TITLE Names . What We Do Question Problem

TITLE Names . What We Do Question Problem

TITLE Names . What We Do Question Problem Methods Conclusions TITLE Maya Amjadi1, Mallary Greenlee-Wacker1 and William M. Nauseef1 1 Inflammation Program and Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine University of Iowa, and Veterans Administration Medical Center, Iowa City, IA 52240 Abstract Overall Hypothesis Methods Summary Future Directions Figure 1. Overall model for the hypothesis that ectosomes from PMN-SA play a role in inflammation. As depicted in Figure 1, PMN-SA produce ectosomes, resist efferocytosis, and eventually lyse. Here we tested the hypothesis that ectosomes isolated from PMN-SA play a proinflammatory role in the local inflammation that accompanies a S. aureus infection. Introduction Figure 2. Method for isolating ectosomes for use in characterization and functional assays. PMN are isolated from 200ml of heparinized blood. Mid-log USA300 is opsonized with 10% pooled human serum, washed, and incubated with PMN for 20 minutes at a 10:1 MOI. Ectosomes are isolated by a series of centrifugations to remove PMN and bacteria and ultracentrifugation to recover ectosomes. Ectosomes are then analyzed for characterization and functional assays. These centrifugation steps are summarized above where supernatant is indicated as S and pellet is indicated as P. Results Ectosomes are positive for CD66b and Phosphatidylserine Ectosomes contain myeloperoxidase

A. A. B. B. Ladder (kDa) MPO Ectosomes Figure 3. Characterizing ectosomes by flow cytometry shows that they are CD66b and Phosphatidylserine (PS) positive. The percentage of cells positive for CD66b and PS was assayed by flow cytometry. Bars represent the average of two experiments standard deviation (A). Shown are histograms from two experiments with unstained ectosomes shown in black and CD66b-stained ectosomes shown in red (B). 2. Determine other functional properties of ectosomes from PMN-SA a. Determine if ectosomes are engulfed by macrophages b. Determine how ectosomes modulate inflammation Acknowledgements Figure 4. Ectosomes contain protein, specifically myeloperoxidase (MPO). Protein was measured and quantified by bicinchoninic acid assay. The average protein concentration from three experiments standard deviation is shown in A. MPO and 50 g of protein from ectosome preparations were resolved on a 5-20% gradient gel. MPO was detected by immunoblotting (B, n = 3).

Ectosomes contain DNA 1. Characterize the macrophage cytokine profile in response to ectosomes from PMN-SA a. Measure the dose response to ectosomes b. Measure the cytokine profile Macrophages treated with ectosomes from PMN-SA release elevated levels of TNF- The authors thank Kevin Leidal, Sally McCormick-Hill, Sara Hinde and Sarah Bloomberg for technical assistance. This work was supported by funding from the Iowa Center for Research by Undergraduates (ICRU), the Dewey Stuit Fund for Undergraduate Research and the Department of Biology at the University of Iowa. The Nauseef lab is also supported by the National Institute of Health Grants AI70958 and AI044642 (to W.M.N) and by a Merit Review award and use of facilities at the Iowa City Department of Veterans Affairs (VA) Medical Center, Iowa City, IA 52246. References Deleo FR, Otto M, Kreiswirth BN, Chambers HF. (2010) Community-associated methicillin-resistant Staphylococcus aureus. Lancet 375, 1557-1568. Gasser, O., and Schifferli, J.A. (2004) Activated polymorphonuclear neutrophils disseminate anti-inflammatory microparticles by ectocytosis. Blood 104, 2543-2548. Gasser, O., Hess, C., Miot, S., Deon, C., Sanchez, J.C., Schifferli, J.A. (2002) Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils. Experimental Cell Research 285, 243-257. Greenlee-Wacker, M.C., Rigby, K.M., Kobayashi, S.D., Porter, A. R., DeLeo, F. R., Nauseef, W.M. (2014) Phagocytosis of Staphylococcus aureus by Human Neutrophils Prevents Macrophage Efferocytosis and Induces Programmed Necrosis. J Immunol 192 4709-4717. Figure 5. Ectosomes isolated from PMN-SA contain nucleic acids. PMN (A) and ectosomes (B) were left unstained (black) or were stained with SYTO13 Green-Fluorescent Nucleic Acid Stain (blue) and analyzed by flow cytometry. DNA was isolated from ectosomes using Dneasy Blood and Tissue kit from Qiagen, and DNA was quantified by nanodrop. DNA [103.0ng] was resolved on a 0.7% agarose gel and stained with Midori Green Advanced DNA stain. Shown is a representative experiment (C, n = 2). Figure 6. Pro-inflammatory cytokine TNF- is released by macrophages treated with PMN-SA ectosomes. Human monocyte-derived macrophages were treated with 0-1g/ml ectosomes and TNF- was quantified by ELISA. Bars represent the average of triplicate wells standard deviation (n=1). Schwartz J, Leidal KG, Femling JK, Weiss JP, Nauseef WM: Neutrophil bleaching of GFP-expressing staphylococci: probing the intraphagosomal fate of individual bacteria. J Immunol 2009; 183, 2632-2641. Timar, C.I., Lorincz, A. M., Cspnyi-Kmi, R., Vlyi-Nagy, A., Nagy, G., Buzs, E.I., Ivnyi, Z., Kittel, ., Powell, D. W., McLeish, K. R., Ligeti, E. (2013) Antibacterial effect of microvesicles released from human neutrophilic granulocytes. Blood. 121, 510-518.

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