Human mannose-binding lectin inhibitor prevents Shiga toxin–induced renal injury
Authors: Masayuki Ozaki, Yulin Kang, Ying Siow Tan, Vasile I. Pavlov, Bohan Liu, Daniel C. Boyle, Rafail I. Kushak, Mikkel-Ole Skjoedt, Eric F. Grabowski, Yasuhiko Taira, Gregory L. Stahl
Journal: Kidney International (2016)

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, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, but a significantly decreased glomerular filtration rate compared with 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. 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, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, but a significantly decreased glomerular filtration rate compared with 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. Hemolytic uremic syndrome (HUS) is a disease characterized by renal failure, thrombocytopenia, and hemolytic anemia.1Tarr P.I. Gordon C.A. Chandler W.L. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome.Lancet. 2005; 365: 1073-1086Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar HUS is divided into the following 2 major classes by its etiologies: Shiga toxin-2 (Stx-2)–producing Escherichia coli HUS (STEC HUS) and atypical HUS (aHUS). Stx, an AB5 toxin produced by bacteria, has been identified as the causative factor in STEC HUS.2O'Brien A.D. LaVeck G.D. Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.Infect Immun. 1983; 40: 675-683PubMed Google Scholar A major source of Stx is ingestion of food contaminated with Stx-producing Escherichia coli or Shigella dysenteriae.3Butler T. Haemolytic uraemic syndrome during shigellosis.Trans R Soc Trop Med Hyg. 2012; 106: 395-399Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Currently there is no proven therapeutic option for STEC HUS other than supportive care.4Bitzan M. Treatment options for HUS secondary to Escherichia coli O157:H7.Kidney Int Suppl. 2009; : S62-S66Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Supportive therapy with dialysis in the acute phase of STEC HUS can be life-saving; however, chronic renal failure and hypertension develop in many patients.5Spinale J.M. Ruebner R.L. 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Tsurugi K. Yutsudo T. et al.Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins.Eur J Biochem. 1988; 171: 45-50Crossref PubMed Scopus (617) Google Scholar In a small number of instances, protein synthesis may be enhanced (e.g., tissue factor).11Grabowski E.F. Liu B. Gerace M.R. et al.Shiga toxin-1 decreases endothelial cell tissue factor pathway inhibitor not co-localized with tissue factor on the cell membrane.Thromb Res. 2015; 135: 1214-1217Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 12Grabowski E.F. Kushak R.I. Liu B. et al.Shiga toxin downregulates tissue factor pathway inhibitor, modulating an increase in the expression of functional tissue factor on endothelium.Thromb Res. 2013; 131: 521-528Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Although these intracellular mechanisms have been elucidated, the mechanisms by which ribosomal inactivation induces the peculiar characteristics of STEC HUS are unknown.13Jandhyala D.M. Thorpe C.M. Magun B. Ricin and Shiga toxins: effects on host cell signal transduction.Curr Top Microbiol Immunol. 2012; 357: 41-65PubMed Google Scholar Involvement of the complement system has been suggested as a possible mechanism in the development of STEC HUS.14Noris M. Mescia F. Remuzzi G. STEC-HUS, atypical HUS and TTP are all diseases of complement activation.Nat Rev Nephrol. 2012; 8: 622-633Crossref PubMed Scopus (284) Google Scholar Low C3 plasma levels in STEC HUS patients have been reported.15Cameron J.S. Vick R. Letter: plasma-C3 in haemolytic-uraemic syndrome and thrombotic thrombocytopenic purpura.Lancet. 1973; 2: 975Abstract PubMed Scopus (51) Google Scholar, 16Robson W.L. Leung A.K. Fick G.H. et al.Hypocomplementemia and leukocytosis in diarrhea-associated hemolytic uremic syndrome.Nephron. 1992; 62: 296-299Crossref PubMed Scopus (67) Google Scholar Other changes in the complement system include increased levels of breakdown products of the 2 components of the alternative pathway C3 convertase, C3 (C3b, C3c, C3d), factor B (Ba and Bb), as well as sC5b-9.17Monnens L. Molenaar J. Lambert P.H. et al.The complement system in hemolytic-uremic syndrome in childhood.Clin Nephrol. 1980; 13: 168-171PubMed Google Scholar, 18Thurman J.M. Marians R. Emlen W. et al.Alternative pathway of complement in children with diarrhea-associated hemolytic uremic syndrome.Clin J Am Soc Nephrol. 2009; 4: 1920-1924Crossref PubMed Scopus (156) Google Scholar, 19Stahl A.L. Sartz L. Karpman D. Complement activation on platelet-leukocyte complexes and microparticles in enterohemorrhagic Escherichia coli-induced hemolytic uremic syndrome.Blood. 2011; 117: 5503-5513Crossref PubMed Scopus (151) Google Scholar These findings imply that inhibiting the complement system may attenuate the cell injury and lead to resolution of renal insufficiency. In children with severe neurological STEC HUS, anti-C5 antibody eculizumab was shown to be clinically effective.20Lapeyraque A.L. Malina M. Fremeaux-Bacchi V. et al.Eculizumab in severe Shiga-toxin-associated HUS.N Engl J Med. 2011; 364: 2561-2563Crossref PubMed Scopus (206) Google Scholar In contrast, other studies have questioned the effectiveness of eculizumab in the treatment of STEC HUS.21Kielstein J.T. Beutel G. Fleig S. et al.Best supportive care and therapeutic plasma exchange with or without eculizumab in Shiga-toxin-producing E. coli O104:H4 induced haemolytic-uraemic syndrome: an analysis of the German STEC-HUS registry.Nephrol Dial Transplant. 2012; 27: 3807-3815Crossref PubMed Scopus (181) Google Scholar, 22Menne J. Nitschke M. Stingele R. et al.Validation of treatment strategies for enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome: case-control study.BMJ. 2012; 345: e4565Crossref PubMed Scopus (238) Google Scholar However, the role of the lectin complement pathway in STEC HUS is unknown. We hypothesized that mannose-binding lectin 2 (MBL2) of the lectin pathway plays a role in the pathogenesis of STEC HUS. MBL2 is an initiating factor of the lectin complement pathway and recognizes pathogens and altered-self cells, resulting in induction of an inflammatory response and thrombogenesis. On MBL2 binding to its target, serine proteases called MBL/ficolin-associated serine proteases (MASP-1, MASP-2, MASP-3) activate C4 and C2 leading to formation of the C3 and C5 convertases and ultimately to formation of the membrane attack complex (e.g., C5b-9). MASPs are also involved in cleavage of prothrombin to thrombin, cleavage of fibrinogen to fibrin, and activation of factor XIII.23Gulla K.C. Gupta K. Krarup A. et al.Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot.Immunology. 2010; 129: 482-495Crossref PubMed Scopus (107) Google Scholar, 24Hess K. Ajjan R. Phoenix F. et al.Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation.PLoS One. 2012; 7: e35690Crossref PubMed Scopus (83) Google Scholar MBL2 and MASPs are involved in the activation of multiple biological pathways involved in thrombotic diseases.25La Bonte L.R. Pavlov V.I. Tan Y.S. et al.Mannose-binding lectin-associated serine protease-1 is a significant contributor to coagulation in a murine model of occlusive thrombosis.J Immunol. 2012; 188: 885-891Crossref PubMed Scopus (71) Google Scholar We have shown that MBL2 is associated with endothelial injury during sterile injury such as ischemia/reperfusion injury and thrombogenesis.26Pavlov V.I. Skjoedt M.O. Siow T.Y. et al.Endogenous and natural complement inhibitor attenuates myocardial injury and arterial thrombogenesis.Circulation. 2012; 126: 2227-2235Crossref PubMed Scopus (61) Google Scholar To investigate the role of MBL2 further in clinically relevant settings, we recently described a mouse model in which human MBL2 is knocked-in (KI) with the absence of murine MBLs (MBL2+/+Mbl1–/–Mbl2–/–; MBL2 KI mouse) and also used a functionally inhibiting monoclonal antibody (mAb) against MBL2, clone 3F827Collard C.D. Vakeva A. Morrissey M.A. et al.Complement activation after oxidative stress: role of the lectin complement pathway.Am J Pathol. 2000; 156: 1549-1556Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 28Zhao H. Wakamiya N. Suzuki Y. et al.Identification of human mannose binding lectin (MBL) recognition sites for novel inhibitory antibodies.Hybrid Hybridomics. 2002; 21: 25-36Crossref PubMed Scopus (24) Google Scholar, 29Pavlov V.I. Tan Y.S. McClure E.E. et al.Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.Am J Pathol. 2015; 185: 347-355Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar to inhibit MBL2 function. To mimic STEC HUS, we modeled Stx-induced renal injury using the MBL2 KI mouse and purified Stx-2. In the current study, we examined the role of MBL2 in the pathogenesis of STEC HUS and explored the effectiveness of 3F8 in this mouse model. We determined the toxicity of Stx-2 in MBL2 KI mice. Dose-response experiments determined that the 50% lethal dose was 625 pg/g. Administration of Stx-2 (625 pg/g) resulted in rapidly progressive weight loss; mice were moribund 3 days later and displayed convulsions and ataxia, indicating impairment of the nervous system. To reproduce more clinically relevant STEC HUS in MBL2 KI mice without death, the Stx-2 dose was reduced to 125 pg/g. We used weight loss as an indicator of toxicity instead of death in these pilot studies because weight loss in rodent STEC HUS models correlates with illness.30Keepers T.R. Psotka M.A. Gross L.K. et al.A murine model of HUS: Shiga toxin with lipopolysaccharide mimics the renal damage and physiologic response of human disease.J Am Soc Nephrol. 2006; 17: 3404-3414Crossref PubMed Scopus (123) Google Scholar, 31Sauter K.A. Melton-Celsa A.R. Larkin K. et al.Mouse model of hemolytic-uremic syndrome caused by endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by anti-Stx2 antibody.Infect Immun. 2008; 76: 4469-4478Crossref PubMed Scopus (68) Google Scholar, 32Petruzziello-Pellegrini T.N. Yuen D.A. Page A.V. et al.The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin-associated hemolytic uremic syndrome in humans and mice.J Clin Invest. 2012; 122: 759-776Crossref PubMed Scopus (73) Google Scholar With a dose of Stx-2 (125 pg/g), various degrees of weight loss developed in MBL2 KI mice, but none exhibited neurological deficits by day 4. This sublethal dose of Stx-2 (125 pg/g) was used in all experimental studies. For sham/controls, mice were injected with either phosphate-buffered saline (PBS) or toxoid (e.g., a mutated form of Stx-2 that is produced and purified in the same manner as Stx-2).33Acheson D.W. Jacewicz M. Kane A.V. et al.One step high yield affinity purification of Shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays.Microb Pathog. 1993; 14: 57-66Crossref PubMed Scopus (35) Google Scholar, 34Wen S.X. Teel L.D. Judge N.A. et al.Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge.Vaccine. 2006; 24: 1142-1148Crossref PubMed Scopus (56) Google Scholar Mice were weighed every 24 hours from day −1 to day 4 (Figure 1). The MBL2 KI mice receiving Stx-2 and an isotype control monoclonal antibody (clone 1C10) began to lose body weight by day 2. On day 4, these mice lost an average of 11% of their baseline body weight. To control for any contaminating lipopolysaccharide or blood volume changes, we injected mice with toxoid or PBS.33Acheson D.W. Jacewicz M. Kane A.V. et al.One step high yield affinity purification of Shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays.Microb Pathog. 1993; 14: 57-66Crossref PubMed Scopus (35) Google Scholar, 34Wen S.X. Teel L.D. Judge N.A. et al.Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge.Vaccine. 2006; 24: 1142-1148Crossref PubMed Scopus (56) Google Scholar Similar to PBS, toxoid-injected mice had no weight loss over the 4-day observation period. MBL2 KI mice treated with 3F8 and receiving Stx-2 had significantly less weight loss at day 4 compared with the 1C10 + Stx-2 group. Functional serum MBL2 levels were evaluated in mice receiving PBS or toxoid and mice receiving Stx-2 and treated with either PBS, 1C10, or 3F8 (Figure 2). All groups had similar levels of circulating MBL2 (e.g., ∼3 μg/ml of sera), which was significantly inhibited in mice receiving 3F8. The main characteristics of STEC HUS are anemia, thrombocytopenia, and renal injury. We observed a small but significant decrease in red blood cell count from 11 ± 1 to 8 ± 1 (106 cells/mm3) after Stx-2 administration compared with control. To better evaluate Stx-2– induced lysis of red blood cells, we investigated free serum hemoglobin as an index of anemia. We observed an increase in free serum hemoglobin in mice treated with PBS or 1C10 and also receiving Stx-2 compared with control mice receiving only PBS or toxoid (Figure 3). Mice treated with 3F8 had significantly lower free serum hemoglobin levels, and these levels were not different from the control mice (e.g., mice receiving only PBS or toxoid). We also observed a significant decrease in platelet counts induced by Stx-2 compared with mice receiving only PBS (Figure 4). (Note: because toxoid did not affect other parameters, the toxoid group was discontinued in the remaining studies.) Although the platelet counts in the 3F8 + Stx-2 group were not significantly different from those in either the PBS only group or the Stx-2 group, there was a trend for platelet counts with 3F8 + Stx-2 to be higher than those in the PBS + Stx-2 group, a trend that might have reached statistical significance with a higher number. These data demonstrate Stx-2– induced anemia and thrombocytopenia in this novel mouse line with anemia blocked by anti-MBL2 treatment.Figure 4Platelet count. Plasma blood samples were analyzed for platelet counts on day 4. Mice treated with PBS + PBS had significantly higher platelet counts compared with those treated with PBS + Stx-2. Treatment with 3F8 + Stx-3 did not significantly alter platelet counts compared with the PBS + PBS group, but they were also not different from the PBS + Stx-2 group. Bars and brackets represent the mean ± SEM, and the numbers in the bars represent number of mice studied. *P < 0.05. ns, not significant; PBS, phosphate-buffered saline; Stx-2, Shiga toxin-2.View Large Image Figure ViewerDownload (PPT) Serum samples were also analyzed for 2 representative markers of renal injury (creatinine [Cr] and cystatin C) (Figure 5). Four days after administration of Stx-2, serum Cr was increased significantly compared with the PBS-only group (Figure 5, top). Serum Cr in the 3F8 + Stx-2 group was significantly lower compared with the group receiving PBS + Stx-2. Similar to the Cr data, serum cystatin C (Figure 5, bottom) was increased in response to Stx-2 compared with the control group (PBS only). Serum cystatin C levels were significantly lower in the 3F8 + Stx-2 group compared with the PBS + Stx-2 group. To evaluate renal function, we determined glomerular filtration rate (GFR). GFR was significantly decreased in the PBS + Stx-2 group compared with the PBS-only group (Figure 6). GFR in the 3F8 + Stx-2 group was significantly higher compared with the PBS + Stx-2 group and not significantly different from the PBS-only group. In summary, Stx-2 induced the following 3 characteristics of STEC HUS: renal failure, anemia, and thrombocytopenia in MBL2 KI mice. In this Stx-2 model, functional inhibition of MBL2 with mAb 3F8 prevented renal failure and anemia, with marginal effects on thrombocytopenia. Renal function (e.g., GFR) was significantly protected from Stx-2–induced injury by MBL2 inhibition. Local synthesis of complement protein C3 from renal epithelial cells is an important mediator for local tissue injury in renal disease.35Sheerin N.S. Risley P. Abe K. et al.Synthesis of complement protein C3 in the kidney is an important mediator of local tissue injury.FASEB J. 2008; 22: 1065-1072Crossref PubMed Scopus (65) Google Scholar We examined renal C3d deposition as an index of complement activation by all complement pathways. As others have demonstrated,35Sheerin N.S. Risley P. Abe K. et al.Synthesis of complement protein C3 in the kidney is an important mediator of local tissue injury.FASEB J. 2008; 22: 1065-1072Crossref PubMed Scopus (65) Google Scholar, 36Zhou W.D. Marsh J.E. Sacks S.H. Intrarenal synthesis of complement.Kidney Int. 2001; 59: 1227-1235Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar we observed local C3d in renal sections from mice given only PBS (Figure 7a; n = 3). Mice (n = 3) injected with PBS + Stx-2 (Figure 7b) had significantly (P = 0.001) greater renal C3d deposition compared with control mice (Figure 7a) (47 ± 6% increase). The 3F8 + Stx-2 group (n = 2; Figure 7c) demonstrated C3d deposition that was not different from that of control mice (Figure 7a) and significantly less than PBS + Stx-2 (P = 0.004) (Figure 7b). These data demonstrate that MBL2 and the lectin complement pathway are responsible for complement activation and C3d deposition after Stx-2 administration. Immunohistochemical staining for fibrin demonstrated fibrin deposition in the glomeruli of PBS + Stx-2 mice (Figure 8a). There was significantly less fibrin deposition in the glomeruli in the 3F8 + Stx-2 group (Figure 8a and b). These data demonstrate the important role of the MBL2 complex in glomeruli fibrin deposition after Stx-2 administration. We developed a murine model with 3 characteristics of STEC HUS (renal injury, anemia, and thrombocytopenia) using MBL2 KI mice injected with Stx-2. We found that the anti-MBL2 mAb 3F8 attenuated complement C3d deposition and the renal injury induced by Stx-2. Although involvement of complement activation, particularly the classic and alternative pathways, has been suggested in STEC HUS, the mechanisms by which this occurs have not been fully elucidated.15Cameron J.S. Vick R. Letter: plasma-C3 in haemolytic-uraemic syndrome and thrombotic thrombocytopenic purpura.Lancet. 1973; 2: 975Abstract PubMed Scopus (51) Google Scholar, 18Thurman J.M. Marians R. Emlen W. et al.Alternative pathway of complement in children with diarrhea-associated hemolytic uremic syndrome.Clin J Am Soc Nephrol. 2009; 4: 1920-1924Crossref PubMed Scopus (156) Google Scholar, 37Porubsky S. Federico G. Muthing J. et al.Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure.J Pathol. 2014; 234: 120-133Crossref PubMed Scopus (46) Google Scholar, 38Paixao-Cavalcante D. Botto M. 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Studying the role of MBL in mouse models of human disease has been limited as wild-type mice have 2 active forms of MBL (e.g., Mbl1 and MBL2) and inhibitors of these 2 functional MBLs are not available. To overcome these limitations, we generated a humanized MBL2-expressing mouse (MBL2+/+ Mbl1−/− Mbl2−/−; MBL2 KI) within the Mbl1 locus.29Pavlov V.I. Tan Y.S. McClure E.E. et al.Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.Am J Pathol. 2015; 185: 347-355Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar These mice produce functional MBL2, no mouse Mbl1 or Mbl2, and mAb 3F8 completely inhibits MBL2 function in vivo.29Pavlov V.I. Tan Y.S. McClure E.E. et al.Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.Am J Pathol. 2015; 185: 347-355Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Using these mice, it is now possible to study the role of the MBL/lectin complement pathway in the pathogenesis of STEC HUS. We evaluated renal injury in our model by measuring GFR. Stx-2 induced a decrease in GFR, and 3F8 was protective against the Stx-2 induced filtration dysfunction. These observations indicate that MBL2 mediates renal injury in this STEC HUS model. Because of the absence of Gb3 on murine glomerular endothelial cells, it has generally been said that microthrombosis in the glomeruli cannot be reproduced in a mouse model of STEC HUS.40Obrig T.G. Escherichia coli Shiga toxin mechanisms of action in renal disease.Toxins (Basel). 2010; 2: 2769-2794Crossref PubMed Scopus (103) Google Scholar Consistent with this conventional theory, microthrombi were not found in the glomeruli of our STEC HUS model. However, we observed glomerular injury indicated by fibrin deposits and decreased GFR in this model. The fibrin deposits were not accompanied by light microscopic thrombi as is typical in the thrombotic microangiopathy in humans. A similar finding was reported in the glomeruli of another mouse STEC HUS model.31Sauter K.A. Melton-Celsa A.R. Larkin K. et al.Mouse model of hemolytic-uremic syndrome caused by endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by anti-Stx2 antibody.Infect Immun. 2008; 76: 4469-4478Crossref PubMed Scopus (68) Google Scholar Fibrin depositions were significantly decreased in the glomeruli of mice treated with the anti-MBL2 mAb 3F8. This effect of 3F8 is similar to the effect that we reported previously in which 3F8 inhibited fibrin deposition after reperfusion of the ischemic myocardium.29Pavlov V.I. Tan Y.S. McClure E.E. et al.Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.Am J Pathol. 2015; 185: 347-355Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar These data suggest that the MBL/MASP complex directly activated the coagulation cascade in this model. A recent study demonstrating the affinity of Stx-2 for glycolipids and glycans suggests the possibility that other cellular components contribute to Stx-2 potency and support our findings that glomerular injury developed without Gb3 expression.41Gallegos K.M. Conrady D.G. Karve S.S. et al.Shiga toxin binding to glycolipids and glycans.PLoS One. 2012; 7: e30368Crossref PubMed Scopus (69) Google Scholar Further, Stx is transferred within microvesicles in a Gb3 receptor–independent manner.42Arvidsson I. Stahl A.L. Hedstrom M.M. et al.Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.J Immunol. 2015; 194: 2309-2318Crossref PubMed Scopus (60) Google Scholar, 43Stahl A.L. Arvidsson I. Johansson K.E. et al.A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.PLoS Pathog. 2015; 11: e1004619Crossref PubMed Scopus (79) Google Scholar Based on these past observations, we believe that Stx-2 induces glomerular injury in our model, even in the absence of Gb3. Moreover, MBL2 can independently induce tubular epithelial cell death without complement activation.44van der Pol P. Schlagwein N. van Gijlswijk D.J. et al.Mannan-binding lectin mediates renal ischemia/reperfusion injury independent of complement activation.Am J Transplant. 2012; 12: 877-887Crossref PubMed Scopus (62) Google Scholar Our findings further support an important role for MBL2-mediated renal injury and extends these findings to include glomerular injury in a murine STEC HUS model. Published findings and observations in the current study led us to propose the following mechanism of MBL2-mediated renal injury in this STEC HUS model (Figure 9). Stx produced by STEC or Shigella dysenteriae enters the bloodstream and is internalized in endothelial cells.42Arvidsson I. Stahl A.L. Hedstrom M.M. et al.Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.J Immunol. 2015; 194: 2309-2318Crossref PubMed Scopus (60) Google Scholar, 43Stahl A.L. Arvidsson I. 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