Complement activation in preterm delivery in mice

Abstracts / Molecular Immunology 47 (2010) 2198–2294

protein (C4BP) and factor H (fH) affect their degradation. Freshly isolated human neutrophils were induced to produce NETs and DNA degradation assays and confocal microscopy were used as read-out systems. We found that C1q decreased the degradation of NETs by DNase-I, whereas C4BP and fH had no affect. C1q bound to a high extent to the NETs, which possibly explains the observation that C1q protects the NETs from degradation by DNase-I. NET-bound C1q/C1 can trigger complement cascade and we found C3b deposits on the NETs incubated with human serum. How the complement activation and deposition from human sera affects degradation is currently under investigation. This study brings two important components of the innate immune system together and will broaden the understanding of how deficiencies in the separate systems affect, and in some cases, lead to human diseases. doi:10.1016/j.molimm.2010.05.228 50 DAF deficiency is protective during atherosclerosis in apoE deficient mice D. Lewis a , Christopher L. Jackson b , Mark J. Perry b , B. Paul Morgan a , Timothy R. Hughes a a b

Cardiff University, Cardiff, UK Bristol University, Bristol, UK

Atherosclerosis, the leading cause of death in the western world, is a disease driven by chronic inflammation within the artery wall. The involvement of complement in atherosclerosis has gradually become evident with human studies showing deposition of complement proteins and activation products within the atherosclerotic plaque. Data generated from animal studies have revealed that different pathways and activation products of complement have conflicting roles within the disease process. Activation of the terminal pathway appears to be proatherogenic as revealed through the use of apoE mice deficient in C6 or the terminal pathway regulator CD59, while the role of the activation pathways still remains unclear with studies on C3 deficient mice implying a protective role for C3. We examined the role of decay accelerating factor (DAF; CD55), a membrane inhibitor of the C3 convertase, in atherosclerosis by backcrossing DAF deficient mice (DAF−/−) onto the apoE deficient (apoE−/−) background. Adult male ApoE−/−DAF−/− mice were fed a high fat diet for 12 weeks. Severity of atherosclerosis was assessed in the brachiocephalic artery and lipid analysis carried out on serum. ApoE−/−/DAF−/− mice had significantly smaller atherosclerotic plaques when compared to apoE−/− controls (cross-sectional area 46 ␮m × 103 ␮m ± 14 × 103 versus 146 ␮m × 103 ␮m ± 14 × 103). Triglyceride levels were significantly lower than those present in control animals before (2.1 ± 0.1 mmol versus 5.1 ± 0.6 mmol) and after (1.667 ± 0.3 mmol versus 3.6 ± 0.5 mmol) high fat diet. Cholesterol levels after high fat feeding were also markedly lower in apoE−/−/DAF−/− mice compared to controls (29.0 ± 1.8 mmol/L versus 38.3 ± 2.5 mmol/L). Conversely, percentage body fat was raised in apoE−/−/DAF−/− mice compared to controls (28.63 ± 4.2% versus 19.5 ± 1.8%). We suggest that these changes are a direct consequence of complement dysregulation and provide evidence in support of this hypothesis. doi:10.1016/j.molimm.2010.05.229

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51 The onset of reperfusion injury: Deposition of antibodies and complement in viable myocardium in a porcine model of acute coronary syndrome Pranitha Kamat a , Anne Broillet b , Alexandre Helbert b , Thierry Tranquart b , Michel Schneider b , Robert Bettinger b , Francois Rieben a a b

University of Bern, Department of Clinical Research, Bern, Switzerland Bracco Research SA, Geneva, Switzerland

Background: Recognition of ischemia-induced neoepitopes by IgM, followed by complement activation, was proposed as pathophysiological mechanism in reperfusion injury. The respective data are from mouse models with considerable reperfusion-induced tissue necrosis. We now assessed the role of antibodies and complement in a porcine model of acute coronary syndrome with short term ischemia and minimal reperfusion-induced myocardial necrosis. Method: Six piglets of 30 kg were used to induce coronary occlusion by a balloon catheter for 20 min, followed by 6 h of reperfusion. The myocardial ischemic area at risk (AAR) was demarcated by Evan’s Blue. Triphenyl tetrazolium chloride incubation was used to discriminate between vital and necrotic parts. Deposition of IgM, IgG and C3b/c were detected by immunofluorescence. Contrast echocardiography with antibody-coated microbubbles was used to non-invasively assess P-selection expression within the myocardium and compared with Immunohistochemistry on paraffin sections. Results: In this short term coronary occlusion model, no reperfusion-induced myocardial necrosis was observed and only 2 out of 6 animals showed increased troponin-I. However, deposition of IgG (P = 0.035) and C3b/c (P = 0.014) was significantly higher in the AAR than in the non-ischemic myocardium. IgM deposition was also apparently higher, but the difference was not significant. P-selectin expression could be observed specifically within the AAR by contrast echocardiography and correlated with immunohistochemical detection. Discussion: The observed deposition of IgG, IgM and C3b/c within the AAR, in the absence of tissue necrosis, suggests that indeed natural antibodies and complement may be essential players in the onset of ischemia/reperfusion injury. The presence of these markers, together with the expression of P-selectin, can therefore be used to identify even short term myocardial ischemia up to several hours after the ischemic event. doi:10.1016/j.molimm.2010.05.230 52 Complement activation in preterm delivery in mice Juan M. Gonzalez a , Claus V. Franzke b , Roberto Romero a , Guillermina Girardi c a

Perinatology Research Branch, NICHD/NIH/DHHS, Bethesda, Maryland and Detroit, MI, USA b Molecular Dermatology,University of Freiburg, Germany c York College, CUNY, New York, NY, USA Preterm labor/delivery (PTD) is the leading cause of perinatal morbidity and mortality worldwide. It is the leading identifiable cause of cerebral palsy and it costs $26 billion per year in the U.S. alone. Infection/inflammation is the only mechanism of disease which has been causally linked to spontaneous preterm labor. The innate immune system plays a key role in parturition; therefore we decided to study the role of complement activation in the

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Abstracts / Molecular Immunology 47 (2010) 2198–2294

mechanism responsible for preterm parturition. We developed an animal model of preterm labor by administering LPS through the vaginal route, which resemble the clinical disorder. Increased C3 deposition was found in the columnar endocervical epithelium. The serum concentrations of C3adesArg in LPS-treated mice with preterm parturition were significantly higher than in the control group. ((ng/ml): LPS (n = 6): 15517 ± 10462, saline (n = 4):66 ± 41). In addition, an increase in C3adesArg was found in amniotic fluid in LPS-treated mice. Complement activation was also observed in preterm labor induced by the administration of a progesterone receptor antagonist (RU 486); an animal model for preterm labor not induced by infection/ inflammation. Complement activation in both models was associated with “cervical remodeling.” A dramatic change in the distensibility of the cervix was observed in preparation for parturition in both models. Matrix metalloproteinases (MMPs), are proteolytic enzymes involved in tissue remodeling. Gelatin substrate gel electrophoresis studies showed increased MMP-9 activity in cervical tissue from LPS-treated mice. In addition, cervical tissue from LPS-treated C5aR deficient mice did not show increased MMP-9 activity, suggesting a role for C5aR in the increased activity of MMP-9 observed in LPS-induced PTD. We propose that complement activation is required for the increase of MMP-9 activity and the subsequent ripening and dilatation of the cervix that leads to premature labor. doi:10.1016/j.molimm.2010.05.231 53 Comparison of expression profiles from wildtype and C6 deficient peripheral nerves after crush injury M. Sta a , K. Fluiter a , V. Ramaglia a , R. Wolterman a , B.P. Morgan c , F. Baas a,b a

Neurogenetics Laboratory, University of Amsterdam, Amsterdam, The Netherlands b Department of Neurology Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK The complement (C) system, highly expressed in nerves, is activated during Wallerian degeneration (WD). Using C6 deficient rats, we showed that the membrane attack complex (MAC) is required for rapid WD after peripheral nerve crush injury. Both genetic and pharmacological inhibition of C activation protected the nerve from axonal and myelin breakdown at 3 days post-injury showing improved regeneration compared to control animals as judged by neuropathology and sensory function. Here we extended this study to transected peripheral nerves, a severe clinical condition from which recovery, if at all, is slow. We show that regeneration of C6 deficient transected peripheral nerves is accelerated compared to wildtypes, as judged by axon diameter, myelin thickness and number of regenerative clusters. We propose that an early destructive C-mediated event during WD hampers the subsequent regeneration. Therefore, to determine the mechanisms of improved regeneration, the RNA isolated from sciatic nerves of wildtype and C6 deficient rats at 48 and 72 h after injury was used for microarray whole genome mRNA profiling. Statistical analysis (ANOVA with Benjamini–Hochberg false discovery rate correction) was used to detect significant differences. For this project we focused on the expression of all genes known to be involved in the C system. In the sciatic nerve we found 18 and 24 C genes differentially regulated at 48 and 72 h post-injury, respectively. Of these, C4 binding protein (C4BP) gene was highly up-regulated whereas C receptor 2 (CR2) and C factor H (CFH)-like 1 genes were highly down-regulated

in C6 deficient nerves compared to wildtypes at 48 h post-injury. Platelet CFH and CFH-like 1 genes were highly upregulated at 72 h post-injury. We conclude that C is highly regulated after peripheral nerve injury and we identify C4BP, CR2, CFH and CFH-like 1 as candidate C genes involved in the mechanisms for improved regeneration of C6 deficient nerves. doi:10.1016/j.molimm.2010.05.232 54 Inhibition of complement activation prevents features of preeclampsia in a mouse model Shari E. Gelber (MD, PhD) a , Patricia B. Redecha b , Stephen Tomlinson (PhD) c , Robin L. Davisson (PhD) a , Jane E. Salmon (MD) b a

Weill Cornell Medical College, New York 10065, NY, United States Hospital for Special Surgery, New York 10021, NY, United States c Medical University of South Carolina, Charleston 29425, SC, United States b

Objective: Preeclampsia (PE) is a major cause of maternal and fetal morbidity and mortality. Studies in women demonstrate an association between complement activation in early pregnancy and development of PE. Mouse models of miscarriage and placental insufficiency show that activation of complement is essential and causative in angiogenic dysregulation and poor pregnancy outcome. Pregnant BPH/5 mice exhibit the cardinal symptoms of PE (late gestational hypertension, proteinuria, growth restriction and fetal loss). We used a targeted inhibitor of mouse C3 (CR2-Crry) to determine whether blockade of complement activation could prevent features of PE in BPH/5 mice. Methods: Virgin BPH/5 mice were mated and treated with CR2Crry (250 ␮g I.V.) on gestational day (GA) 5 and 10 of pregnancy. Controls were treated with normal saline solution (NSS). Mice were sacrificed at GA 15.5, pups were weighed; placentas and plasma were collected for histology and analysis of VEGF levels, respectively. In separate experiments, mice were mated and tissue collected for histology at GA 8. Results: At GA 8 there was evidence of increased complement deposition in the ectoplacental cone of the BPH/5 animals compared to control C57/bl6 mice. Treatment with CR2-Crry was associated with a decrease in resorption frequency (fetal losses/total pregnancies) (CR2-Crry 6 ± 4%, n = 6; NSS 21 ± 5%, p < 0.05), an increase in average pup weight (CR2-Crry: 331 ± 7 mg; NSS 310 ± 13 mg; p < 0.05), normalization of the size of the junctional zone of the placenta and an increase in VEGF levels (CR2-Crry 35 ± 3 pg/ml; NSS 23 ± 3 pg/ml). Conclusion: Our findings implicate complement activation in the pathogenesis of fetal loss and growth restriction in a mouse model of PE. Inhibition of complement activation with a novel targeted inhibitor results in an increase in fetal weight, restoration of normal placental architecture, and decrease in angiogenic imbalance and suggests that complement inhibition may be a treatment for PE. doi:10.1016/j.molimm.2010.05.233