title: Alternative pathway activation of complement by Shiga toxin promotes exuberant C3a formation that triggers microvascular thrombosis.
abstract: Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti-P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.
authors.affiliation: Mario Negri Institute for Pharmacological Research, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, 24126 Bergamo, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Morigi
authors.forename: Miriam
authors.initials: M
authors.lastname: Galbusera
authors.forename: Sara
authors.initials: S
authors.lastname: Gastoldi
authors.forename: Monica
authors.initials: M
authors.lastname: Locatelli
authors.forename: Simona
authors.initials: S
authors.lastname: Buelli
authors.forename: Anna
authors.initials: A
authors.lastname: Pezzotta
authors.forename: Chiara
authors.initials: C
authors.lastname: Pagani
authors.forename: Marina
authors.initials: M
authors.lastname: Noris
authors.forename: Marco
authors.initials: M
authors.lastname: Gobbi
authors.forename: Matteo
authors.initials: M
authors.lastname: Stravalaci
authors.forename: Daniela
authors.initials: D
authors.lastname: Rottoli
authors.forename: Francesco
authors.initials: F
authors.lastname: Tedesco
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Remuzzi
authors.forename: Carlamaria
authors.initials: C
authors.lastname: Zoja
mesh_terms: D000818:Animals; D002460:Cell Line; D015926:Complement C3a / Q000096:biosynthesis / Q000378:metabolism / Q000633:toxicity*; D011415:Complement Factor B / Q000172:deficiency / Q000235:genetics; D003170:Complement Pathway, Alternative / Q000276:immunology*; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D004927:Escherichia coli Infections / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D019453:Escherichia coli O157 / Q000276:immunology / Q000472:pathogenicity; D006463:Hemolytic-Uremic Syndrome / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007678:Kidney Glomerulus / Q000098:blood supply / Q000276:immunology / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008833:Microcirculation / Q000276:immunology; D019007:P-Selectin / Q000502:physiology; D011485:Protein Binding / Q000276:immunology; D022622:Shiga Toxin 1 / Q000633:toxicity*; D022641:Shiga Toxin 2 / Q000633:toxicity*
chemical_list: D019007:P-Selectin; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D015926:Complement C3a; D011415:Complement Factor B