Volume 23, Issue 1, Pages 121-133.e4 (January 2018) Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis  Esther K.S. Lee, Mark R. Gillrie, Lu Li, Jason W. Arnason, Jung Hwan Kim, Liane Babes, Yuefei Lou, Amir Sanati-Nezhad, Stephen K. Kyei, Margaret M. Kelly, Christopher H. Mody, May Ho, Bryan G. Yipp  Cell Host & Microbe  Volume 23, Issue 1, Pages 121-133.e4 (January 2018) DOI: 10.1016/j.chom.2017.11.009 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2018 23, 121-133. e4DOI: (10. 1016/j. chom. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 The Lung Is a Site of PMN-Mediated Host Responses against Bloodstream C. albicans (A) Organ CFU at 1 hr or 24 hr of candidemia. Unpaired Student’s t test within each organ. (B) Flow cytometry of Ly6G+ Syto9+ C. albicans containing PMN 5 min after i.v. injection. Blue contour plots are from uninfected controls. (C) Quantification of Ly6G+ Syto9 C. albicans+ cells from (B). One-way ANOVA, Tukey’s multiple comparison. (D and E) PMN-depleted mice or isotype control had C. albicans CFU determined after (D) 1 hr or (E) 24 hr of fungemia. Red lines highlight the delta mean change. Two-tailed Mann-Whitney test. (F and G) (F) Clinical sepsis severity scores and (G) hypothermia were determined in mice pre-depleted of PMN following 24 hr of candidemia. Two-tailed Mann-Whitney test. (H) Syto9+ C. albicans were visualized in real time following intravenous administration using resonant scanning pulmonary intravital microscopy. (I) The percentage of adherent and tethering C. albicans was determined within the pulmonary circulation. (J) Intravital imaging and tracking of lung PMN (blue) in response to live Syto9+ C. albicans (green). Scale bar represents 33 μm. (K) Quantification of PMN targeting each individual C. albicans within the pulmonary vasculature was quantified. Symbols represent n values for (A) and (D)–(G). Error bars represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Cell Host & Microbe 2018 23, 121-133.e4DOI: (10.1016/j.chom.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

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 μm. (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 × 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 × 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. Cell Host & Microbe 2018 23, 121-133.e4DOI: (10.1016/j.chom.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Intravascular PMN Swarming Leads to Clusters in the Lung Circulation In Vivo (A) Clusters were observed using pulmonary intravital microscopy. PMN cluster (blue) and live Syto9+ C. albicans (green). (B) Top: PMN cluster growth (blue) and PMN crawling tracks (white lines). Captured Syto9+ C. albicans are observed. Bottom: Vector diagrams demonstrate the magnitude and direction of movement of the cluster forming PMN (red arrows) and the leading edge of the cluster (black arrow). (C) PMN were manually tracked and divided into swarming (white tracks) versus non-swarming PMN (red tracks). (D) PMN displacement was compared between swarming and non-swarming PMN. Unpaired two-tailed t test. (E) PMN (white) are shown just prior to and 1 hr following i.v. live C. albicans. Scale bar represents 200 μm. (F) PMN clusters were quantified. One-way ANOVA, Dunnett’s multiple comparison. (G and H) Mean cluster area (G) and cluster max skeletal length (H) were determined. Unpaired two-tailed t test. n = at least 3 for each panel. Error bars represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Cell Host & Microbe 2018 23, 121-133.e4DOI: (10.1016/j.chom.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

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 μm. (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 μm. (H and I) (H) Mean cluster area and (I) max. skeletal length of PMN clusters after C. albicans (1 hr). (J–L) 1 × 107 A488 zymosan or 1 × 107 A488 E. coli bioparticles (green) were injected i.v. during lung imaging. (J) Intravital images are shown at 60 min. Scale bar, 100 μm. 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. Cell Host & Microbe 2018 23, 121-133.e4DOI: (10.1016/j.chom.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 LTB4 Is Not Required for Host Defense but Drives PMN Clustering, Pulmonary Hemorrhage, Hypoxemia, and Worse Clinical Outcomes (A) Quantification of C. albicans phagocytosed during the initial 10 min of infection. (B) The number of PMN chemotaxing to individual C. albicans. (C) PMN displacement during chemotaxis was quantified. (D) Fungal CFU in the organs of mice 1 hr and 24 hr after C. albicans infection. Unpaired two-tailed t test. (E) Pulmonary blood flow was visualized using fluorescent dextran (green) i.v. without or 1 hr after C. albicans. Scale bar represents 75 μm. (F) Hypoxemia was determined using pulse oximetry and (G) pulmonary hemorrhage was quantified in the BAL using flow following 4 hr of intravenous C. albicans. (H) Histology following 4 hr of infection or control. (I) Hypothermia and (G) clinical severity scores were measured 24 hr after C. albicans infection. Unpaired two-tailed t test. For (A)–(C), (E), and (H) represent a minimum of n = 3. For other panels, n = the symbols shown. Error bars represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. Cell Host & Microbe 2018 23, 121-133.e4DOI: (10.1016/j.chom.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 C. albicans Induces Rapid Human PMN Phagocytic Activity, Clustering, and Vascular Occlusion within a Human Pulmonary Microvasculature Model (A) Micrographs of C. albicans, leukocytes (CD45), PMN (CD15), and DNA (Höescht 33342, blue) by epifluorescence confocal microscopy. (B) Real-time C. albicans-leukocyte interactions on endothelial cells were assessed by spinning-disc confocal microscopy. (C) Quantification of recruited cells. (D) Percentage of immobilized C. albicans that were phagocytosed by PMN at 30 min. (E and F) (E) C. albicans was quantified within or outside of clusters and (F) recruited PMN associated in a cluster versus not in a cluster is shown at 30 min. (G) Cluster formation in an ibidi flow chamber. (H) C. albicans-induced PMN clusters at 30 min in a human microvascular PDMS microfluidic device. All data are expressed as mean ± SEM for n = 5–7. Student’s paired t test was used to compare data between two groups, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. All scale bars represent 20 μm. Cell Host & Microbe 2018 23, 121-133.e4DOI: (10.1016/j.chom.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

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 μg/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 μm. Cell Host & Microbe 2018 23, 121-133.e4DOI: (10.1016/j.chom.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions