condition. Black line = median. One-way ANOVA: ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, ∗∗∗∗: p < 0.0001.

Article Snippet: Human Mesothelial Cells , ZenBio , N/A.

Techniques: Staining, Control

Journal: iScience

Article Title: Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

doi: 10.1016/j.isci.2024.109950

Figure Lengend Snippet: Mesothelial cell contractility is impaired following treatment with forskolin and PKA inhibition restores baseline contractile function (A) Kinetics of collagen gel substrate displacement fields in control and forskolin-treated (20μm) ZTGFP mesothelial cells. Scale bar = 500μm. (Β) Quantification of the average bead displacement shown in panel A imaged at 4h intervals. Data is mean ± SEM in n ≥ 37 cells pooled from N = 3 biological replicates. T-test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (C) Representative displacement fields of collagen substrates seeded with ZTGFP mesothelial cells 24h following treatment with each condition: control (red), forskolin (yellow), PKA inhibitor (gray) and their combination (cyan). Scale bar = 500μm. (D) Dot plot of bead displacements by ZTGFP mesothelial cells ( n = 195, 252, 108, 246 cells for each condition). N = 3 biological replicates. Black line = median. One-way ANOVA: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Human Mesothelial Cells , ZenBio , N/A.

Techniques: Inhibition, Control

Journal: iScience

Article Title: Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

doi: 10.1016/j.isci.2024.109950

Figure Lengend Snippet: Hypercontractile mesothelial cells exhibit increased clearance efficiency across multiple cancer cell models with impaired cell-cell junction organization and increased actin stress fibers (A) (Top) Schematic of clearance assay and pre-treatment schedule with calyculin A. (Bottom) Representative images of OVCAR8 spheroids clearing a ZTGFP mesothelial barrier treated with calyculin A. Scale bar = 500μm. (B) Dynamics of normalized clearance areas through ZTGFP mesothelial monolayers (green) treated with calyculin A (0.5 nM) for 10 min and washed prior to spheroid addition. Data is mean ± SEM in N = 5 biological replicates. T-test: ∗: p < 0.05; ∗∗: p < 0.01. (C) Kinetics of collagen gel substrate displacement fields in control and calyculin A-treated ZTGFP mesothelial cells. Scale bar = 500μm. (D) Quantification of ZTGFP mesothelial cell-induced collagen substrate displacements following treatment with control (red) and calyculin A (pink). N > 35 cells for each time frame. Data is mean ± SEM in N = 3 biological replicates. T-test: ∗∗∗∗: p < 0.0001. (E) Representative images of ZTGFP mesothelial cell-cell junction staining for β-Catenin (red), ZO-1 (magenta) and DAPI cell nuclei (blue). Scale bar = 20μm. (F) Quantification of ZO-1 staining cluster density. See for Equation 4 . Black line = median. N = 3 biological replicates. Control n = 94 and calyculin A n = 90 junctions. Mann-Whitney test: ∗: p < 0.05; ∗∗: p < 0.01. (G) Representative images of staining for actin cytoskeleton (magenta) and cell nuclei (blue) of ZTGFP mesothelial cells. Scale bar = 20μm. (H) Quantification of actin fluorescence intensity per cell. Data is mean ± SEM in N = 3 biological replicates. T-test ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Human Mesothelial Cells , ZenBio , N/A.

Techniques: Control, Staining, MANN-WHITNEY, Fluorescence

Journal: iScience

Article Title: Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

doi: 10.1016/j.isci.2024.109950

Figure Lengend Snippet:

Article Snippet: Human Mesothelial Cells , ZenBio , N/A.

Techniques: Recombinant, Software, Blocking Assay

Journal: PLoS ONE

Article Title: Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice

doi: 10.1371/journal.pone.0027564

Figure Lengend Snippet: C57BL/6 mice (WT) or C57BL/6 mice infected with N. brasiliensis ( A ) 2 or ( B ) 4 weeks earlier ( Nippo mice) were injected with 150 CFU of K. pneumoniae i.p. and survival was monitored. Mice infected with N. brasiliensis 2 but not 4 weeks before bacterial infection are more likely to survive septic peritonitis than unparasitized control mice. (n = 25 mice/group, ** P = 0.0018).

Article Snippet: To decipher the mechanism by which N. brasiliensis enhances K. pneumoniae clearance, peritoneal inflammatory cells were quantified in Nippo mice at baseline and 4 and 24 h after K. pneumoniae infection.

Techniques: Infection, Injection, Control

Journal: PLoS ONE

Article Title: Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice

doi: 10.1371/journal.pone.0027564

Figure Lengend Snippet: C57BL/6 (WT) and C57BL/56 mice infected 2 weeks earlier with N. brasiliensis ( Nippo Mice) were injected i.p. with 150 CFU of K. pneumoniae . Dilutions of peritoneal lavage fluid obtained 4 (A) and 24 h (B) after K. pneumoniae injection were cultured on agar plates and bacterial colonies were counted. (n = 7–9 mice/group, * P <0.05).

Article Snippet: To decipher the mechanism by which N. brasiliensis enhances K. pneumoniae clearance, peritoneal inflammatory cells were quantified in Nippo mice at baseline and 4 and 24 h after K. pneumoniae infection.

Techniques: Infection, Injection, Cell Culture

Journal: PLoS ONE

Article Title: Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice

doi: 10.1371/journal.pone.0027564

Figure Lengend Snippet: C57BL/6 (WT) and C57BL/6 mice infected 2 weeks earlier with N. brasiliensis ( Nippo mice) were euthanized at baseline ( A ), 4 h ( B ) and 24 h ( C ) after i.p. injection of 150 CFU of K. pneumoniae and inflammatory cells were recovered by peritoneal lavage. Total cells were counted using a hemocytometer and differential cell counts were determined on cytospun cells stained with Diff-Quik. (n = 7–9 mice/group, ** P <0.01, *** P <0.001 for Nippo compared to unparasitized control mice).

Article Snippet: To decipher the mechanism by which N. brasiliensis enhances K. pneumoniae clearance, peritoneal inflammatory cells were quantified in Nippo mice at baseline and 4 and 24 h after K. pneumoniae infection.

Techniques: Infection, Injection, Staining, Diff-Quik, Control

Journal: PLoS ONE

Article Title: Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice

doi: 10.1371/journal.pone.0027564

Figure Lengend Snippet: Levels of ( A ) TNF-α, ( B ) IL-6, and ( C ) IL-1β were measured by ELISA in peritoneal lavage fluid from WT or Nippo mice at baseline, 4 and 24 h after i.p. injection of 150 CFU of K. pneumoniae . (n = 7–9 mice/time point, * P <0.05 for Nippo mice compared to unparasitized control mice).

Article Snippet: To decipher the mechanism by which N. brasiliensis enhances K. pneumoniae clearance, peritoneal inflammatory cells were quantified in Nippo mice at baseline and 4 and 24 h after K. pneumoniae infection.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Control

Journal: PLoS ONE

Article Title: Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice

doi: 10.1371/journal.pone.0027564

Figure Lengend Snippet: ( A ) Mast cell-deficient Kit W-sh /Kit W-sh mice ( Wsh , n = 25) or Wsh mice infected with N. brasiliensis 2 weeks earlier ( Wsh+Nippo mice, n = 24) were injected with 150 CFU of K. pneumoniae i.p. and survival was monitored. ( B ) pmc-WT (25 mice) and pmc-WT mice infected with N. brasiliensis 2 weeks earlier (pmc-WT- Nippo Mice, 25 mice), were injected with 150 CFU of K. pneumoniae i.p. and survival monitored (* P <0.012). ( C ) Expulsion of N. brasiliensis is normal in Kit W-sh /Kit W-sh mice. WT and Kit W-sh /Kit W-sh mice (n = 5/group) were infected with 500 N. brasiliensis larvae and intestinal worm burden assessed 7 and 14 d after infection.

Article Snippet: To decipher the mechanism by which N. brasiliensis enhances K. pneumoniae clearance, peritoneal inflammatory cells were quantified in Nippo mice at baseline and 4 and 24 h after K. pneumoniae infection.

Techniques: Infection, Injection

Journal: PLoS ONE

Article Title: Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice

doi: 10.1371/journal.pone.0027564

Figure Lengend Snippet: ( A ) IL-4 +/+ mice (WT), IL-4 +/+ mice infected with N. brasiliensis 2 weeks earlier ( Nippo mice), IL-4 −/− mice, or IL-4 −/− mice infected with N. brasiliensis 2 weeks earlier (IL-4 −/− + Nippo) were injected with 150 CFU of K. pneumoniae i.p and survival was monitored. (n = 35 mice/group, * P = 0.02 comparing WT vs. Nippo mice and P = 0.13 comparing IL-4 −/− vs. IL-4 −/− + Nippo). ( B ) Wsh mice (Wsh) were reconstituted by i.p. injection of 125,000 WT BMCMC (Wsh+WT) or BMCMC preconditioned for 7 d with 50 ng/mL IL-4 (Wsh+WT+IL4). 24 h later, mice were injected with 150 CFU of K. pneumoniae i.p. Mice reconstituted with BMCMC conditioned with IL-4 were more likely to survive than controls (n = 35 mice/group). ( C ) IL-4 enhances mast cell-dependent neutrophil recruitment during K. pneumoniae septic peritonitis. Wsh+WT and Wsh+IL4 mice were injected with 150 CFU of K. pneumoniae i.p. Inflammatory cells in peritoneum of uninfected Wsh -WT (black bar) or Wsh -IL-4 (white bar) and infected Wsh -WT ( Wsh -WT-kleb, hatched bar) and infected Wsh -IL-4 ( Wsh -IL-4-kleb, boxed bar) mice 4 h after infection with Klebsiella . (* P <0.05 Wsh-WT vs. Wsh-WT (kleb) neut, and ** P <0.01 Wsh-IL4-kleb vs. Wsh-WT-kleb neuts). ( D ) IL-4 conditioned mast cells produced greater amounts of IL-6. BMCMC cultured for 7 d in the absence (cont) or presence of 50 ng/mL IL-4 (IL-4) were stimulated with heat-killed K. pneumoniae and the amount of IL-6 released into the culture media quantified by ELISA. (** P <0.01).

Article Snippet: To decipher the mechanism by which N. brasiliensis enhances K. pneumoniae clearance, peritoneal inflammatory cells were quantified in Nippo mice at baseline and 4 and 24 h after K. pneumoniae infection.

Techniques: Infection, Injection, Produced, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Antitumor effects of plasma-activated sodium acetate solution on gastric cancer cells

doi: 10.1038/s41598-025-04977-3

Figure Lengend Snippet: ( a ) The experimental system employed to produce plasma-activated liquids. L represents the distance between the plasma source and medium, and V represents the volume of the irradiated medium. In this experiment, L was fixed at 3 mm, and V at 6 ml. ( b ) Antitumor effects of PAL, PAA, 1% PASA, 3% PASA, and 5% PASA on GC cell lines. PASA had stronger antitumor effects at T = 0.5, 1, and 3 min compared with PAL. ( c ) Effects of PAL, PAA, 3% PASA, and 5% PASA on human peritoneal mesothelial cells. PAA and 3% PASA caused much less damage to normal peritoneal mesothelial cells compared with PAL. Error bars indicate standard deviation.

Article Snippet: Human GC cell lines MKN1-Luc (RRID: CVCL_J261) and MKN45-Luc (RRID: CVCL_J262) were purchased from the Japanese Cancer Research Resources Bank (Tokyo, Japan), and human peritoneal mesothelial cells were from KAC Co., Ltd. (Kyoto, Japan).

Techniques: Clinical Proteomics, Irradiation, Standard Deviation

Journal: Scientific Reports

Article Title: Antitumor effects of plasma-activated sodium acetate solution on gastric cancer cells

doi: 10.1038/s41598-025-04977-3

Figure Lengend Snippet: ( a ) Apoptosis assay of normal peritoneal mesothelial cells and GC cells treated with 3% PASA. The percentages of apoptotic plus dead MKN1-Luc and MKN45-Luc cells increased within T = 3 min. ( b ) Morphological change induced by 3% PASA treatment. Morphological changes in MKN45-Luc cells by treatment with 3% sodium acetate solutions without plasma exposure (control group) or 3% PASA for T = 10 min (treatment group) were observed using time-lapse photography. In the treatment group, numerous blebs, indicative of apoptosis, were observed around the cells (arrow).

Article Snippet: Human GC cell lines MKN1-Luc (RRID: CVCL_J261) and MKN45-Luc (RRID: CVCL_J262) were purchased from the Japanese Cancer Research Resources Bank (Tokyo, Japan), and human peritoneal mesothelial cells were from KAC Co., Ltd. (Kyoto, Japan).

Techniques: Apoptosis Assay, Clinical Proteomics, Control

Journal: Frontiers in Immunology

Article Title: Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

doi: 10.3389/fimmu.2018.02458

Figure Lengend Snippet: Mice that lack regulatory T cells have elevated peritoneal and macrophage counts and cytokine/chemokine levels . (A) Total and differential counts of peritoneal exudate (PE) cells isolated from scurfy (Sf) mice or littermate controls (Wt), fixed with cytospin centrifuge, and stained with May-Grünwald/Giemsa ( n > 15 per group). (B) Average cell diameter in PE determined by automated cell analyser ( n = 9–10). (C) Chipcytometry stainings of omentum majus fixed on ZellSafe™ chips; scale bar = 100 μm. (D) Percentages ( n = 8–9) total cell numbers ( n = 5–6) of CD115 + CD11b + F4/80 + peritoneal macrophages (PM) determined by flow cytometry and automated cell analyser. (E) Chemokine and cytokine levels in peritoneal fluid, measured by bead-based multiplex immunoassay ( n = 5 samples, each pooled from 2–3 mice). Statistical analyses were performed using unpaired Student's t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. n.s., not significant; Mac, macrophages; Eos, eosinophils; Lymph, lymphocytes; Neutr, neutrophils; Baso, basophils.

Article Snippet: Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany).

Techniques: Isolation, Staining, Flow Cytometry, Multiplex Assay

Journal: Frontiers in Immunology

Article Title: Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

doi: 10.3389/fimmu.2018.02458

Figure Lengend Snippet: Peritoneal macrophages in scurfy mice acquire state of activation and exhaustion. (A) In vivo proliferation ( n = 4, one representative experiment out of three) and ( B) apoptosis (Annexin V staining; n = 9–13 per time-point) of peritoneal macrophages (PM) in control (Wt) and scurfy (Sf) mice measured by flow cytometry. (C) ATP amount in purified PM quantified by CellTiter-Glo ( n = 3–5). Flow cytometry analysis of (D) autophagy ( n = 4–5, one representative experiment out of two) and (E) phagocytosis in vivo ( n = 5) in Wt and Sf PM. (F) Nitrogen oxide (NO) secretion by purified PM measured by Griess assay ( n = 4–8). Statistical analyses were performed using unpaired Student's t -test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany).

Techniques: Activation Assay, In Vivo, Staining, Control, Flow Cytometry, Purification, Griess Assay

Journal: Frontiers in Immunology

Article Title: Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

doi: 10.3389/fimmu.2018.02458

Figure Lengend Snippet: Immunophenotyping by single-cell chipcytometry and transcriptomics reveal plasticity of peritoneal macrophage activation profiles . (A) Schematic outline of the experimental protocol, (B) exemplary surface marker stainings of the cells fixed on chip, and (C) immunophenotyping of scurfy (Sf) and unaffected (Wt) peritoneal macrophages (PM) from the untreated mice and after their transfer to Wt or Sf peritoneal cavity (“Sf PM Wt mice” and “Wt PM Sf mice”), respectively, performed by single-cell chipcytometry. Data are depicted as mean fluorescent intensities (MFI) for each surface marker expressed by single cells and shown as heatmaps (middle) and bar graphs (bottom; n~60 cells per group, pooled from more than 8 donor mice). (D) Microarray-based transcriptome analysis of gene expression in PM from untreated Sf and Wt mice and after their intraperitoneal (i.p.) transfer to Wt or Sf mice, respectively (“Sf PM Wt mice” and “Wt PM Sf mice”; two independent experiments, each experimental group contains pooled cells from more than 5 mice). Statistical analysis was performed using one-way ANOVA, *** p < 0.001.

Article Snippet: Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany).

Techniques: Activation Assay, Marker, Microarray, Transcriptome Wide Gene Expression

Journal: Frontiers in Immunology

Article Title: Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

doi: 10.3389/fimmu.2018.02458

Figure Lengend Snippet: Injection of Wt CD4 + T cells to Sf mice restores PM number and inflammatory cytokine expression. (A) Total peritoneal exudate (PE) cells and percentages of peritoneal macrophages (PM) in controls (Wt), scurfy mice (Sf), and adoptively transferred Sf mice with Wt CD4 + cells (Sf+CD4), analyzed by automated cell counter and flow cytometry, respectively ( n ≥ 5). (B) Heatmap, linear discriminant analysis, and bar graphs of cell surface markers measured by chipcytometry on gated CD115 + CD11b + F4/80 + PM ( n = 200 cells per group, pooled from more than 3 mice). (C) Impact of adoptive Wt CD4 + T cell transfer on cytokine mRNA expression by PM and peritoneal CD4 + T cells, measured by qPCR ( n = 3–4). (D) Percentages of PM in PE of Sf mice after in vivo treatment with anti-M-CSF antibody (Ab) or isotype (Iso) control ( n = 5 mice, data are pooled from two independent experiments). Statistical analyses were performed using one-way ANOVA (A-C) or unpaired Student's t -test (D), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany).

Techniques: Injection, Expressing, Flow Cytometry, In Vivo, Control

Journal: Frontiers in Immunology

Article Title: Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

doi: 10.3389/fimmu.2018.02458

Figure Lengend Snippet: Peritoneal macrophage subsets and their effector functions are skewed due to the absence of regulatory T cells. (A) Gating strategy used to identify large peritoneal macrophages (LPM) and small peritoneal macrophages (SPM) by flow cytometry. Cells were FACS sorted and stained with May-Grünwald/Giemsa. Data shown are representative from >10 experiments. (B) Percentages ( n = 8) and (C) total cell numbers ( n = 5–6) of CD115 + CD11b high F4/80 high LPM and CD115 + CD11b int F4/80 int SPM in the peritoneal exudate of scurfy (Sf) and littermate control (Wt) mice determined by flow cytometry. (D) Percentages of LPM and SPM in Sf mice within PE after injection of anti-M-CSF antibody (Ab) or isotype (Iso) control antibody ( n = 5 mice, data are pooled from two independent experiments). Flow cytometry analyses of (E) in vivo proliferation ( n = 4, one representative experiment out of three), (F) apoptosis (Annexin V staining; n = 8–11 per time-point), (G) autophagy ( n = 5-6), and (H) phagocytosis rate in vivo ( n = 5) of LPM and SPM in Sf and Wt mice. Statistical analyses were performed using unpaired Student's t -test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany).

Techniques: Flow Cytometry, Staining, Control, Injection, In Vivo

Journal: Frontiers in Immunology

Article Title: Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

doi: 10.3389/fimmu.2018.02458

Figure Lengend Snippet: Large and small peritoneal macrophages have distinct immune profiles in steady-state and inflammatory conditions. (A) Quantitative PCR analysis of gene expression in large and small peritoneal macrophages (LPM and SPM), isolated from scurfy (Sf) and control (Wt) mice ( n = 4–7 samples per group, each sample contains pooled cells from 3 to 7 mice). (B) Expression pattern of cell surface markers involved in immune response in LPM and SPM analyzed by single-cell chipcytometry. Data are depicted as mean fluorescent intensities (MFI) for each surface marker expressed by single cells ( n = 28–62 cells per experimental group, each sample contains pooled cells from more than 8 donor mice).

Article Snippet: Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Isolation, Control, Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages

doi: 10.3389/fimmu.2018.02458

Figure Lengend Snippet: Microenviroment shapes immune signatures of large and small peritoneal macrophages. (A) Schematic outline of the experimental protocol and (B,C) immunophenotyping by single-cell chipcytometry of control (Wt) and scurfy (Sf) large and small peritoneal macrophages (LPM and SPM), isolated from either non-treated mice or following PM transfer to Sf and Wt mice, respectively. Data are depicted as MFI for each surface marker expressed by single cells and shown as heatmap (B) or bar graphs (C) . n = 28–62 cells per experimental group, each sample contains pooled cells from more than 8 donor mice. Statistical analyses were performed using one-way ANOVA, *** p < 0.001.

Article Snippet: Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany).

Techniques: Control, Isolation, Marker