Journal: Cell host & microbe

Article Title: Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

doi: 10.1016/j.chom.2017.11.009

Figure Lengend Snippet: Figure 2. Rapid PMN Intravascular Chemotaxis to Sequestered C. albicans Is Complement Dependent (A) Lung C5a levels were determined using ELISA in control and C3/ animals, following i.v. C. albicans. Two-way ANOVA, Sidak’s multiple comparison. (B) Using intravital microscopy, PMN chemotactic behaviors in C3/ and anti-C5aR mAb-treated mice were observed. Images shown are 10 min after C. albicans injection. Red arrows highlight yeast not recognized by PMN. Scale bar represents 30 mm. (C) Quantification of PMN chemotaxing to pathogens in C3/ and C5a receptor-blocked mice. One-way ANOVA, Dunnett’s multiple comparison. (D) The percentage of C. albicans phagocytosed during the initial 10 min of intravenous administration in control, C3/, and anti-C5aR mAb-treated mice using pulmonary intravital microscopy. t tests were used to compare C57BL/6 and anti-C5aR mAb treated (*) or C3/ (#) mouse time points. (E and F) C. albicans CFU was quantified in organs at (E) 1 hr and (F) 24 hr after injection with 1 3 106 C. albicans in mice treated with anti-C5a receptor antibodies or isotype. (G) Clinical sepsis scores of isotype and anti-C5aR mAb antibody treated mice 24 hr after injection with 1 3 106 C. albicans. A score of 21 is the clinical endpoint for euthanization. For (A)–(F), at least three individual experiments were performed; for (G) three individual experiments were performed. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Ly6G (clone 1A8) BioLegend RRID: AB_2563207 (BioLegend Cat. No. 127636) Anti-CD31 (clone MEC13.3) BioLegend RRID: AB_2161030 (BioLegend Cat. No. 102515) Anti-CD45 (clone 30-F11) BioLegend RRID: AB_493532 (BioLegend Cat. No. 103121) Anti-CD49b (clone HMa2) BD Bioscience Catalog No. 558295 Anti-C5aR (CD88, clone 20/70) BioLegend RRID: AB_2067286 (BioLegend Cat. No. 135804) Purified anti-mouse Ly6G (clone 1A8) BioXCell BP0075-1 Anti-TER119 Biolegend RRID: AB_528961 (BioLegend Cat. No. 116218) Anti-human CD15 (clone W6D3) BioLegend RRID: AB_756018 (BioLegend Cat. No. 323012) Anti-human CD45 (clone 2D1) R&D Systems Catalog # FAB1430G Anti-human C5aR (CD88, clone S5/1) BioLegend RRID: AB_2259318 (BioLegend Cat. No. 344302) Bacterial and Virus Strains C. albicans (strain ATCC58716) ATCC LUMC-101 Chemicals, Peptides, and Recombinant Proteins Zymosan A (S. cerevisiae) BioParticles ThermoFisher Z23373 Escherichia coli (K-12 strain) BioParticles ThermoFisher E13231 SYTO 9 Green Fluorescent Nucleic Acid Stain ThermoFisher S34854 Compstatin Tocris Cat. No. 2585 LY293111 Cayman Chemical Company CAS N 161172-51-6 Recombinant mouse C5a R&D Systems 2150-C5-025 Critical Commercial Assays LTB4 Parameter Assay Kit R&D Systems KGE006B EasySep Mouse Biotin Positive Selection Kit STEMCELL Technologies Catalog # 18556 Mouse Complement C5a PicoKine ELISA Kit Boster Biological Technology EK0987 Polydimethylsiloxane (PDMS) microfluidic devices SynvivoBio Cat # 104001 Experimental Models: Cell Lines human lung microvascular endothelial cells HLMEC Lonza CC-2527 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Stock No: 000664 LTB4R knockout (B6.129S4-Ltb4r1tm1Adl/J) The Jackson Laboratory Stock No: 008102 C3 / ; (B6;129S4-C3tm1Crr/J) Gift from Paul Kubes N/A CD11b / ; (B6.129S4-Itgamtm1Myd/J) Gift from Paul Kubes N/A CD11a / ; (B6.129S7-Itgaltm1Bll/J) Gift from Paul Kubes N/A LysM-GFP Gift from Paul Kubes N/A Ly6G-cre (C57BL/6-Ly6g(tm2621(CretdTomato)Arte)) Gift from Matthias Gunzer N/A Software and Algorithms Volocity PerkinElmer N/A Leica LAS Leica N/A Prism 7 software GraphPad V7.02 FlowJo FlowJo V10.1 e1 Cell Host & Microbe 23, 1–13.e1–e4, January 10, 2018

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Control, Comparison, Intravital Microscopy, Injection

Journal: Cell host & microbe

Article Title: Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

doi: 10.1016/j.chom.2017.11.009

Figure Lengend Snippet: Figure 4. PMN Vascular Clustering during Candidemia Is Dependent on LTB4 In Vivo (A) PMN clustering (white) is compared among C57BL/6, C3/, and anti-C5aR mAb-treated mice following 1 hr of i.v. live C. albicans. Scale bar, 200 mm. (B and C) (B) Mean cluster area and (C) max skeletal length of clusters were compared. One-way ANOVA, Holm-Sidak’s multiple comparison. (D and E) Lung LTB4 levels of C57BL/6 and C3/ mice (D), two-way ANOVA, Holm-Sidak’s multiple comparison, and following anti-C5aR mAb-treated mice (E). (F) LTB4 in isolated pulmonary PMN with or without C. albicans infection (1 hr). Unpaired two-tailed t test. (G) A representative image of PMN clusters (white) in LTB4R/ mice following C. albicans (1 hr). Scale bar, 200 mm. (H and I) (H) Mean cluster area and (I) max. skeletal length of PMN clusters after C. albicans (1 hr). (J–L) 1 3 107 A488 zymosan or 1 3 107 A488 E. coli bioparticles (green) were injected i.v. during lung imaging. (J) Intravital images are shown at 60 min. Scale bar, 100 mm. Clusters were quantified as (K) max skeletal length and (L) mean cluster area. ANOVA was performed for panels with more than two groups, while unpaired two-tailed t test were used to compare two groups. n = at least 3 for each panel. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Ly6G (clone 1A8) BioLegend RRID: AB_2563207 (BioLegend Cat. No. 127636) Anti-CD31 (clone MEC13.3) BioLegend RRID: AB_2161030 (BioLegend Cat. No. 102515) Anti-CD45 (clone 30-F11) BioLegend RRID: AB_493532 (BioLegend Cat. No. 103121) Anti-CD49b (clone HMa2) BD Bioscience Catalog No. 558295 Anti-C5aR (CD88, clone 20/70) BioLegend RRID: AB_2067286 (BioLegend Cat. No. 135804) Purified anti-mouse Ly6G (clone 1A8) BioXCell BP0075-1 Anti-TER119 Biolegend RRID: AB_528961 (BioLegend Cat. No. 116218) Anti-human CD15 (clone W6D3) BioLegend RRID: AB_756018 (BioLegend Cat. No. 323012) Anti-human CD45 (clone 2D1) R&D Systems Catalog # FAB1430G Anti-human C5aR (CD88, clone S5/1) BioLegend RRID: AB_2259318 (BioLegend Cat. No. 344302) Bacterial and Virus Strains C. albicans (strain ATCC58716) ATCC LUMC-101 Chemicals, Peptides, and Recombinant Proteins Zymosan A (S. cerevisiae) BioParticles ThermoFisher Z23373 Escherichia coli (K-12 strain) BioParticles ThermoFisher E13231 SYTO 9 Green Fluorescent Nucleic Acid Stain ThermoFisher S34854 Compstatin Tocris Cat. No. 2585 LY293111 Cayman Chemical Company CAS N 161172-51-6 Recombinant mouse C5a R&D Systems 2150-C5-025 Critical Commercial Assays LTB4 Parameter Assay Kit R&D Systems KGE006B EasySep Mouse Biotin Positive Selection Kit STEMCELL Technologies Catalog # 18556 Mouse Complement C5a PicoKine ELISA Kit Boster Biological Technology EK0987 Polydimethylsiloxane (PDMS) microfluidic devices SynvivoBio Cat # 104001 Experimental Models: Cell Lines human lung microvascular endothelial cells HLMEC Lonza CC-2527 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Stock No: 000664 LTB4R knockout (B6.129S4-Ltb4r1tm1Adl/J) The Jackson Laboratory Stock No: 008102 C3 / ; (B6;129S4-C3tm1Crr/J) Gift from Paul Kubes N/A CD11b / ; (B6.129S4-Itgamtm1Myd/J) Gift from Paul Kubes N/A CD11a / ; (B6.129S7-Itgaltm1Bll/J) Gift from Paul Kubes N/A LysM-GFP Gift from Paul Kubes N/A Ly6G-cre (C57BL/6-Ly6g(tm2621(CretdTomato)Arte)) Gift from Matthias Gunzer N/A Software and Algorithms Volocity PerkinElmer N/A Leica LAS Leica N/A Prism 7 software GraphPad V7.02 FlowJo FlowJo V10.1 e1 Cell Host & Microbe 23, 1–13.e1–e4, January 10, 2018

Techniques: In Vivo, Comparison, Isolation, Infection, Two Tailed Test, Injection, Imaging

Journal: Cell host & microbe

Article Title: Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

doi: 10.1016/j.chom.2017.11.009

Figure Lengend Snippet: Figure 7. Anti-fungal Activity and Clustering of Human PMN Is Complement and LTB4 Dependent (A) Images of C5aR inhibition on PMN and C. albicans interactions in a human lung microvascular chamber (anti-C5aR-antibody 20 mg/mL). (B) C. albicans phagocytosis by PMN. (C) Residual immobilized but non-phagocytosed yeast PMN. (D and E) (D) Cluster formation and (E) the number of clustered PMN. (F) Images of LTB4R inhibition on PMN and C. albicans interactions in a human lung microvascular chamber. (G) Quantification of immobilized but non-phagocytosed yeast at 30 min. (H and I) (H) Clusters per field of view (FOV) and (I) PMN that were clustered per FOV were determined at 30 min. All data are expressed as mean ± SEM for n = 5–7. Student’s t tests were used to compare data between two groups. *p < 0.05 and **p < 0.01. All scale bars represent 20 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Ly6G (clone 1A8) BioLegend RRID: AB_2563207 (BioLegend Cat. No. 127636) Anti-CD31 (clone MEC13.3) BioLegend RRID: AB_2161030 (BioLegend Cat. No. 102515) Anti-CD45 (clone 30-F11) BioLegend RRID: AB_493532 (BioLegend Cat. No. 103121) Anti-CD49b (clone HMa2) BD Bioscience Catalog No. 558295 Anti-C5aR (CD88, clone 20/70) BioLegend RRID: AB_2067286 (BioLegend Cat. No. 135804) Purified anti-mouse Ly6G (clone 1A8) BioXCell BP0075-1 Anti-TER119 Biolegend RRID: AB_528961 (BioLegend Cat. No. 116218) Anti-human CD15 (clone W6D3) BioLegend RRID: AB_756018 (BioLegend Cat. No. 323012) Anti-human CD45 (clone 2D1) R&D Systems Catalog # FAB1430G Anti-human C5aR (CD88, clone S5/1) BioLegend RRID: AB_2259318 (BioLegend Cat. No. 344302) Bacterial and Virus Strains C. albicans (strain ATCC58716) ATCC LUMC-101 Chemicals, Peptides, and Recombinant Proteins Zymosan A (S. cerevisiae) BioParticles ThermoFisher Z23373 Escherichia coli (K-12 strain) BioParticles ThermoFisher E13231 SYTO 9 Green Fluorescent Nucleic Acid Stain ThermoFisher S34854 Compstatin Tocris Cat. No. 2585 LY293111 Cayman Chemical Company CAS N 161172-51-6 Recombinant mouse C5a R&D Systems 2150-C5-025 Critical Commercial Assays LTB4 Parameter Assay Kit R&D Systems KGE006B EasySep Mouse Biotin Positive Selection Kit STEMCELL Technologies Catalog # 18556 Mouse Complement C5a PicoKine ELISA Kit Boster Biological Technology EK0987 Polydimethylsiloxane (PDMS) microfluidic devices SynvivoBio Cat # 104001 Experimental Models: Cell Lines human lung microvascular endothelial cells HLMEC Lonza CC-2527 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Stock No: 000664 LTB4R knockout (B6.129S4-Ltb4r1tm1Adl/J) The Jackson Laboratory Stock No: 008102 C3 / ; (B6;129S4-C3tm1Crr/J) Gift from Paul Kubes N/A CD11b / ; (B6.129S4-Itgamtm1Myd/J) Gift from Paul Kubes N/A CD11a / ; (B6.129S7-Itgaltm1Bll/J) Gift from Paul Kubes N/A LysM-GFP Gift from Paul Kubes N/A Ly6G-cre (C57BL/6-Ly6g(tm2621(CretdTomato)Arte)) Gift from Matthias Gunzer N/A Software and Algorithms Volocity PerkinElmer N/A Leica LAS Leica N/A Prism 7 software GraphPad V7.02 FlowJo FlowJo V10.1 e1 Cell Host & Microbe 23, 1–13.e1–e4, January 10, 2018

Techniques: Activity Assay, Inhibition

Journal: Molecular Neurobiology

Article Title: High-Fat Diet-Induced Excessive Accumulation of Cerebral Cholesterol Esters and Microglial Dysfunction Exacerbate Alzheimer's Disease Pathology in APP NL−G−F mice

doi: 10.1007/s12035-025-05052-8

Figure Lengend Snippet: HFD feeding reduced TREM2 expression in microglia of APP NL–G–F mice brain. ( a - b ) Representative images of somatosensory cortex and hippocampus from APP NL–G–F mice fed an ND or an HFD for 27 weeks. Sections were stained with BAN50 (blue), IBA1 (green) and TREM2 (red). Scale bar = 50 μm. ( c - d ) Quantification of TREM2 immunofluorescence intensity of cortex and hippocampus from APP NL–G–F mice. Each dot represents an analyzed mouse, and 35 images were taken from each mouse brain for cortical analysis and 10 images were taken from each mouse brain for hippocampal analysis ( n = 4 mice per group). ( e – f ) Mean fluorescence intensity (MFI) graph of TREM2 + microglia isolated from cortex and hippocampus tissue samples from APP NL–G–F mice fed with ND or HFD for 27 weeks, as determined by flow cytometry with gating on CD45 and CD11b. ( g , j ) Percentage of CD45 + CD11b + cell population among all living single cells from the cortex and hippocampus tissue samples of APP NL–G–F mice. ( h , k ) Percentage of TREM2 + cell population within CD45 + CD11b + cell population from the cortex and hippocampus tissue samples of APP NL–G–F mice. ( i , l ) MFI of TREM2 in the CD45 + CD11b + population from the cortex and hippocampus tissue samples of APP . NL–G–F mice. Values are average ± SEM. Statistical analyses were performed using an unpaired t-test (* p < 0.05, ** p < 0.01; ns, not significant)

Article Snippet: After removing red blood cells and debris, cells were blocked with Fc Receptor Blocking Reagent (1: 100) for 15 min at room temperature and then stained with PE anti-mouse/human CD11b Antibody (1:50, BioLengend, Cat. No. 101208, Clone M1/70), Alexa Fluor 488 anti-mouse CD45 Antibody (1:50, BioLengend, Cat. No. 103122, Clone 30-F11) and Human/Mouse TREM2 APC-conjugated Antibody (R&D Systems, Catalog #FAB17291 A) for 30 min at room temperature.

Techniques: Expressing, Staining, Immunofluorescence, Fluorescence, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.

doi: 10.1016/j.celrep.2020.108073

Figure Lengend Snippet: Figure 6. BIRC2 Knockdown in B16F10 Cells Increases Anti-tumor Immunity by Increasing CXCL9 Expression (A) NTC and BIRC2-KD subclones were implanted into C57BL/6 mice. When BIRC2-KD tumors became palpable, mice were treated with anti-CXCL9 or IgG every 3 days. Tumor volumes were determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). (B–E) Tumors were harvested on day 35, and the percentage of CD8+ T cells (relative to CD45+ population) (B), CD8+/CD44+/CD69+ T cells (C), CD3/NK1.1+ NK cells (D), and CD11b+/CD11c+/F4/80 DCs (E) was determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). All immune cell populations (except B) were calculated as a percentage of the total live cells (based on forward and side scatter). (F–H) The Pearson correlation test was performed to compare CXCL9 mRNA expression with CD8+ T cell score (F), NK cell score (G), and DC score (H), using TCGA data from 481 human melanomas. See also Figures S3C and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020

Techniques: Knockdown, Expressing