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