Background The involvement of complement system in brain injury has been scarcely investigated. Here we document the pivotal role of mannose binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. Methods and Results Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessels occlusion). We first observed that MBL is deposited on ischemic vessels up to 48h after injury and that functional MBL/MASP2 complexes are increased. Next we demonstrated that: 1) MBL−/− mice are protected from both transient and permanent ischemic injury; 2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24h after ischemia in mice; 3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18h after ischemia, as assessed following 28d in rats. Conclusions Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.

Decay-accelerating factor (DAF) is a cell surface regulator that accelerates the dissociation of C3/C5 convertases and thereby prevents the amplification of complement activation on self cells. In the context of transplantation, DAF has been thought to primarily regulate antibody-mediated allograft injury, which is in part serum complement-dependent. Based on our previously delineated link between DAF and CD4 T cell responses, we evaluated the effects of donor Daf1 (the murine homolog of human DAF) deficiency on CD8 T cell-mediated cardiac allograft rejection. MHC-disparate Daf1−/− allografts were rejected with accelerated kinetics compared with wild-type grafts. The accelerated rejection predominantly tracked with DAF’s absence on bone marrow-derived cells in the graft and required allograft production of C3. Transplantation of Daf1−/− hearts into wild-type allogeneic hosts augmented the strength of the anti-donor (direct pathway) T cell response, in part through complement-dependent proliferative and pro-survival effects on alloreactive CD8 T cells. The accelerated allograft rejection of Daf1−/− hearts occurred in recipients lacking anti-donor Abs. The results reveal that donor DAF expression, by controlling local complement activation on interacting T cell APC partners, regulates the strength of the direct alloreactive CD8+ T cell response. The findings provide new insights into links between innate and adaptive immunity that could be exploited to limit T cell-mediated injury to an allograft following transplantation.

The first line of host defense is the innate immune system that includes coagulation factors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulation factor-like

Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. While it’s known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. Ferric chloride (FeCl3) treatment resulted in intra-arterial occlusive thrombogenesis within 10min in wild-type (WT) and C2/fB null mice. In contrast, MBL null and MASP-1/-3 KO mice had significantly decreased FeCl3-induced thrombogenesis. Reconstitution with rhMBL restored FeCl3-induced thrombogenesis in MBL null mice to levels comparable to WT mice, suggesting a significant role of the MBL-MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL-MASP complex-dependent platelet aggregation. In vitro, MBL-MASP complexes were captured on mannan-coated plates and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/fB null, MBL-A−/− or MBL-C−/− sera, however MBL null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. Recombinant human (rh)MBL alone failed to cleave S2238, however cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL-MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.

Background Coagulation disorders and reperfusion of ischemic myocardium are major causes of morbidity and mortality. Lectin pathway initiation complexes are composed of multimolecular carbohydrate recognition subcomponents and three lectin pathway specific serine proteases. We have recently shown that the lectin pathway specific carbohydrate recognition subcomponent mannose-binding lectin (MBL) plays an essential role in the pathophysiology of thrombosis and ischemia/reperfusion injury. Thus, we hypothesized that the endogenous MBL associated protein, MAP-1, that inhibits complement activation in vitro also could be an in vivo regulator by attenuating myocardial schema/reperfusion injury and thrombogenesis when used at pharmacologic doses in wild type mice. Methods and Results MAP-1, in two mouse models, preserves cardiac function, decreases infarct size, decreases C3 deposition, inhibits MBL deposition and prevents thrombogenesis. Further, we also demonstrate that MAP-1 displaces MASP-1, MASP-2 and MASP-3 from the MBL complex. Conclusions Our results suggest that the natural, endogenous inhibitor, MAP-1effectively inhibits lectin pathway activation in vivo. MAP-1 at pharmacologic doses represents a novel therapeutic approach for human diseases involving the lectin pathway and its associated MASPs.

Complement activation has been shown to play an important role in the inflammation and tissue injury following myocardial ischemia and reperfusion (MI/R). Several recent studies from our laboratory demonstrated the importance of mannose-binding lectin (MBL) as the initiation pathway for complement activation and the resulting pathological effects following MI/R. However, other studies from the past suggest an important role of the classical pathway and perhaps natural antibodies. In the present study, we used newly generated genetically modified mice that lack secreted IgM (sIgM), MBL-A, and MBL-C (sIgM/MBL null) in a plasma reconstitution mouse model of MI/R. Following 30 min of ischemia and 4 h of reperfusion, left ventricular ejection fractions were significantly higher in sIgM/MBL null mice reconstituted with MBL null or sIgM/MBL null plasma compared with reconstitution with wild-type (WT) plasma or WT mice reconstituted with WT plasma following MI/R. Serum troponin I concentration, myocardial polymorphonuclear leukocyte infiltration, and C3 deposition were dependent on the combined presence of sIgM and MBL. These results demonstrate that MI/R-induced complement activation, inflammation, and subsequent tissue injury require both IgM and MBL. Thus MBL-dependent activation of the lectin pathway may not be completely antibody independent in I/R models.

BackgroundMesenchymal stem cell–derived extracellular vesicles (EVs) are believed to be cardioprotective in myocardial infarct. The objective of this study was to examine the effects of human mesenchymal cell–derived EV injection on cardiac function, myocardial blood flow, and vessel density in the setting of chronic myocardial ischemia.Methods and ResultsTwenty‐three Yorkshire swine underwent placement of an ameroid constrictor on their left circumflex artery. Two weeks later, the animals were split into 2 groups: the control group (CON; n=7) and the EV myocardial injection group (MVM; n=10). The MVM group underwent myocardial injection of 50 μg of EVs in 2 mL 0.9% saline into the ischemic myocardium. Five weeks later, the pigs underwent a harvest procedure, and the left ventricular myocardium was analyzed. Absolute blood flow and the ischemic/nonischemic myocardial perfusion ratio were increased in the ischemic myocardium in the MVM group compared with the CON group. Pigs in the MVM group had increased capillary and arteriolar density in the ischemic myocardial tissue compared with CON pigs. There was an increase in expression of the phospho–mitogen‐activated protein kinase/mitogen‐activated protein kinase ratio, the phospho–endothelial nitric oxide synthase/endothelial nitric oxide synthase ratio, and total protein kinase B in the MVM group compared with CON. There was an increase in cardiac output and stroke volume in the MVM group compared with CON.ConclusionsIn the setting of chronic myocardial ischemia, myocardial injection of human mesenchymal cell–derived EVs increases blood flow to ischemic myocardial tissue by induction of capillary and arteriolar growth via activation of the protein kinase B/endothelial nitric oxide synthase and mitogen‐activated protein kinase signaling pathways resulting in increased cardiac output and stroke volume.

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2 expressing mice (MBL2 KI) that lack murine Mbls (MBL2+/+Mbl1−/−Mbl2−/−), a novel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, thrombocytopenia, and increased serum creatinine, free serum hemoglobin, and cystatin C levels, but a significantly decreased glomerular filtration rate all compared to control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition in glomeruli in Stx-2 injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels, and significantly attenuated Stx-2 induced renal injury, free serum hemoglobin levels, renal C3d and fibrin deposition, and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can be used to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2 induced renal injury.