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C1q is associated with a reduced inflammatory response of decidual endothelial cells
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Abstracts / Molecular Immunology 48 (2011) 1666–1733
immunity in allergic asthma. For this purpose, we adoptively transferred GM-CSF-differentiated bone marrow (BM)-derived mDCs from wildtype (wt), C3aR−/−, C5aR−/− and C3aR−/−/C5aR−/− mice that had been pulsed with crude extract from house dust mite (HDM) intra-tracheally into wt mice. We found that mDCs from wt and C3aR-deficient mice induced a strong asthmatic response, characterized by a marked increase in airway hyperresponsiveness (AHR), strong Th2 but minor Th17 and Th1 cytokine production and a mixed eosinophilic and neutrophilic infiltration of the lung. In contrast, AHR, Th2 cytokine production and eosinophilic inflammation were substantially decreased following adoptive transfer of C5aR−/− or C3aR−/−/C5aR−/− mDCs. However, mice treated with such mDCs suffered from high pulmonary neutrophil infiltration and increased production of IL-17 and INF? from pulmonary cells. Our data suggest that C5aR signaling biases mDCs driven adaptive immunity towards Th2 cytokine production and eosinophilic inflammation but prevents Th17 and Th1 cytokine production and neutrophilic inflammation. The role of C3aR signaling in mDC driven Th cell differentiation and pulmonary inflammation appears to be minor. doi:10.1016/j.molimm.2011.06.299 P80 C1q is associated with a reduced inflammatory response of decidual endothelial cells E. Masat a,∗ , R. Bulla a , F. Bossi a , C. Agostinis b , F. De Seta b , M. Tonon b , F. Tedesco a a
University of Trieste, Trieste, Italy b IRCCS Burlo Garofolo, Trieste, Italy The endothelial cells (ECs) localized in various districts of the vascular tree represent an heterogeneous population of cells in terms of functional activity and play a fundamental role in the control of inflammation. We have investigated the response of ECs isolated from human decidual ECs (DECs) and from adult dermal microvasculature (ADMECs) to inflammatory stimuli. Interestingly, DECs exhibited a markedly reduced response to LPS than ADMECs as evaluated by the degree of expression of adhesion molecules and cytokines secretion. This finding was associated with a lower expression level of TLR4, MD2 and MyD88. A distinct feature of DECs acquired during pregnancy is the ability to synthesize and express C1q on the cell surface (Bulla et al., 2008). Since C1q was shown to suppress LPS-induced IL-12p40 production in bone marrowderived DC (BMDC), we hypothesised that C1q may play a role in the control of DECs response to LPS. We therefore investigated the expression of TLR4, MD-2, and MyD88 in resting and LPSstimulated ADMECs pre-incubated for 2 h with C1q. Our results showed that C1q induced a reduced mRNA expression of TLR4, MD-2, and MyD88 which was particularly evident in resting cells suggesting that C1q may act as a down-regulator of ECs inflammatory response at foetal–maternal interface. doi:10.1016/j.molimm.2011.06.300 P81 The role of the component C5 in murine models of fatty liver disease: An inflammatory analysis L.B. Bavia ∗ , L.I. Isaac University of São Paulo/Biomedical Sciences Institute, São Paulo, Brazil The complement system plays an important role in the development of Alcoholic Fatty Liver (AFLD) and Non Alcoholic Fatty
Liver (NAFLD) diseases. It is known that complement activation can trigger an important inflammatory response in hepatic tissue contributing to the pathogenesis of these diseases. C3a and C5a are crucial for liver regeneration after toxic injury. Alternatively, these fragments can be associated with hepatic triglyceride (TG) accumulation, liver injury and ethanol-induced inflammation. Here, we have investigated the role of C5 in murine models of AFLD and NAFLD. Eight- to twelve-week-old male C57Bl/6 (C5 sufficient) and A/J (C5 deficient) mice were fed with Liber-DeCarli diet containing ethanol (AFLD), glucose (NAFLD) or maltose dextrin (pair-fed control) for 6, 8 or 10 weeks. We then performed plasma quantification of ALT and AST, histopathological analysis, and evaluated the hepatic TG, liver TNF-alpha, IL-6, IL-1beta, IL-10, IL-12, IL-17 and NO. Both C57Bl/6 and A/J mice developed ethanol-induced steatosis after 6, 8 and 10 weeks, but apparently, A/J is more resistant to the development of the AFLD model. Also, A/J mice produces more IL-10 than pro-inflammatory cytokines, greater NO production and lower fibrosis than C57Bl/6 mice. Conversely, in the NAFLD model, C57Bl/6 mice present exacerbated hepatic TG while A/J mice are resistant after 10 weeks. The C57Bl/6 mice present increased IL-6 level and less fibrosis than A/J mice. Our study shows the importance of C5 for the balance of pro- and anti-inflammatory response to the development of inflammation, steatosis, and fibrosis in fatty liver disease models. doi:10.1016/j.molimm.2011.06.301 P82 Complement-mediated acute induction of endothelial-tomesenchymal transition (EndMT) in a swine model of renal ischemia/reperfusion > (I/R) injury G.C. Castellano a,∗ , C. Curci a , M. Carielloa a , S. Simone a , V. Montinaro a , E. Van Amersfoort b , B. Oortwijn b , L. Gesualdo a , F.P. Schena a , G. Grandaliano a a b
University of Bari, Bari, Italy Pharming B.V., Leiden, Netherlands
EndMT is a pathogenic process recently described in cancer, chronic kidney disease and cardiac fibrosis. To date, little is known on the molecular mechanisms regulating EndMT. In a swine model of renal I/R injury, 30 of warm ischemia led to an intense C4d and C5b-9 deposition on peritubular and glomerular EC. By confocal microscopy, we observed a 50% reduction of CD31+EC, with a 90% increased expression of alpha-SMA, a myofibroblast marker. We demonstrated the presence of CD31+/alpha-SMA+ cells in peritubular capillaries and renal interstitium. Considering the deposition of Complement on EC in vivo, we investigated whether C3a was able to induce EndMT in vitro. Western blotting and microscopy analysis demonstrated that C3a induced an increase in alpha-SMA (0.770.38; ctr 0.420.1; pixel-ratio; p value < 0.05) and a decrease in von Willebrand-Factor expression (4.850.89; basal 7.260.57 pixel-ratio; p value < 0.03) with 35% reduction in EC survival. Activated EC showed an increased phosporilation of AKT; specific AKT-inhibition significantly limited the C3a-induced EndMT in vitro. These data suggest a critical role for Complement in the acute induction of the EndMT process via the AKT pathway. Therapeutic inhibition of these systems may be essential to prevent the development of vascular-derived tissue fibrosis in different human diseases where EndMT occurs. doi:10.1016/j.molimm.2011.06.302
Abstracts / Molecular Immunology 48 (2011) 1666–1733
immunity in allergic asthma. For this purpose, we adoptively transferred GM-CSF-differentiated bone marrow (BM)-derived mDCs from wildtype (wt), C3aR−/−, C5aR−/− and C3aR−/−/C5aR−/− mice that had been pulsed with crude extract from house dust mite (HDM) intra-tracheally into wt mice. We found that mDCs from wt and C3aR-deficient mice induced a strong asthmatic response, characterized by a marked increase in airway hyperresponsiveness (AHR), strong Th2 but minor Th17 and Th1 cytokine production and a mixed eosinophilic and neutrophilic infiltration of the lung. In contrast, AHR, Th2 cytokine production and eosinophilic inflammation were substantially decreased following adoptive transfer of C5aR−/− or C3aR−/−/C5aR−/− mDCs. However, mice treated with such mDCs suffered from high pulmonary neutrophil infiltration and increased production of IL-17 and INF? from pulmonary cells. Our data suggest that C5aR signaling biases mDCs driven adaptive immunity towards Th2 cytokine production and eosinophilic inflammation but prevents Th17 and Th1 cytokine production and neutrophilic inflammation. The role of C3aR signaling in mDC driven Th cell differentiation and pulmonary inflammation appears to be minor. doi:10.1016/j.molimm.2011.06.299 P80 C1q is associated with a reduced inflammatory response of decidual endothelial cells E. Masat a,∗ , R. Bulla a , F. Bossi a , C. Agostinis b , F. De Seta b , M. Tonon b , F. Tedesco a a
University of Trieste, Trieste, Italy b IRCCS Burlo Garofolo, Trieste, Italy The endothelial cells (ECs) localized in various districts of the vascular tree represent an heterogeneous population of cells in terms of functional activity and play a fundamental role in the control of inflammation. We have investigated the response of ECs isolated from human decidual ECs (DECs) and from adult dermal microvasculature (ADMECs) to inflammatory stimuli. Interestingly, DECs exhibited a markedly reduced response to LPS than ADMECs as evaluated by the degree of expression of adhesion molecules and cytokines secretion. This finding was associated with a lower expression level of TLR4, MD2 and MyD88. A distinct feature of DECs acquired during pregnancy is the ability to synthesize and express C1q on the cell surface (Bulla et al., 2008). Since C1q was shown to suppress LPS-induced IL-12p40 production in bone marrowderived DC (BMDC), we hypothesised that C1q may play a role in the control of DECs response to LPS. We therefore investigated the expression of TLR4, MD-2, and MyD88 in resting and LPSstimulated ADMECs pre-incubated for 2 h with C1q. Our results showed that C1q induced a reduced mRNA expression of TLR4, MD-2, and MyD88 which was particularly evident in resting cells suggesting that C1q may act as a down-regulator of ECs inflammatory response at foetal–maternal interface. doi:10.1016/j.molimm.2011.06.300 P81 The role of the component C5 in murine models of fatty liver disease: An inflammatory analysis L.B. Bavia ∗ , L.I. Isaac University of São Paulo/Biomedical Sciences Institute, São Paulo, Brazil The complement system plays an important role in the development of Alcoholic Fatty Liver (AFLD) and Non Alcoholic Fatty
Liver (NAFLD) diseases. It is known that complement activation can trigger an important inflammatory response in hepatic tissue contributing to the pathogenesis of these diseases. C3a and C5a are crucial for liver regeneration after toxic injury. Alternatively, these fragments can be associated with hepatic triglyceride (TG) accumulation, liver injury and ethanol-induced inflammation. Here, we have investigated the role of C5 in murine models of AFLD and NAFLD. Eight- to twelve-week-old male C57Bl/6 (C5 sufficient) and A/J (C5 deficient) mice were fed with Liber-DeCarli diet containing ethanol (AFLD), glucose (NAFLD) or maltose dextrin (pair-fed control) for 6, 8 or 10 weeks. We then performed plasma quantification of ALT and AST, histopathological analysis, and evaluated the hepatic TG, liver TNF-alpha, IL-6, IL-1beta, IL-10, IL-12, IL-17 and NO. Both C57Bl/6 and A/J mice developed ethanol-induced steatosis after 6, 8 and 10 weeks, but apparently, A/J is more resistant to the development of the AFLD model. Also, A/J mice produces more IL-10 than pro-inflammatory cytokines, greater NO production and lower fibrosis than C57Bl/6 mice. Conversely, in the NAFLD model, C57Bl/6 mice present exacerbated hepatic TG while A/J mice are resistant after 10 weeks. The C57Bl/6 mice present increased IL-6 level and less fibrosis than A/J mice. Our study shows the importance of C5 for the balance of pro- and anti-inflammatory response to the development of inflammation, steatosis, and fibrosis in fatty liver disease models. doi:10.1016/j.molimm.2011.06.301 P82 Complement-mediated acute induction of endothelial-tomesenchymal transition (EndMT) in a swine model of renal ischemia/reperfusion > (I/R) injury G.C. Castellano a,∗ , C. Curci a , M. Carielloa a , S. Simone a , V. Montinaro a , E. Van Amersfoort b , B. Oortwijn b , L. Gesualdo a , F.P. Schena a , G. Grandaliano a a b
University of Bari, Bari, Italy Pharming B.V., Leiden, Netherlands
EndMT is a pathogenic process recently described in cancer, chronic kidney disease and cardiac fibrosis. To date, little is known on the molecular mechanisms regulating EndMT. In a swine model of renal I/R injury, 30 of warm ischemia led to an intense C4d and C5b-9 deposition on peritubular and glomerular EC. By confocal microscopy, we observed a 50% reduction of CD31+EC, with a 90% increased expression of alpha-SMA, a myofibroblast marker. We demonstrated the presence of CD31+/alpha-SMA+ cells in peritubular capillaries and renal interstitium. Considering the deposition of Complement on EC in vivo, we investigated whether C3a was able to induce EndMT in vitro. Western blotting and microscopy analysis demonstrated that C3a induced an increase in alpha-SMA (0.770.38; ctr 0.420.1; pixel-ratio; p value < 0.05) and a decrease in von Willebrand-Factor expression (4.850.89; basal 7.260.57 pixel-ratio; p value < 0.03) with 35% reduction in EC survival. Activated EC showed an increased phosporilation of AKT; specific AKT-inhibition significantly limited the C3a-induced EndMT in vitro. These data suggest a critical role for Complement in the acute induction of the EndMT process via the AKT pathway. Therapeutic inhibition of these systems may be essential to prevent the development of vascular-derived tissue fibrosis in different human diseases where EndMT occurs. doi:10.1016/j.molimm.2011.06.302