IL 10 drives the re establishment of peritoneal macrophage populations in bacterial peritonitis

IL-10 drives the re-establishment of peritoneal macrophage populations in bacterial peritonitis Inauguraldissertation Zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr rer nat.) an der Mathematisch-Naturwissenschaftlichen Fakultaet der Ernst-Moritz-Arndt-Universitaet Greifswald Vorgelegt von Huu Hung Nguyen Geboren am 24.07.1981 In Ho Chi Minh city, Vietnam Greifswald, den 08.08.2011 Dekan: Prof Klaus Fesser Gutachter: Prof Barbara Bröker Gutachter: Prof Steffen Jung Tag der Promotion: 18/11/2011 CONTENTS ! ABBREVIATIONS !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! "! LIST OF TABLES!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! "#! LIST OF FIGURES !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!#! INTRODUCTION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $! Blood monocytes are precursors of several phagocyte populations !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $! 1.1 Blood monocyte populations""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" #! 1.2 Monocyte half-life """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" $! 1.3 Blood monocytes give rise to macrophages and dendritic cells in peripheral tissue"""""""""""""""""""""" %! Macrophages in the peritoneal cavity!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! %! Changes in the peritoneal macrophage populations induced by inflammation !!!!!!!!!!!!!!!!!!!!!!!!!!! &! Animal models to study polymicrobial sepsis: CLP, CASP, and FIP !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '! IL-10 is a regulatory factor of immune response in infection !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! (! 5.1 IL-10 and its receptor""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" &! 5.2 IL-10 is involved in regulating monocyte differentiation in infection"""""""""""""""""""""""""""""""""""""""""""""""" &! Aim of the study !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! )! MATERIALS AND METHODS!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!$*! Instruments !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $*! Buffers and cell culture medium !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $*! Reagents !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $$! Antibodies and antibody-conjugated Microbeads!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $+! Mice!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $,! Inflammatory model!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $,! Sample preparation for FACS analysis !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $,! 7.1 Blood """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""#%! 7.2 Peritoneal wash cells""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""#'! 7.3 Omentum """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""#(! 7.4 Parathymic lymph nodes """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""#(! Bacterial count!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $-! Intracellular staining for iNOS !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $&! 10 Blood monocyte isolation !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $'! 11 Peritoneal macrophage isolation !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $'! 11.1 Peritoneal macrophages from uninfected mice"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""#)! 11.2 Peritoneal macrophages from infected mice """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""#&! 12 Morphological analysis!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $(! ! 13 T cell priming assay in vitro!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $(! 14 Cytokine assays !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $)! 15 Phagocytosis assay in vivo !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +*! 16 Adoptive cell transfer experiments !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +$! 16.1 Monocyte transfer to the peritoneum """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""$#! 16.2 Peritoneal cell transfer """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""$#! 17 IL-10 application!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +$! 18 Statistical analyses !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +$! RESULTS!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!++! Macrophages in the peritoneal cavity of normal mice!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ++! 1.1 Flow cytometry analysis """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""$$! 1.2 Functional analysis: T cell priming assay in vitro """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""$$! 1.3 Functional analysis: phagocytosis assay in vivo """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""$'! Turnover of R1 and R2 cell populations!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +-! Maintainance of R1 macrophage integrity by blood monocytes !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +&! Resident peritoneal macrophage populations rapidly disappear in polymicrobial sepsis – they associate with the omentum !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +(! Re-formation of peritoneal macrophage populations after infection !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ,$! 5.1 Flow cytometry analysis """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""%#! 5.2 Functional analysis: T cell priming assay in vitro """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""%#! 5.3 Functional analysis: phagocytosis assay in vivo """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""%$! Peritoneal macrophage populations after infection in CX3CR-1+/GFP mice!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ,,! 6.1 CX3CR-1 expression shows that the R2-like macrophage population is heterogeneous """"""""""""%%! 6.2 R2-like B macrophages not differ from R2-like A macrophages in phagocytosing apoptotic cells in vivo """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""%'! 6.3 R2-like B macrophages produce less iNOS in vivo than R2-like A macrophages""""""""""""""""""%(! 6.4 Cytokine and chemokine profiles of R2-like B macrophages differ from those of R2-like A macrophages""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""%*! Peritoneal macrophage populations are derived from blood monocytes in infection!!!!!!!!!!!!!! ,(! 7.1 Re-establishment of peritoneal macrophage populations from blood monocytes"""""""""""""""""""""""""%&! 7.2 Monocyte populations have non-identical differentiation potentials in infection """""""""""""""""""""""""'+! The re-established macrophage populations rapidly leave the peritoneum in the later phase of infection !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! %$! Blood monocytes are continueously recruited to the infected peritoneum in the later phase of infection but they change their differentiation program to give rise to R1-like macrophages !! %%! 10 The formation of R2-like macrophages during infection correlates in time with the production of IL-10!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! %&! 11 IL-10 signalling is required for the rapid re-establishment of R2-like macrophages !!!!!!!!!!! %'! 11.1 Peritoneal wash macrophage populations in IL-10-/- mice """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""')! ! 11.2 Peritoneal wash macrophage populations in IL-10R1-/- mice""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""',! 12 IL-10 targets monocytes to drive their differentiation into R2-like macrophages !!!!!!!!!!!!!!!!! %)! 13 The IL-10R1+/+ monocytes have the possibility to drive the signalling incompetent IL-10R1-/monocytes into the R2-like phenotype in infection !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! -$! 14 Administration of exogenous IL-10 alone is sufficient to drive the generation of R2 macrophages from monocytes in untreated mice!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! -,! DISCUSSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! Dynamics of peritoneal resident macrophages under steady state conditions !!!!!!!!!!!!!!!!!!!!!!!!!! ! Dynamics of peritoneal macrophages in wild type mice after bacterial infection!!!!!!!!!!!!!!!!!!!! -&! Re-establishment of the peritoneal macrophage populations !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! -(! The R2-like macrophage population in CX3CR-1+/GFP mice!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! -)! The re-established macrophage populations undergo a second phase of re-organization !!!!! -)! Mechanisms which regulate the choice of macrophage fate in the peritoneum!!!!!!!!!!!!!!!!!!!!!!!! -)! SUMMARY!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!&,! REFFERENCES!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!&-! ERKLÄRUNG!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'$! CURRICULUM VITAE !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'+! PUBLICATIONS!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!',! ACKNOWLEDGMENTS!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'%! ! ! ABBREVIATIONS AF647 AlexaFluor 647nm APC Antigen-presenting cell APC (fluorochrome) Allophycocyanin BAL Bronchoalveolar lavage BMQC 2,3,6,7-tetrahydro-9-bromomethyl-1H,5H-quinolizino(9,1-gh)coumarin CASP Colon ascendens stent peritonitis CBA Cytometry based assay CC Ceacal content CCL-2 CC motif chemokine ligand CCR-2 CC motif chemokine receptor CD Cluster of differentiation CFSE Carboxyfluorescein diacetate succinimidyl ester cfu Colony forming unit CLP Ceacal ligation and puncture CO2 Carbon dioxide CSF-1 Colony-stimulating factor CSF-1R Colony-stimulating factor receptor CX3CL-1 CX3C motif chemokine ligand or fractalkine CX3CR-1 CX3C motif chemokine receptor CXCL CXC motif chemokine ligand Cy Cyanine DAMP Damage-associated molecular pattern DAPI 4’,6-diamidino-2-phenylidole dihydrochloride DC Dendritic cell DMSO Dimethyl sulfoxide DSS Dextran sulphate sodium EDTA Ethylenediaminetetraacetic acid eF450 eFluor 450nm FACS Fluorescence-activated cell sorting FCS Fetal calf serum FIP Fecal-induced peritonitis FITC Fluorescein isothiocyanate GFP Green fluorescent protein GM-CSF Granulocyte-macrophage colony stimulating factor ! i i.p Intraperitoneally i.v Intravenously ICAM Intercellular adhesion molecule IFN-! Interferon gamma IL Interleukin IL-R Interleukin receptor iNOS Inducible NO synthase KC Keratinocyte chemoattractant LFA-1 Lymphocyte function-associated antigen LN Lymph node LPM Large peritoneal macrophage LPS Lipopolysaccharide M-CSF Macrophage colony stimulating factor MACS Magnetic-activated cell sorting MCP-1 Monocyte-chemoattractant protein MDP Macrophage/dendritic cell progenitors MDR Macrophage disappearance reaction MFI Mean fluorescent intensity MHC-II Major histocompatibility complex class II MIP Macrophage inflammatory protein MSP-R Macrophage-stimulating protein receptor NLR NOD-like receptors NO Nitric oxide OVA Ovalbumin PAMP Pathogen-associated molecular pattern PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline PCR Polymerase chain reaction PE Phycoerythrin PerCP Peridinin chlorphyll protein PHK-1 Paul Karl Horan PI Propidium iodide pLN Parathymic lymph node rmIL Recombinant mouse interleukin RPMI Roswell Park Memorial Institute SEM Standard error of the mean SPM Small peritoneal macrophage ! ii TCR T cell receptor TG Thioglycolate TGF-" Transforming growth factor beta Tip-DC TNF-a and inducible NO synthase-producing dendritic cell TLR Toll-like receptors TNF-# Tumor necrosis factor alpha V450 Violet 450 ! iii LIST OF TABLES Table 1: List of antibodies used for flow cytometry analysis 12 Table 2: List of antibody or streptavidin conjugated Microbeads used for cell sorting by MACS 13 Table 3: Total numbers of CD45+ leukocytes recovered from the peritoneum, omentum, and parathymic LNs of uninfected and infected BALB/c mice 29 ! iv LIST OF FIGURES Figure 1: Macrophage populations in the peritoneum of normal BALB/c mice 23 Figure 2: T cell priming assay in vitro for peritoneal macrophage populations under steady state conditions 24 Figure 3: In vivo phagocytosis activity of peritoneal macrophage populations in the steady state 25 Figure 4: Stability of leukocyte populations in the normal peritoneum 26 Figure 5: Blood monocytes differentiate into R1 macrophages in the normal peritoneum 27 Figure 6: The number of aecrobes (CFU) recovered from the peritoneum day after i.p injection of 15mgCC 28 Figure 7: Macrophage populations in the peritoneum, omentum, and parathymic lymph nodes 30 Figure 8: T cell priming assay in vitro for peritoneal macrophage populations formed days after infection 32 Figure 9: In vivo phagocytosis activity of peritoneal macrophage populations formed days after infection 33 Figure 10: Macrophage populations in CX3CR-1+/GFP transgenic mice 34 Figure 11: Comparison of the phagocytic activity of peritoneal R2-like A and R2-like B macrophages 35 Figure 12: iNOS expression in peritoneal macrophage populations in CX3CR-1+/GFP transgenic mice 36 Figure 13: Cytokine and chemokine production in peritoneal R2-like A and R2-like B macrophages 37 Figure 14: The fate of resident leukocyte populations after infection 38 Figure 15: Blood monocytes differentiate into R2 macrophages in the infected peritoneum 39 Figure 16: Distinct differentiation potentials of monocyte subsets in the infected peritoneum 40 Figure 17: The relationship between macrophage populations after infection 42 Figure 18: The second phase of macrophage disappearance after days of infection 44 Figure 19: Apoptosis in peritoneal macrophage populations after infection 45 Figure 20: IL-10 production in the peritoneal cavity following infection 46 Figure 21: Circulating monocyte subsets and peritoneal macrophage populations in BALB/c IL10-/- mice 48 Figure 22: Circulating monocyte subsets and peritoneal macrophage populations in C57BL/6 IL10R1-/- mice 49 Figure 23: IL-10R1 expression on the blood monocyte subsets 50 Figure 24: IL-10R1 on monocytes is necessary to drive R2-like macrophage generation 51 ! v Huu Hung Nguyen DISCUSSION the signalling incompetent grafted monocytes generated R2 cells Indeed, in this case the IL10R1-/- grafted cells were converted to R2 cells with even higher efficiency than were the IL10R1+/+ cells shown in Figure 24B This shows that the essential interaction of IL-10 with its receptor does not need to take place on the cell which will thereafter be directed to the R2 niche The grafted monocytes in Figure 25A develop efficiently and in an IL-10 dependent manner into R2 cells even though they are unable to respond to IL-10 Figure 27: A predicted mechanism of IL-10 directed R2 macrophage generation For this reason the model had to be modified by assigning IL-10 an essential but less direct role in directing the differentiation of monocytes to R2 cells In its modified form (Fig 27), IL-10 interacts with the monocytes in the peritoneum and causes them to release a mediator X In analogy to IL-2 signalling in T cell activation (Malek, 2008) we propose that factor X will then bind to its receptor on monocytes to initiate the R2 differentiation program As is the situation with IL-2 on T cells, factor X may operate not only to stimulate the cell which produced it in an autocrine fashion, but also – albeit with reduced efficiency – to stimulate IL-10R1-/- deficient bystander cells in a paracrine fashion This provides an explanation for the generation of IL-10R1+/+ monocyte derived R2 cells in an IL10R1-/- host in Figure 24B In this case factor X can only be produced by the small number of grafted monocytes The factor will therefore be limiting and, although the autocrine activation loop functions, it is not sufficient to drive all of the grafted cells to R2 Not surprisingly, the level of ! 61 Huu Hung Nguyen DISCUSSION factor X is also too low to stimulate the host cells in a paracrine fashion In contrast, in the experiment shown in Figure 25A the grafted monocytes cannot themselves respond to IL-10 but all of the host monocytes can The large number of host cells produce factor X which becomes non-limiting and so all monocytes – host and graft derived – are efficiently driven in the direction of R2 This model was confirmed by the experiment shown in Figure 25B that increasing the number of grafted IL-10R1+/+ monocyte results in an increase in the concentration of factor X in the peritoneum and now the host IL-10R1-/- monocytes can be driven into the R2 direction The weakness of this model is that we are forced to propose the existence of factor X, a mediator whose molecular nature is quite unknown However, the basic outline of the model can still be tested because it makes the clear prediction that differentiation, even of IL-10R-/monocytes, to the R2 state can only take place in the presence of a sufficient level of IL-10 As shown in Figure 20, the IL-10 concentration in the infected peritoneum peaks day after infection and falls off rapidly thereafter Were our model to be correct then the generation of R2 cells by the IL-10 dependent pathway would be operative only over the first few days of infection This is exactly what we see As shown in Figure 18, 10 days after infection, when the IL-10 level in the peritoneum has returned to background levels, R2 cells can no longer be efficiently formed Because of this we hypothesised that in a normal peritoneum, where no detectable IL-10 is present, the injection of rmIL-10 would stimulate the generation of R2 cells This is indeed the case (Fig 26) In this protocol the grafted monocytes are directed along the R2 differentiation pathway even in a non-inflammatory environment In this work we have delineated the processes by which macrophage populations in the mouse peritoneum are formed and maintained These experiments open up new questions of which the most pressing is certainly the nature of Factor X Further work will be needed to pursue this point Possibly micro-array anaylsis might reveal potential mediators produced by monocytes in the inflamed peritoneum at early stages of the infection which would bring us the next crucial step forward.! ! 62 Huu Hung Nguyen SUMMARY SUMMARY ! The aim of this thesis work was to explore the physiological and functional properties of peritoneal macrophage populations in both the steady state and in inflammatory conditions In the steady state there are two populations of macrophages in the peritoneum which I refer to as the R1 and R2 populations The R1 cells are a rapidly turning over population which constitute around 20% of the peritoneal macrophages I show that these cells have the capacity to efficiently present peptides on MHC-II to CD4+ T cells but that they are poor at phagocytosis Monocytes transferred into the un-infected peritoneum give rise almost exclusively to this R1 population, suggesting that the R1 fate is the default pathway of monocyte development under steady state conditions In contrast, the R2 population in the peritoneum turns over very slowly in the steady state and is composed of cells which are poor at the presentation of peptide to T cells but which are efficient at phagocytosis Both of these populations are lost from the peritoneum within an hour of the induction of a poly-microbial peritonitis A large fraction of the R2 population relocates from the peritoneal wash fraction to the omentum, the fate of the R1 population is less clear Over the course of the next three days, the macrophage populations in the peritoneum are re-established Transfer experiments using genetically marked cell populations demonstrated that neither the R1 nor the R2 populations which “disappeared” one hour after infection contributes to the reestablished peritoneal wash fraction macrophage pool at day While the re-established R1 population retains the functional properties and the FACS phenotype of the steady state R1 cells, the re-established R2-like population is clearly not identical to the R2 cells present in the pre-infection environment In particular, this R2-like population can be split into two subpopulations which have non-identical functional properties In this inflammatory situation monocytes transferred into the peritoneum now acquire the capacity to differentiate not only into R1-like cells but also into R2-like macrophages I looked for the molecular basis driving this change of monocyte differentiation in the infected peritoneum by using a solid phase cytometry based ELISA procedure to examine the spectrum of cytokines produced in the peritoneum in response to poly-microbial infection One of the most prominent cytokines produced early in infection is IL-10 To determine whether IL10 is directly involved in assigning monocyte fate in the peritoneum I looked at the ability of mice carrying a targeted deficiency of either the IL-10 gene or of the IL-10 receptor gene to form the R2-like cells after infection Neither mouse strain efficiently generates the R2-like ! 63 Huu Hung Nguyen SUMMARY population after infection Adoptive transfer of genetically marked wild type or mutant monocytes into appropriate hosts demonstrated that the effect of IL-10 is not direct Rather, the IL-10 responding cell produces a mediator which then directs monocyte fate Thus, the bystander IL-10R deficient monocytes are driven by the mediator produced by wild type monocytes to generate R2 cells with high efficiency The crucial role of this IL-10 dependent pathway was underscored by supplementation experiments Mice carrying a targeted deficiency of the IL-10 gene fail to generate the R2 population during peritonitis However, injection of IL-10 into these animals rescues the capacity to form the R2 population In addition the normal default pathway of monocyte development in un-infected animals which leads to the R1 population is modulated by injection of IL-10 so that the monocytes can now differentiate into the R2 population The work presented in this thesis describes the steady state populations of phagocytes in the un-infected peritoneum and the dynamics of these populations during the induction of peritonitis It also uncovers an IL-10 dependent pathway which regulates the choice of monocyte developmental fate within the peritoneum.! 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67 Huu Hung Nguyen REFFERENCES Moore, K.W., Vieira, P., Fiorentino, D.F., Trounstine, M.L., Khan, T.A., and Mosmann, T.R (1990) Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI Science 248, 1230-1234 Muller, W (2006) Dissecting the cytokine network Cell Immunol 244, 162-164 Murphy, K.M., Heimberger, A.B., and Loh, D.Y (1990) Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo Science 250, 1720-1723 Niess, J.H., Brand, S., Gu, X., Landsman, L., Jung, S., McCormick, B.A., Vyas, J.M., Boes, M., Ploegh, H.L., Fox, J.G., et al (2005) CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance Science 307, 254-258 Ouyang, W., Rutz, S., Crellin, N.K., Valdez, P.A., and Hymowitz, S.G (2011) Regulation and functions of the IL-10 family of cytokines in inflammation and disease Annu Rev Immunol 29, 71109 Pils, M.C., Pisano, F., Fasnacht, N., Heinrich, J.M., Groebe, L., Schippers, A., Rozell, B., Jack, R.S., and Muller, W (2010) Monocytes/macrophages and/or neutrophils are the target of IL-10 in the LPS endotoxemia model Eur J Immunol 40, 443-448 Quah, B.J., Warren, H.S., and Parish, C.R (2007) Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester Nat Protoc 2, 2049-2056 Rajakariar, R., Lawrence, T., Bystrom, J., Hilliard, M., Colville-Nash, P., Bellingan, G., Fitzgerald, D., Yaqoob, M.M., and Gilroy, D.W (2008) Novel biphasic role for lymphocytes revealed during resolving inflammation Blood 111, 4184-4192 Randolph, G.J., Beaulieu, S., Lebecque, S., Steinman, R.M., and Muller, W.A (1998) Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking Science 282, 480-483 Rangel-Moreno, J., Moyron-Quiroz, J.E., Carragher, D.M., Kusser, K., Hartson, L., Moquin, A., and Randall, T.D (2009) Omental milky spots develop in the absence of lymphoid tissue-inducer cells and support B and T cell responses to peritoneal antigens Immunity 30, 731-743 Rice, L., Orlow, D., Ceonzo, K., Stahl, G.L., Tzianabos, A.O., Wada, H., Aird, W.C., and Buras, J.A (2005) CpG oligodeoxynucleotide protection in polymicrobial sepsis is dependent on interleukin-17 J Infect Dis 191, 1368-1376 Robben, P.M., LaRegina, M., Kuziel, W.A., and Sibley, L.D (2005) Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis J Exp Med 201, 1761-1769 Roers, A., Siewe, L., Strittmatter, E., Deckert, M., Schluter, D., Stenzel, W., Gruber, A.D., Krieg, T., Rajewsky, K., and Muller, W (2004) T cell-specific inactivation of the interleukin 10 gene in mice results in enhanced T cell responses but normal innate responses to lipopolysaccharide or skin irritation J Exp Med 200, 1289-1297 Schopf, L.R., Hoffmann, K.F., Cheever, A.W., Urban, J.F., Jr., and Wynn, T.A (2002) IL-10 is critical for host resistance and survival during gastrointestinal helminth infection J Immunol 168, 2383-2392 Schulz, O., Jaensson, E., Persson, E.K., Liu, X., Worbs, T., Agace, W.W., and Pabst, O (2009) Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions J Exp Med 206, 3101-3114 Secher, T., Vasseur, V., Poisson, D.M., Mitchell, J.A., Cunha, F.Q., Alves-Filho, J.C., and Ryffel, B (2009) Crucial role of TNF receptors and in the control of polymicrobial sepsis J Immunol 182, 7855-7864 ! 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69 Huu Hung Nguyen REFFERENCES Varol, C., Landsman, L., Fogg, D.K., Greenshtein, L., Gildor, B., Margalit, R., Kalchenko, V., Geissmann, F., and Jung, S (2007) Monocytes give rise to mucosal, but not splenic, conventional dendritic cells J Exp Med 204, 171-180 Wang, Z., Rui, T., Yang, M., Valiyeva, F., and Kvietys, P.R (2008) Alveolar macrophages from septic mice promote polymorphonuclear leukocyte transendothelial migration via an endothelial cell Src kinase/NADPH oxidase pathway J Immunol 181, 8735-8744 Zhang, X., Majlessi, L., Deriaud, E., Leclerc, C., and Lo-Man, R (2009) Coactivation of Syk kinase and MyD88 adaptor protein pathways by bacteria promotes regulatory properties of neutrophils Immunity 31, 761-771 ! ! ! 70 Huu Hung Nguyen ACKNOWLEDGMENTS ! ERKLÄRUNG ! Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der MathematischNaturwissenschaftlichen 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 08.08.2011 Huu Hung Nguyen ! 71 Huu Hung Nguyen ACKNOWLEDGMENTS ! CURRICULUM VITAE Personal infromation Name: Huu Hung Nguyen Address: C204/179A Doan Van Bo Str., District 4, Ho Chi Minh city, Vietnam Gender: Male Date of birth: July 24th 1981 Place of birth: Ho Chi Minh city, Vietnam Nationality: Vietnamese Education and work experience 2006-2011 Ph.D student at the Institute of Immunology and Transfusion medicine, University of Greisfwald, Germany 2004-2006 Diploma equivalence in Life science, Joint educational Training Center Hanoi-Greifswald program 2003-2004 Researcher and teaching assistant, Department of animal and human physiology, University of natural sciences, Ho Chi Minh city, Vietnam 1999-2003 Bachelor of science in Biotechology, University of Natural Sciences, Ho Chi Minh city, Vietnam Greifswald, August 08th, 2011 Huu Hung Nguyen ! 72 Huu Hung Nguyen ACKNOWLEDGMENTS ! PUBLICATIONS ! Manuscript in preparation Nguyen, H H., Tran, B T., Chandode, R., Hildebrandt, P., Mueller, W., Broeker, B., and Jack, R., IL-10 drives the re-establishment of peritoneal macrophage populations in bacterial peritonitis Posters Nguyen, H H and Jack, R., March 2009 The origin and turnover of peritoneal phagocyte populations 5th Spring school on Immunology Ettal, Bavaria, Germany 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 Nguyen, H H., Jack, R., June 2010 IL-10 regulates macrophage formation in acute peritonitis Conference: “Host-pathogen interactions in bacterial infections” Greifswald, Germany Greifswald, August 08th, 2011 Huu Hung Nguyen ! 73 Huu Hung Nguyen ACKNOWLEDGMENTS ! ACKNOWLEDGMENTS ! The success of the thesis would have been impossible without the support of many people Firstly, I would like to express my deep gratitude to my supervisors Prof Barbara Broeker and Prof Robert Jack for giving me a chance to work in the Department of Immunology and guiding me through my study For financial support, I would like to express my thanks to: The Ministry of Education and Training (MOET) of Vietnam Deuscher Akademischer Austausch Dienst/German Academic Exchange Service (DAAD) Prof Fritz Scholz, Institute for Biochemistry, University of Greifswald, and the Graduate school “Studies of the interation of free oxygen radicals with molecules at electrodes and applications to biochemical and medical systems” Prof Barbara Broeker, Institute of Immunology and Transfusion medicine, University of Greifswald and Graduate school “Host-pathogen interactions in generalized bacterial infections” (Graduiertenkolleg GRK 840/3) I would like to thank Prof Uwe Voelker and Dr Petra Hildebrandt, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, for permitting me to use the FACSaria sorter I am grateful to thank Dr Van Trung Chu, Dr Claudia Berek, and Jenny Kirsch, Deutsche Rheuma Forschungs Zentrum Berlin, for supplying DO11.10 mice and FACS training I wish to thank Prof Werner Mueller, University of Manchester, Prof Lars Nitschke, University of Erlangen-Nuernberg, and Prof Reinhold Forster, University of Hannover, for supplying C57BL/6 IL-10R1-/- mice, BALB/c CD45.1 mice, and BALB/c CX3CR1+/GFP transgenic mice, respectively I would like to thank the Visceral, Thoracic and Vascular Surgery Group, Clinic for General Surgery, University of Greifswald, for sharing FACS and cytokine measurement reagents I wish to thank my colleague Bich Thu Tran for sharing her omental cell preparation protocol, reagent organization, and useful disscussion and Rakesh Chandode for sharing IL-10-/- mice ! 74 Huu Hung Nguyen ACKNOWLEDGMENTS ! and for his PCR protocol I would like to express my thank to all Vietnamese friends for enjoying and spending studenttime together in Greifswald Finally, the heartiest thank to my family, my mother Thi Mai Nguyen, and my engaged partner Thi Truc Nga Vo for the motivation of success.! ! 75 [...]... can, in turn, be targets of IL- 10 since they all constitutively express the IL- 10 receptor (IL- 10R) This receptor is composed of two subunits: the IL- 10R1 (" subunit) which specifically binds to IL- 10 and IL- 10R2 (! subunit) which acts as a signal transducing element for the IL- 10R1 as well as for the receptors of other members of the IL- 10 family Thus the IL2 2 receptor consists of IL- 22R1 and IL- 10R2,... distinct populations and this raises the question of whether both populations follow the same kinetics of turnover 3 Changes in the peritoneal macrophage populations induced by inflammation Infection in the peritoneal compartment is called peritonitis Two dominant effects on the cellular components are seen during peritonitis: the disappearance of resident leukocyte populations and the recruitment of. .. peritoneal macrophages were destroyed Transplantation of bone marrow cells led to the reestablishment of the macrophage populations in this comparment but the cells are of graftorigin This data demonstrates that peritoneal macrophages can be derived from precursors in the bone marrow However, the mechanisms which contribute to the re- establishment are still not answered The dynamics of peritoneal macrophage. .. and, in ways which are not well understood, to orchestrate resolution of the inflammation and initiate tissue repair by the secretion of TGF-!1 (Arnold et al., 2007) Since the current findings suggest that the two peritoneal macrophage populations have distinct functions in peritoneal immunity (Ghosn et al., 2 010) , it will be important to better understand how these macrophage populations are re- established... days of culture in the presence of granulocyte -macrophage colony stimulating factor (GM-CSF) and IL- 13 In contrast, when the monocytes were cultured in the presence of macrophage colony stimulating factor (M-CSF), they differentiated into cells which have a macrophage phenotype (express CD14, CD68, and show enhanced phagocytosis activity) However, when IL- 10 was added into the DC-differentiation culture... for 5min and the cells then stained with different appropriate combinations of fluorchrome-conjugated antibodies (see above, section 16.1) TruCount beads were added for cell counting The cells were then washed once and analysed by FACS.! 17 IL- 10 application 100 ng or 200ng of recombinant mouse IL- 10 prepared in 100 µl of PBS was injected ip Two applications were made, the first at the same time of cell... least 5 days (Melnicoff et al., 1989) This study suggested that on day 7 after inflammation the labelled macrophages re- appeared which was evidenced by an increased number of PKH-1+ cells relative to that in untreated animals Since the PKH-1 dye intensity in the cells recovered on day 7 was only barely distinguishable from background, the results were interpreted as suggesting that these macrophages have... of the peritoneal phagocyte populations present ! '! Huu Hung Nguyen INTRODUCTION after resolution of inflammation The finding that the peritoneal macrophage population is heterogeneous makes a re- examination of their dynamics during peritonitis necessary After the loss of resident peritoneal macrophages, peritonitis is characterized by the accumulation of granulocytes from the circulation and of monocytes... 0.2ml of cell preparations was loaded in the cytospin cuvettes and centrifuged at 800 x g for 3min Slides were dried and stained with Giemsa according to the manufacturer’s protocol Briefly, the slides were fixed in methanol for 10min, air dried, and immersed in Giemsa solution for 45min The slides were then rinsed in distilled water, air dried, and the cells were examined with an AXIO Imager using a 100 X... monocytes may be recruited to different inflamed tissues by a similar mechanism involving the local production of MCP-1 and CX3CL-1, but once the monocytes enter the inflamed tissue then their fate depends on tissue specific factors The mechanisms which control the fate of monocytes in these various comparments, in particular in the peritoneum, requires further investigation 2 Macrophages in the peritoneal