Role of neutrophil NADPH oxidase derived reactive oxygen species (ROS) in innate immune responses

Role of neutrophil NADPH oxidase derived reactive oxygen species (ROS) in innate immune responses Inauguraldissertation zur Erlangung des akademischen Grades doctor rerum nuturalium (Dr rer nat.) and der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von TRAN Bich Thu geboren am 06.12.1980 in Ho Chi Minh Stadt, Viet Nam Greifswald, den 29 März 2012 Dekan: Prof Dr Klaus Fesser Gutachter: Prof Dr Reinhard Walther Gutachter: Prof Fritz Ulrich Schade Tag der Promotion: Greifswald, July 20th 2012 Table of contents SUMMARY INTRODUCTION INNATE IMMUNE SURVEILLANCE 1.1 Sentinel systems of innate immunity 1.2 Innate system effector cells PRODUCTION OF ROS OF CELLS OF THE INNATE IMMUNE SYSTEM 2.1 What are ROS? 2.2 Source in mitochondria 2.3 NADPH oxidase 10 FUNCTIONS OF ROS OF CELLS OF THE INNATE IMMUNE SYSTEM 12 3.1 Role(s) in killing bacteria 12 3.2 Detection of biologically relevant ROS 14 3.3 ROS as a signalling component 14 OBJECTIVES OF THIS WORK 16 4.1 Bacterial killing 16 4.2 ROS as signalling elements 17 MATERIALS AND METHODS 18 MATERIALS 18 1.1 Instruments 18 1.2 Laboratory equipment 18 1.3 Reagents 19 1.4 Buffers and solutions 20 1.5 Antibodies 21 1.6 Software 22 1.7 Mice 22 METHODS 22 2.1 Gold preparation 22 2.2 Anaesthetics 22 2.3 Gold implantation 23 2.4 Organ sampling in mice 23 2.5 Inflammatory models 24 2.6 Effect of chemokines on the recruitment of neutrophils into the peritoneal cavity 24 2.7 Sample preparation for flow cytometry analysis 24 2.8 Isolation and subsequent analysis of murine mononuclear cells attaching to gold implants 27 RESULTS 29 DETECTION OF ROS IN PHAGOCYTES 29 1.1 Detection of extracellular ROS in vivo 29 1.2 Detection of intracellular ROS ex vivo 29 EXTRACELLULAR ROS 31 2.1 The effect of extracellular phagocyte derived ROS on the surface of gold implants in vivo 31 CELL POPULATIONS IN THE PERITONEAL CAVITY AND THEIR ASSOCIATION WITH THE IMPLANT 32 3.1 Cell populations in the peritoneal cavity of untreated mice 32 3.2 Implant associated phagocyte populations 33 3.3 Peritoneal wash phagocyte population after implantation 35 3.4 Implants as inducers of peritoneal inflammation 38 ROS AND MECHANISMS OF LEUKOCYTE EXTRAVASATION AFTER INFLAMMATORY STIMULI 41 4.1 Expression of cell adhesion molecules on neutrophils 41 4.2 Chemokines 45 4.3 ROS production and the ability to extravasate 46 4.4 In vivo competiton assay for gp91phox-deficient and wild type neutrophil recruitment to the peritoneum 48 4.5 gp91phox-deficient and wild type neutrophil recruitment in peritoneal inflammation 52 4.6 gp91phox deficient and wild type neutrophil recruitment: chemokine application 53 4.7 gp91phox deficient and wild type neutrophil recruitment in sterile peritoneal inflammation 54 DISCUSSION 57 REFFERENCES 62 ERKLÄRUNG 68 CURRICULUM VITAE ERROR! BOOKMARK NOT DEFINED PUBLICATIONS 71 ACKNOWLEDGMENTS 72 Doctoral dissertation List of Abbreviations ABS: Anti-lock braking system AFM: Atomic force microscopy APC: Allophycocyanin ATP: Adenosine triphosphate BPI: Bactericidal/permeability-increasing protein BSA: Bovine serum albumin CC: Ceacal contents CD: Cluster of differentiation CG: Cathepsin G CGD: Chronic granulomatous disease CRMP-2: Collapsin response mediator protein DAPI: 4',6-diamidino-2-phenylindole DHR: Dihydrorhodamine 123 DNA: Deoxyribonucleic acid DMSO: Dimethyl sulfoxide Duox: Dual oxidase EDTA: Ethylene diamine tetra acetic acid EGF: Epidermal growth factor ESAM: Endothelial cell selective adhesion molecule FACS: Fluorescent activated cell sorting FcR: Fc receptor FCS: Foetal calf serum FITC: Fluorescein isothiocyanate fMLP: N-formyl-methionine-leucine-phenylalanine FP: Fenton polished FPR: Formyl methionyl peptide receptor GAG: Glucose amino glycans GPx: Glutathione peroxidase HBSS: Hanks' balanced salt solution ICAM: Intercellular adhesion molecule IL: Interleukin IMP: Implantation JAM: Junctional adhesion molecule List of abbreviations Doctoral dissertation List of abbreviations KC: Keratinocyte chemo-attractant K/o: Knock out LDL: Low density lipoprotein LFA-1: Lymphocyte-associated functional antigen-1 LIX: LPS-induced CXC chemokine LPS: Lipopolysaccharide MCP-1: Monocyte chemotactic protein-1 MIP-2: Macrophage inflammatory protein-2 MP: Mechanically polished MPO: Myeloperoxidase NALP3: NACHT, LRR and PYD domains-containing protein-3 NE: Neutrophil elastase NET: Neutrophil extracellular traps NGAL: Neutrophil gelatinase–asssociated lipocalin PAF: Platelet activating factor PBS: Phosphate buffered saline PCR: Polymerase chain reaction PDGF: Platelet derived growth factor PE: Phycoerythrin PECAM-1: Platelet/endothelial cell adhesion molecule-1 PerCp: Peridinin chlorphyll protein PMA: Phorbol-12-myristate-13-acetate Phox: Phagocyte oxidase PR3: Proteinase PRR: Pathogen Recognition Receptor PSGL-1: P-selectin glycoprotein ligand-1 Rh-123: Rhodamine-123 ROS: Reactive oxygen species SDS: Sodium dodecyl sulphate SOD: Superoxide dismutase TG: Thioglycollate TNF: Tumour necrosis factor TLR: Toll like receptor VCAM: Vascular cell adhesion molecule VLA: Very late antigen Doctoral dissertation List of figures List of figures Figure The tripartite area code Figure The electron transport chain in the mitochondrion Figure Regulation of the phagocyte NADPH oxidase complex 11 Figure Atomic force micrographs of a polished gold surface 29 Figure Rhodamine generation by blood neutrophils 30 Figure The effect of extracellular phagocyte derived ROS on the surface of gold implants in vivo 32 Untreated C57BL6 peritoneal cell wash contains various immune cell populations 33 Figure Cell populations attaching to the gold surfaces 34 Figure Cell populations in the peritoneal wash fraction 14 days after implantation 35 Figure 10 Comparison of cell populations in the peritoneal cell wash and on the mechanically polished gold surfaces 36 Ex vivo ROS production of phagocyte populations in the peritoneal cell wash after implantation 37 Comparison of cell populations in the peritoneal cell wash of wild type mice at hours and 14 days after implantation 38 Figure 13 Quantitation of granulocyte populations early in inflammation 39 Figure 14 Kinetics of granulocyte population change in inflammation 40 Figure 15 Peritoneal cell wash of wild-type BALB/c and of congenic eosinophil ablated mice 18h after surgery 40 Figure 16 Adhesion molecule expression on blood neutrophils from untreated mice 42 Figure 17 Expression of α7 and β2 integrins on neutrophils from wild type and gp91phox knock-out mice after being challenged with commensal flora for hours 43 Expression of α7 and β2 integrins on neutrophils from wild type and gp91phox knock-out mice after being challenged with thioglycollate for hours 44 MFI of α7 and β2 integrins of blood and peritoneal neutrophils from wild type and gp91phox knock-out hours after treatment 45 Figure 20 KC, LIX or MIP-2 induce neutrophil recruitment into the peritoneal cavity 46 Figure 21 Ex vivo ROS production of neutrophils in blood of untreated and in the blood and peritoneal cell wash 3h after caecal content induced peritonitis 47 (A) C57BL6 peritoneal cell wash 6h after implantation (IMP) and caeceal content induced peritonitis (CC); (B) Ex vivo ROS production of peritoneal neutrophils 6h after implantation and caecal content induced peritonitis 47 Comparison of cell populations in the peritoneal wash of wild-type and gp91phox knock-out mice hours after caecal content induced peritonitis 48 Figure Figure 11 Figure 12 Figure 18 Figure 19 Figure 22 Figure 23 Doctoral dissertation List of figures Figure 24 Assay for the identification of mosaic neutrophil subpopulations in gp91+/ko heterozygous mice Figure 25 +/ko Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 Figure 31 Flow cytomectric analysis of peripheral blood and bone marrow of gp91 heterozygous mice 49 50 phox The ratio of wild type to gp91 deficient neutrophil subpopulations of +/ko one gp91 heterozygous female mouse determined on day 1, and 51 phox Distribution of wild type and gp91 -deficient blood neutrophil subpopulations in gp91+/ko heterozygous female mice 51 Wild type neutrophils have an advantage in entering the peritoneal cavity early after induction of inflammation 52 phox Wild type and gp91 deficient neutrophils have the same ability to enter the peritoneum after being treated with chemokines 54 phox Wild type and gp91 deficient neutrophils have the same ability to enter the peritoneum in a sterile peritoneal inflammation (thioglycollate model) 55 (A) MFI of α7 and β2 integrins of peritoneal neutrophils from wild type and gp91 knock-out mice hours after treatment (B) In the gp91+/ko heterozygous female mice, wild type neutrophils have an advantage in entering the peritoneal cavity in the CC but not in the TG inflammatory models 56 Doctoral dissertation List of tables List of tables Table List of antibodies used for flow cytometry analysis 20 Doctoral dissertation Summary SUMMARY I have investigated the role played by reactive oxygen species (ROS) generated by the phagocyte NADPH oxidase system in the innate immune response I first looked at effector functions by asking whether ROS released from phagocytes might be effective in the killing of extracellular bacteria Since bacteria can be killed in many other ways – for example by proteases or by cationic peptides – I made use of the recently demonstrated capacity of ROS to remove discontinuities from the surface of gold as the basis of an in vivo assay for extracellular ROS Unlike bacterial killing, this readout system is not affected by enzymes, cationic peptides or other biological anti-bacterial agents By this means I was able to use wild type mice and a congenic strain which lacks the gene coding for the gp91 subunit of the phagocyte NADPH oxidase to demonstrate that ROS generated by the NADPH oxidase system are indeed found outside the cells during an inflammation in vivo and that their principle source is neutrophil granulocytes rather than tissue macrophages Since ROS released by these cells will be non-specific in its action it is to be expected that the releasing cell will itself suffer considerable damage This fits well to the known short life of activated neutrophils and may explain the established fact that their death is dependent on the NADPH oxidase system The long lived macrophages, in contrast, restrict their production of extracellular ROS ROS are increasingly being found to be involved in both intra and intercellular signalling processes I looked for an involvement of NADPH oxidase derived ROS in the recruitment of neutrophils to sites of inflammation in vivo Since the gene coding for the gp91 subunit of the NADPH oxidase is on the X chromosome I made use of a mosaic expression strategy based on X chromosomal inactivation The results show that indeed ROS serves as a component of the neutrophil recruitment process in the critical early stages of an infection Possible mechanisms are explored Doctoral dissertation Discussion search for an involvement of ROS in neutrophil recruitment in mice a sensitive mosaic assay was developed which makes use of the fact that the gene for the gp91 subunit of the phagocyte NADPH oxidase is on the X chromosome Because of random X chromosome inactivation in females mice heterozygous for an inactivating mutation of this gene are mosaic and will produce two types of neutrophils – those which express the wild type copy of the gene and those which express the mutant As with other such genes – for example the X chromosome located PigA gene responsible for the formation of GPI anchors – the fraction of mutant and wild type cells is not necessarily : (Keller, Tremml et al 1999) The reason for this is believed to be that the randomness of the X chromosome inactivation is a statistical truth, but the size of the stem cell pools which give rise to the various blood cell lineages is quite small and may permit deviation from the ideal Nevertheless the ratio of wild type to mutant cells is sufficiently stable within one animal to permit a mosaic analysis at least over short periods of time Using this assay I found clear evidence for a requirement of the NADPH oxidase system for the recruitment of neutrophils into the infected peritoneum such that NADPH oxidase competent cells were preponderant at early time points However, in contrast to the situation in the zebra fish the requirement for ROS in the mouse is not in the target tissue but rather in the responding neutrophils These responding neutrophils flood into the peritoneum in very large numbers to combat a bacterial infection and the early phase of the infection may be crucial in determining the outcome Interestingly the advantage of the ROS generating system for the recruitment of the neutrophils appears to be restricted to infection situations since it is not apparent when a sterile inflammation is initiated using thioglycollate treatment This suggests that the detection of bacterial products by sentinel cells of the peritoneal cavity via so−called “pathogen recognition receptors” may be necessary to initiate this response By intravenous adoptive transfer of isolated neutrophils from both C57BL/6 wild type and gp91phox - deficient mice, into thioglycollate treated recipients, Hattori and colleagues noted a reduced peritoneal recruitment of the gp91phox knock-out compared with the wild type neutrophils (Hattori, Subramanian et al 2010) Using the same model, we did not observe any differences in the peritoneal recruitment of gp91phoxdeficient and wild type neutrophil subpopulations in the gp91+/ko heterozygous female 59 Doctoral dissertation Discussion mice The differences may come from the isolation and ex vivo manipulation of neutrophils since neutrophils isolated by density gradient centrifugation may induce ROS production in neutrophils and lead to high backgrounds (Rebecchi, Ferreira Novo et al 2000; Fuchs, Abed et al 2007) An alternative explanation may be that the thioglycollate medium used to induce the sterile inflammation is not a pure substance and the medium once prepared must - for reasons that remain unclear - be aged before use Though widely used, thioglycollate treatment is not an optimally reproducible inflammatory system Nevertheless, the results reported by Hattori et al certainly go in the same direction as my results The NADPH oxidase system is inactive in resting cells and an inflammatory signal such as that delivered through the TNFR1 is required to trigger the assembly of the active complex on the membrane At some point in the extravasation process this complex must be activated and must underlie the recruitment advantage of the NADPH competent neutrophils which is detected at hours post infection The precise role of ROS in this process remains to be elucidated but it is noteworthy that the recruitment advantage is no longer seen at 12 hours after infection The reasons for this are unclear and given the complexities of the extravasation process they may be difficult to pin down Two simple none mutually exclusive scenarios may explain this observation The first makes use of the fact that the NADPH oxidase is required not only for the influx of the cells to the site of inflammation, but it also is required for their death by netosis shortly after activation (Honda, Kano et al.; Brinkmann, Reichard et al 2004; Fuchs, Abed et al 2007) Thus, while cells expressing the wild type allele may have an initial advantage in their rate of entry, the mutant cells will have an extended life time and hence build up in the peritoneal neutrophil population This may explain the apparent loss of the extravasation advantage at the 12 hour time point A second alternative type of explanation might be that the loss of the preference for cells expressing the wild type allele of the NADPH oxidase gp91 gene may simply be a kinetic effect resulting from the massive neutrophil influx into the peritoneum Over the first hours the average rate of neutrophil entry into the peritoneum is x 104 neutrophils per minute but this figure fails to take account of the fact that at the start of this time period only a small number of cells is involved but this number rises rapidly over the ensuing period It is conceivable that an initial ROS dependent step, necessary to open an extravasation 60 Doctoral dissertation Discussion “door”, results in the advantage for the cells expressing the wild type allele in the initial phase but that the system is rapidly swamped by the large number of cells entering the peritoneum so that the mutant cells can now enter an “open door” by a bystander effect Since the NADPH oxidase system is inactive in resting cells and must be assembled in response to activation, my results indicate that neutrophils have achieved a degree of activation prior to their entry into the peritoneum How is this activation achieved – and where? Recruitment of these cells requires the action of chemokines and so I looked at the ability of murine chemokines known to be involved in neutrophil extravasation to differentially attract the wild type and mutant cells Of those tested − KC, LIX and MIP2 − all functioned to promote neutrophil extravasation but none preferentially attracted the NADPH oxidase competent wild type cells This lack of an extravasation advantage for NADPH oxidase competent cells in response to purified chemokine application is reminiscent of the situation in which a sterile inflammatory response is induced in the peritoneal cavity Here too the presence of an active NADPH oxidase system provides no advantage in the in vivo mosaic assay Unlike the situation after a bacterial peritonitis, no special contribution of ROS is required for the response to these chemokines However, a flow cytometry analysis of integrin expression on neutrophils during the early phase of infection demonstrated that the NADPH competent, but not the mutant cells, up regulated the surface expression of both the α7 and β2 integrins This correlative observation may provide a starting point for a future analysis of the molecular mechanism of ROS dependent differential extravasation In summary this study demonstrates that extracellular ROS are released from neutrophils in amounts which may be significant in terms of direct extracellular bacterial killing It further provides evidence for an involvement of ROS in the process of neutrophil extravasation in response to infection 61 Doctoral dissertation Refferences REFFERENCES Akira, S., S Uematsu, et al (2006) "Pathogen recognition and innate immunity." 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Nature 464(7285): 104-7 67 Doctoral dissertaion Erklärung ERKLÄRUNG Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Mathematisch Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde Ferner erkläre ich, daß ich diese Arbeit selbständig verfasst und keine anderen als die darin angegebenen Hilfsmittel und Hilfen benutzt und keine Textabschnitte eines Dritten ohne Kennzeichnung übernommen habe Greifswald, den 29 März 2012 TRAN Bich Thu 68 Doctoral dissertaion Curriculum Vitae Curriculum Vitae TRAN Bich Thu Address : Geschwister Scholl Straße 11d, Zi 225 17491 Greifswald Telephone : 49(0) 176-61260182 Email : bichthu80@yahoo.com PERSONAL DATA Date of Birth : December, 06th, 1980 Place of Birth : Ho Chi Minh City Nationality : Vietnam Marital status : Single Gender : Female EDUCATION 11/2006 − 06/2012 PhD student at the Institute of Medical Biochemistry and Institute of Immunology and Transfusion Medicine, University of Greisfwald, Germany PhD thesis "Role of neutrophil NADPH oxidase derived reactive oxygen species (ROS) in innate immune responses" under the guidance of Prof R.S.Jack and Prof R Walther 02/2005 − 12/2005 Participant of the "Diploma Equivalent" program in Life Science, Joint Educational Training Center Ha Noi − Greifswald (Antrag der Universität Greifswald zur künftigen Lehr− und Forschungskooperation mit wissenschaftlichen Einrichtungen in 69 Doctoral dissertaion Curriculum Vitae Ha Noi bei DAAD−Export−Programm Deutscher Studienangebote) 09/1998 − 09/2002 Bachelor student at the Faculty of Biology, College of Natural Sciences, Viet Nam National University − Ho Chi Minh City RESEARCH EXPERIENCE 01/2009 − 06/2012 University of Greisfwald: Re-routing of innate immune cells in response to peritoneal inflammation 11/2006 − 12/2008 University of Greisfwald: Proteome analysis of lymphoblastic B cell lymphomas from iMycEà mice and their normal counterparts PROFESSIONAL SKILLS ã Mouse in vivo model • Immunological techniques: MACS, FACS, ELISA, CBA • 2D-Electrophoresis • Relevant software: Flowjo, GraphPad Prism, Delta 2D PROFESSIONAL EXPERIENCE 09/2002 − 10/2006 Assistant lecturer and researcher at the Department of Animal Physiology, Faculty of Biology, College of Natural Sciences, Viet Nam National University − Ho Chi Minh City Greifswald, July 23rd 2012 TRAN Bich Thu 70 Doctoral dissertaion Publications PUBLICATIONS Research paper Nguyen, H-H., Tran, B-T., Muller, W., Jack, S.R 2012 IL-10 acts as a developmental switch guiding monocyte differentiation to macrophages during a murine peritoneal infection J Immunology (in press) Poster Nguyen, H H., Hildebrandt, P., Tran, B T., Chandode, R., and Jack, R., September 2009 Altered monocyte differentiation in acute peritonitis 2nd European congress of Immunology Berlin, Germany Greifswald, July 23rd 2012 TRAN Bich Thu 71 Doctoral dissertaion Acknowledgements ACKNOWLEDGMENTS This thesis would not have been written without the financial support from Vietnam through the Ministry of Education and Training (MOET), from Germany through German Academic Exchange Service (DAAD) and Alfried Krupp Kolleg Greifswald via the Graduate college "Studies of the interaction of free oxygen radicals with molecules at electrodes and applications to biochemical and medical systems" Though this dissertation is an individual work, many people have contributed to its completion I am indebted to all who have made it possible and more than that they have made my time and experience in Greifswald become memorable Above all, I am grateful to my two supervisors Prof Reinhard Walther and Prof Robert Smail Jack who gave me the opportunity to work on this project, and then stood by my side during the whole period They always gave me freedom to explore on my own and at the same time the guidance to overcome obstacle and crisis situations They also spent a lot of time discussing my results and contemplating my ideas In addition, I highly appreciate Prof Jack for wielding his gifted red pen to elevate and improve the content of this manuscript I would like to thank Prof Fritz Scholz and his group in the Institute of Biochemistry for their great cooperation In particular, my thanks go to Prof Fritz Scholz, Dr Ulrich Hasse and Katja Vahl for their valuable discussion, AFM measurements, gold preparation and data analysis on the smoothness of gold surfaces I believe I gained knowledge through oral communication just as from all the papers and books that I read My gratitude in this respect goes to Prof Barbara Bröker who put a lot of effort in organising journal clubs, technical seminars, guest lectures and softskill training 72 Doctoral dissertaion Acknowledgements I owe my gratitude to Prof Ivo Steinmetz and Dr Antje Bast in the Institute of Medical Microbiology for the gp91phox knock-out mice Equally indispensable were the support from Dr Van Trung Chu and Dr Claudia Berek who generously provide us the mice carrying a deletion in the GATA-1 promoter I also extend my thanks to Huu Hung Nguyen for sharing his experience in the field of flow cytometry as well as useful discussion along the work I am thankful to all my friends with whom I have shared the best and worst moments of my graduate journey especially Saskia Erttmann I am not exaggerating to say that without her it would have been quite a challenge for me to manage the first few months in Greifswald Finally and most importantly, none of this would have happened without my family especially my parents whom I owe everything for who I am today I am blessed to have my parents and my brother with their unconditional love They are also my source of encouragement, support and strength for all these years 73