- Email: [email protected]
Formyl peptide receptors from the innate immune system and the vomeronasal organ recognize pathogen derived peptides
Abstracts
cells (PBMC). Depletion of CD14+ myeloid cells from PBMC diminished the adiponectin-dependent T cell activation mediated by ADS-MS sera. We suggest that abnormally elevated levels of adiponectin could contribute to enhanced inflammatory states of both innate and adaptive immune responses implicated in early disease mechanisms of pediatriconset MS. doi:10.1016/j.jneuroim.2014.08.242
288 Dispensable role for TLRs 2,3,4,7,9 in the mouse of multiple sclerosis Pushpalatha Palle, Thorsten Buch Institute for Medical Microbiology, Immunology and Hygiene, Technische Universitat of Munich, Munich, Germany Multiple sclerosis (MS) is a demyelinating inflammatory disorder of the central nervous system (CNS), which involves autoimmune responses against myelin antigens. MS is usually considered to be a predominantly T cell mediated autoimmune disease. To understand the initiation phase of the disease it is necessary to study danger signal pathways that may be responsible for driving the immune response. Therefore, we have started to focus on innate immune mechanisms. Toll-like receptors (TLRs) are key components of the innate immune system and play a crucial role in interplay between adaptive immunity and innate immunity. They recognize unique molecular patterns associated with different classes of pathogens and additionally, also recognize some self-proteins and endogenous nucleic acids. Recent studies predominantly from animal models of autoimmune disease and circumstantial data from human patients suggest that inappropriate activation of TLR pathways by endogenous or exogenous ligands may lead to the initiation and perpetuation of autoimmune responses. We have been studying the role of TLRs for the initiation of anti-CNS autoimmunity in the mouse model of the disease, experimental autoimmune encephalomyelitis. We could not confirm a crucial role for TLR9 and for the endosomal TLRs 3, 7, and 9. Further we did not find an effect of absence of TLRs 2,3,4,7 and 9 on the development of anti-CNS autoimmunity. To confirm that TLRs play no role in disease initiation we are currently generating a mouse model lacking signaling by all TLRs. In light of the emerging evidence for the role of the microbiome in initiating and driving autoimmune disease, we consider our observations to be highly relevant to understand how MS is triggered. doi:10.1016/j.jneuroim.2014.08.243
380 Microglia show an intermediate activation status in early lesion formation in multiple sclerosis Laura Peferoena, Daphne Vogela,b, Kimberley Ummenthuma, Marjolein Breurb, Priscilla Heijnenb, Wouter Gerritsena, Paul Van Der Valka, Christine Dijkstrab, Sandra Amora a
Pathology Department, VU Medical Centre, Amsterdam, Netherlands; MCBI, VU Medical Centre, Amsterdam, Netherlands
b
Microglia play an important role during lesion formation in multiple sclerosis (MS). Well before the peripheral immune systems is involved
91
and any sign of myelin damage is noticeable, foci of activated microglia appear in the normal appearing white matter (NAWM). Many of these so called preactive lesions are suggested to spontaneously resolve rather than developing into destructive lesions. This suggest that intrinsic regulation exists, which can stop lesion progression at an early stage. Activated microglia fulfil a broad range of functions both beneficial and harmful to the environment. Environmental signals are widely considered to contribute to the ‘switch’ between an immunoregulatory (M2) and a pro-inflammatory (M1) phentotype, in preactive lesions such signal might be the key to the developemt of active MS lesions. To characterize microglia phenotypes in preactive lesions, we screened for distinctive markers between M1 and M2 microglia, in vitro. This revealed that CD40, CD80 and CD74 were the most distinctive markers for M1, and manose receptor (MR) for M2. Additionally, M1 microglia produce higher levels of pro-inflammatory chemokines CXCL10 and CCL2, where M2 have an induced production of CCL22. Using the panel of M1 and M2 markers we next examined the activation status of microglia in pre-active MS lesions, remyelinating areas, NAWM and healthy control samples. Our data show that all preactive lesions have an intermediate expression pattern; no pre-active lesions were specifically M1 or M2 phenotype. All activated microglia abundantly express M1 markers CD40, CD86, CD74 and the M2 cytokine CCL22. This indicates an intermediate activation status of microglia in preactive lesions. Furthermore, we showed that polarization of human microglia, in vitro, is not static, but a dynamic process in which microglia can switch from a M1 to a M2 phenotype and vice versa. Taken together, our data delineate the versatility of microglia, both in vitro and in vivo, in response to inflammatory signals from the CNS, already in early MS lesion formation. doi:10.1016/j.jneuroim.2014.08.244
215 Formyl peptide receptors from the innate immune system and the vomeronasal organ recognize pathogen derived peptides Timo Schumann, Bernd Bufe, Frank Zufall Department of Physiology, Saarland University/Medical School Homburg, Homburg, Germany The formyl peptide receptor (Fpr) family is well known for its contribution to immune defense against pathogens in human and rodent leukocytes. Recently, several structurally related members of these receptors were discovered in sensory neurons of the mouse vomeronasal organ (VNO), key detectors of pheromones and related semiochemicals. Although the biological role of vomeronasal Fprs is not yet clear, the known contribution of other Fprs to host innate immune defense suggested that they could contribute to vomeronasal pathogen sensing. To determine the function of vomeronasal Fprs, precise knowledge about their agonist properties of is required. In order to investigate the pharmacological properties of the FPRs, we used high throughput calcium imaging comparing responses of Fprs of the immune and VNO system to more than 100 selected agonists. Our experiments revealed a new class of agonists that is capable of activating both, FPRs of the immune system and the vomeronasal organ. Intriguingly, motifs of these agonists are predominately found in bacteria and distinct pathogenic microorganisms. These data are consistent with the assumed role of FPRs in VNO mediated pathogen detection. Therefore, we propose that FPRs provide a common mechanism for the detection of pathogens by the immune system and the pheromone sensing system.
92
Abstracts
This work was supported by a Graduate Scholarship of the Saarland University for Timo Schumann
496 Role of TREM2 and CD200R in modulating microglial response in astrocyte targeted IL10Tg mice following perforant pathway transection.
doi:10.1016/j.jneuroim.2014.08.245 Kalpana Shrivastavaa, Mireia Recasensa, Beatriz Almoldaa, Iain Campbellb, Bernardo Castellanoa, Berta Gonzaleza 182 Reduced release of nitric oxide and different cytokines/ chemokines by aged microglial cells upon activation of Toll-like receptors 2 and 4 Sandra Schützea,b, Annika Kaufmannb, Sandra Ribesb, Jörg Scheffelb, Sandra Redlichb, Uwe-karsten Hanischb, Wolfgang Brücka, Roland Naub a
Department of Geriatrics, Agaplesion Diakonissen Krankenhaus, Frankfurt am Main, Germany; bInstitute of Neuropathology, University Medical Center, Göttingen, Germany Objectives: The incidence of bacterial infections of the central nervous system (CNS) in adults increases with age, and the clinical outcome in elderly individuals is worse than in young persons. Death in the acute phase of the infection and neurologic or neuropsychologic deficits are common complications, and better therapies are needed. Since microglial cells play a key role for the defence of the brain against bacterial infections, we investigated age-related functional changes of these cells in vitro. Methods: Primary microglial cultures were obtained from brains of C57BL/6-N mice aged 2 months (young) and 18 months (aged). For activation, cells cultured in 96-well-plates were treated with agonists of Toll-like receptor (TLR) 1/2 [Tripalmitoyl-S-glycerylcysteine (Pam3CSK4); 0.1 μg/ml] and TLR 4 [endotoxin (LPS) from Escherichia coli serotype 026:B6; 0.01 μg/ml] for 24 h in the presence of interferon-gamma (IFN-gamma; 100 U/ml). Non-activated control cells were treated with IFN-gamma only. Young and aged microglial cells were compared regarding their release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and CXCL1 (KC) into the cell culture supernatant. Results: Upon stimulation with agonists of TLR 2 and 4, aged microglial cells released significantly less NO and cytokines/ chemokines than young microglial cells: nitrite upon Pam3CSK4 treatment: 6.97 (5.96/8.28) vs. 18.40 (8.78/20.89) μM, p = 0.008; TNF-alpha upon LPS treatment: 284.10 (262.40/300.50) vs. 681.20 (468.50/1024.00) pg/ml, p = 0.002; IL-6 upon Pam3CSK4 treatment: 85.70 (17.86/144.30) vs. 924.90 (82.00/937.90) pg/ml, p = 0.04; KC upon LPS treatment: 15.63 (15.63/24.23) vs. 75.00 (44.10/117.40) pg/ml, p = 0.007 [representative results, concentrations in the cell culture supernatant indicated as median (25./75. percentile), comparison aged vs. young by Mann–Whitney-U-test, n = 5–8]. Conclusion: Our in-vitro data show a reduced release of NO and inflammatory cytokines/chemokines by aged microglial cells upon activation with bacterial TLR agonists. The age-related decline of microglial functions might contribute to the higher susceptibility of elderly individuals to bacterial CNS infections. Strategies to improve the functions of aged microglial cells appear promising for prevention and treatment of CNS infections in elderly patients.
doi:10.1016/j.jneuroim.2014.08.246
a
Unit of Histology, Dept. of Cell Biology, Physiology and Immunology, Institute of Neuroscience, Autonomous University of Barcelona, Barcelona, Spain; bSchool of Molecular Biosciences, University of Sydney, Sydney, NSW, Australia One of the endogenous mechanisms regulating inflammatory cell activation following brain injury is the expression of modulator and/or inhibitor membrane receptors. Recent studies demonstrate that regulatory receptor TREM2-mediated phagocytic function of microglia/macrophages (MM) is required for debris clearance and inhibitory receptor CD200R-mediated deactivation of microglia is necessary for maintenance of CNS tissue homeostasis. Also, antiinflammatory Interleukin-10 (IL-10) has been demonstrated in activated astrocytes and microglia after injury in the CNS but its specific role still remains ambiguous. Hence, the objective of this study was to characterize the cells expressing TREM2 and CD200R as an effect of local astrocyte-targeted production of IL-10 using the axonal anterograde degeneration paradigm. For this purpose, unilateral perforant pathway transection (PPT) was performed on adult GFAP-IL10 transgenic (Tg) animals and corresponding wild types (WT) littermates. Animals were intracardially perfused at 3,7,14 and 21 days post-lesion using 4% paraformaldehyde, brains frozen and sections cut in a cryostat. We performed immunohistochemistry for Iba-1 (microglia) CD200R and TREM2. Our results showed a basal low expression of TREM2 in MM of Tg animal in contrast to WT where TREM2 was mostly absent. An increase in TREM2+CD68+ phagocytizing MM was noted in the ipsilateral hemisphere of the Tg animal especially in the molecular layer of the dennervated dentate gyrus and was always higher than that of its respective WT. A very low CD200R expression was observed in the dennervated zone at all the time points studied in both WT and Tg. In conclusion, this study demonstrates that local production of IL-10 by astrocytes in the CNS may modify the microglial response associated with PPT by modulating endogenous regulatory receptors as TREM2. Future studies are concentrated on evaluating the role of these receptors in axonal sprouting in WT and Tg animals following PPT. Supported by Ministry of Science and Innovation (BFU2011-27400) doi:10.1016/j.jneuroim.2014.08.247
308 Dendritic cells are specifically affected by different immunomodulatory treatment strategies in multiple sclerosis Katja Thomas, Tony Sehr, Undine Hainke, Thorsten Schultheiß, Tjalf Ziemssen Department of Neurology, Neuroimmunological Lab, University Hospital, Dresden, Germany Objective: To evaluate innate immune system as relevant target in multiple sclerosis (MS) treatment regimes. Background: Dendritic cells (DC) and monocytes (Mo) are professional antigen presenting cells (APC) orchestrating relevant processes in innate
cells (PBMC). Depletion of CD14+ myeloid cells from PBMC diminished the adiponectin-dependent T cell activation mediated by ADS-MS sera. We suggest that abnormally elevated levels of adiponectin could contribute to enhanced inflammatory states of both innate and adaptive immune responses implicated in early disease mechanisms of pediatriconset MS. doi:10.1016/j.jneuroim.2014.08.242
288 Dispensable role for TLRs 2,3,4,7,9 in the mouse of multiple sclerosis Pushpalatha Palle, Thorsten Buch Institute for Medical Microbiology, Immunology and Hygiene, Technische Universitat of Munich, Munich, Germany Multiple sclerosis (MS) is a demyelinating inflammatory disorder of the central nervous system (CNS), which involves autoimmune responses against myelin antigens. MS is usually considered to be a predominantly T cell mediated autoimmune disease. To understand the initiation phase of the disease it is necessary to study danger signal pathways that may be responsible for driving the immune response. Therefore, we have started to focus on innate immune mechanisms. Toll-like receptors (TLRs) are key components of the innate immune system and play a crucial role in interplay between adaptive immunity and innate immunity. They recognize unique molecular patterns associated with different classes of pathogens and additionally, also recognize some self-proteins and endogenous nucleic acids. Recent studies predominantly from animal models of autoimmune disease and circumstantial data from human patients suggest that inappropriate activation of TLR pathways by endogenous or exogenous ligands may lead to the initiation and perpetuation of autoimmune responses. We have been studying the role of TLRs for the initiation of anti-CNS autoimmunity in the mouse model of the disease, experimental autoimmune encephalomyelitis. We could not confirm a crucial role for TLR9 and for the endosomal TLRs 3, 7, and 9. Further we did not find an effect of absence of TLRs 2,3,4,7 and 9 on the development of anti-CNS autoimmunity. To confirm that TLRs play no role in disease initiation we are currently generating a mouse model lacking signaling by all TLRs. In light of the emerging evidence for the role of the microbiome in initiating and driving autoimmune disease, we consider our observations to be highly relevant to understand how MS is triggered. doi:10.1016/j.jneuroim.2014.08.243
380 Microglia show an intermediate activation status in early lesion formation in multiple sclerosis Laura Peferoena, Daphne Vogela,b, Kimberley Ummenthuma, Marjolein Breurb, Priscilla Heijnenb, Wouter Gerritsena, Paul Van Der Valka, Christine Dijkstrab, Sandra Amora a
Pathology Department, VU Medical Centre, Amsterdam, Netherlands; MCBI, VU Medical Centre, Amsterdam, Netherlands
b
Microglia play an important role during lesion formation in multiple sclerosis (MS). Well before the peripheral immune systems is involved
91
and any sign of myelin damage is noticeable, foci of activated microglia appear in the normal appearing white matter (NAWM). Many of these so called preactive lesions are suggested to spontaneously resolve rather than developing into destructive lesions. This suggest that intrinsic regulation exists, which can stop lesion progression at an early stage. Activated microglia fulfil a broad range of functions both beneficial and harmful to the environment. Environmental signals are widely considered to contribute to the ‘switch’ between an immunoregulatory (M2) and a pro-inflammatory (M1) phentotype, in preactive lesions such signal might be the key to the developemt of active MS lesions. To characterize microglia phenotypes in preactive lesions, we screened for distinctive markers between M1 and M2 microglia, in vitro. This revealed that CD40, CD80 and CD74 were the most distinctive markers for M1, and manose receptor (MR) for M2. Additionally, M1 microglia produce higher levels of pro-inflammatory chemokines CXCL10 and CCL2, where M2 have an induced production of CCL22. Using the panel of M1 and M2 markers we next examined the activation status of microglia in pre-active MS lesions, remyelinating areas, NAWM and healthy control samples. Our data show that all preactive lesions have an intermediate expression pattern; no pre-active lesions were specifically M1 or M2 phenotype. All activated microglia abundantly express M1 markers CD40, CD86, CD74 and the M2 cytokine CCL22. This indicates an intermediate activation status of microglia in preactive lesions. Furthermore, we showed that polarization of human microglia, in vitro, is not static, but a dynamic process in which microglia can switch from a M1 to a M2 phenotype and vice versa. Taken together, our data delineate the versatility of microglia, both in vitro and in vivo, in response to inflammatory signals from the CNS, already in early MS lesion formation. doi:10.1016/j.jneuroim.2014.08.244
215 Formyl peptide receptors from the innate immune system and the vomeronasal organ recognize pathogen derived peptides Timo Schumann, Bernd Bufe, Frank Zufall Department of Physiology, Saarland University/Medical School Homburg, Homburg, Germany The formyl peptide receptor (Fpr) family is well known for its contribution to immune defense against pathogens in human and rodent leukocytes. Recently, several structurally related members of these receptors were discovered in sensory neurons of the mouse vomeronasal organ (VNO), key detectors of pheromones and related semiochemicals. Although the biological role of vomeronasal Fprs is not yet clear, the known contribution of other Fprs to host innate immune defense suggested that they could contribute to vomeronasal pathogen sensing. To determine the function of vomeronasal Fprs, precise knowledge about their agonist properties of is required. In order to investigate the pharmacological properties of the FPRs, we used high throughput calcium imaging comparing responses of Fprs of the immune and VNO system to more than 100 selected agonists. Our experiments revealed a new class of agonists that is capable of activating both, FPRs of the immune system and the vomeronasal organ. Intriguingly, motifs of these agonists are predominately found in bacteria and distinct pathogenic microorganisms. These data are consistent with the assumed role of FPRs in VNO mediated pathogen detection. Therefore, we propose that FPRs provide a common mechanism for the detection of pathogens by the immune system and the pheromone sensing system.
92
Abstracts
This work was supported by a Graduate Scholarship of the Saarland University for Timo Schumann
496 Role of TREM2 and CD200R in modulating microglial response in astrocyte targeted IL10Tg mice following perforant pathway transection.
doi:10.1016/j.jneuroim.2014.08.245 Kalpana Shrivastavaa, Mireia Recasensa, Beatriz Almoldaa, Iain Campbellb, Bernardo Castellanoa, Berta Gonzaleza 182 Reduced release of nitric oxide and different cytokines/ chemokines by aged microglial cells upon activation of Toll-like receptors 2 and 4 Sandra Schützea,b, Annika Kaufmannb, Sandra Ribesb, Jörg Scheffelb, Sandra Redlichb, Uwe-karsten Hanischb, Wolfgang Brücka, Roland Naub a
Department of Geriatrics, Agaplesion Diakonissen Krankenhaus, Frankfurt am Main, Germany; bInstitute of Neuropathology, University Medical Center, Göttingen, Germany Objectives: The incidence of bacterial infections of the central nervous system (CNS) in adults increases with age, and the clinical outcome in elderly individuals is worse than in young persons. Death in the acute phase of the infection and neurologic or neuropsychologic deficits are common complications, and better therapies are needed. Since microglial cells play a key role for the defence of the brain against bacterial infections, we investigated age-related functional changes of these cells in vitro. Methods: Primary microglial cultures were obtained from brains of C57BL/6-N mice aged 2 months (young) and 18 months (aged). For activation, cells cultured in 96-well-plates were treated with agonists of Toll-like receptor (TLR) 1/2 [Tripalmitoyl-S-glycerylcysteine (Pam3CSK4); 0.1 μg/ml] and TLR 4 [endotoxin (LPS) from Escherichia coli serotype 026:B6; 0.01 μg/ml] for 24 h in the presence of interferon-gamma (IFN-gamma; 100 U/ml). Non-activated control cells were treated with IFN-gamma only. Young and aged microglial cells were compared regarding their release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and CXCL1 (KC) into the cell culture supernatant. Results: Upon stimulation with agonists of TLR 2 and 4, aged microglial cells released significantly less NO and cytokines/ chemokines than young microglial cells: nitrite upon Pam3CSK4 treatment: 6.97 (5.96/8.28) vs. 18.40 (8.78/20.89) μM, p = 0.008; TNF-alpha upon LPS treatment: 284.10 (262.40/300.50) vs. 681.20 (468.50/1024.00) pg/ml, p = 0.002; IL-6 upon Pam3CSK4 treatment: 85.70 (17.86/144.30) vs. 924.90 (82.00/937.90) pg/ml, p = 0.04; KC upon LPS treatment: 15.63 (15.63/24.23) vs. 75.00 (44.10/117.40) pg/ml, p = 0.007 [representative results, concentrations in the cell culture supernatant indicated as median (25./75. percentile), comparison aged vs. young by Mann–Whitney-U-test, n = 5–8]. Conclusion: Our in-vitro data show a reduced release of NO and inflammatory cytokines/chemokines by aged microglial cells upon activation with bacterial TLR agonists. The age-related decline of microglial functions might contribute to the higher susceptibility of elderly individuals to bacterial CNS infections. Strategies to improve the functions of aged microglial cells appear promising for prevention and treatment of CNS infections in elderly patients.
doi:10.1016/j.jneuroim.2014.08.246
a
Unit of Histology, Dept. of Cell Biology, Physiology and Immunology, Institute of Neuroscience, Autonomous University of Barcelona, Barcelona, Spain; bSchool of Molecular Biosciences, University of Sydney, Sydney, NSW, Australia One of the endogenous mechanisms regulating inflammatory cell activation following brain injury is the expression of modulator and/or inhibitor membrane receptors. Recent studies demonstrate that regulatory receptor TREM2-mediated phagocytic function of microglia/macrophages (MM) is required for debris clearance and inhibitory receptor CD200R-mediated deactivation of microglia is necessary for maintenance of CNS tissue homeostasis. Also, antiinflammatory Interleukin-10 (IL-10) has been demonstrated in activated astrocytes and microglia after injury in the CNS but its specific role still remains ambiguous. Hence, the objective of this study was to characterize the cells expressing TREM2 and CD200R as an effect of local astrocyte-targeted production of IL-10 using the axonal anterograde degeneration paradigm. For this purpose, unilateral perforant pathway transection (PPT) was performed on adult GFAP-IL10 transgenic (Tg) animals and corresponding wild types (WT) littermates. Animals were intracardially perfused at 3,7,14 and 21 days post-lesion using 4% paraformaldehyde, brains frozen and sections cut in a cryostat. We performed immunohistochemistry for Iba-1 (microglia) CD200R and TREM2. Our results showed a basal low expression of TREM2 in MM of Tg animal in contrast to WT where TREM2 was mostly absent. An increase in TREM2+CD68+ phagocytizing MM was noted in the ipsilateral hemisphere of the Tg animal especially in the molecular layer of the dennervated dentate gyrus and was always higher than that of its respective WT. A very low CD200R expression was observed in the dennervated zone at all the time points studied in both WT and Tg. In conclusion, this study demonstrates that local production of IL-10 by astrocytes in the CNS may modify the microglial response associated with PPT by modulating endogenous regulatory receptors as TREM2. Future studies are concentrated on evaluating the role of these receptors in axonal sprouting in WT and Tg animals following PPT. Supported by Ministry of Science and Innovation (BFU2011-27400) doi:10.1016/j.jneuroim.2014.08.247
308 Dendritic cells are specifically affected by different immunomodulatory treatment strategies in multiple sclerosis Katja Thomas, Tony Sehr, Undine Hainke, Thorsten Schultheiß, Tjalf Ziemssen Department of Neurology, Neuroimmunological Lab, University Hospital, Dresden, Germany Objective: To evaluate innate immune system as relevant target in multiple sclerosis (MS) treatment regimes. Background: Dendritic cells (DC) and monocytes (Mo) are professional antigen presenting cells (APC) orchestrating relevant processes in innate