Shiga Toxin Activates Complement and Binds Factor H: Evidence for an Active Role of Complement in Hemolytic Uremic Syndrome

Complement activation occurs during enterohemorrhagic Escherichia coli (EHEC) infection and may exacerbate renal manifestations. In this study, we show glomerular C5b-9 deposits in the renal biopsy of a child with EHEC-associated hemolytic uremic syndrome. The role of the terminal complement complex, and its blockade as a therapeutic modality, was investigated in a mouse model of E. coli O157:H7 infection. BALB/c mice were treated with monoclonal anti-C5 i.p. on day 3 or 6 after intragastric inoculation and monitored for clinical signs of disease and weight loss for 14 d. All infected untreated mice (15 of 15) or those treated with an irrelevant Ab (8 of 8) developed severe illness. In contrast, only few infected mice treated with anti-C5 on day 3 developed symptoms (three of eight, p < 0.01 compared with mice treated with the irrelevant Ab on day 3) whereas most mice treated with anti-C5 on day 6 developed symptoms (six of eight). C6-deficient C57BL/6 mice were also inoculated with E. coli O157:H7 and only 1 of 14 developed disease, whereas 10 of 16 wild-type mice developed weight loss and severe disease (p < 0.01). Complement activation via the terminal pathway is thus involved in the development of disease in murine EHEC infection. Early blockade of the terminal complement pathway, before the development of symptoms, was largely protective, whereas late blockade was not. Likewise, lack of C6, and thereby deficient terminal complement complex, was protective in murine E. coli O157:H7 infection.

Section: Discussionmentioning

confidence: 99%

Early Terminal Complement Blockade and C6 Deficiency Are Protective in Enterohemorrhagic Escherichia coli–Infected Mice

Arvidsson

Rebetz

Loos

et al. 2016

“…High plasma levels of Bb and C5b-9 were recently measured in 17 children with D+HUS, indicating C activation via alternative pathway during the onset of D+HUS (28). Another study reported that Stx2 activated C in the fluid phase in vitro (29). However, the use of huge amounts of Stx2, together with the observation of no active Stx in plasma of patients with D+HUS (30), raises concerns about the pathophysiologic meaning of these results.…”

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confidence: 92%

Alternative Pathway Activation of Complement by Shiga Toxin Promotes Exuberant C3a Formation That Triggers Microvascular Thrombosis

Morigi

Galbusera

Gastoldi

et al. 2011

210

201

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.

“…Among the toxin actions on endothelial cells involved in HUS pathogenesis, damage to DNA, ribotoxic stress, endoplasmic reticulum stress, activation of the apoptotic program, stimulation of the production of proinflammatory cytokines, and upregulation of adhesion molecules have been described (17)(18)(19)(20)(21)(22)(23). Moreover, activation of complement, formation of platelet-leukocyte complexes, and production of tissue factor-bearing microparticles in blood by Stx have been demonstrated in vitro and in the blood from patients with HUS (24)(25)(26). In this context, several lines of evidence indicate that Stx bind in vitro to platelets through Gb3Cer lipoforms similar to those on target endothelial cells (27)(28)(29).…”

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confidence: 99%

Identification of TLR4 as the Receptor That Recognizes Shiga Toxins in Human Neutrophils

Brigotti

Carnicelli

Arfilli

et al. 2013

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin–producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.