Role of Tumor Necrosis Factor Alpha in Disease Using a Mouse Model of Shiga Toxin-Mediated Renal Damage

Mice have been extensively employed as an animal model of renal damage caused by Shiga toxins. In this study, we examined the role of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) in the development of toxin-mediated renal disease in mice. Mice pretreated with TNF-α and challenged with Shiga toxin type 1 (Stx1) showed increased survival compared to that of mice treated with Stx1 alone. Conversely, mice treated with Stx1 before TNF-α administration succumbed more quickly than mice given Stx1 alone. Increased lethality in mice treated with Stx1 followed by TNF-α was associated with evidence of glomerular damage and the loss of renal function. No differences in renal histopathology were noted between animals treated with Stx1 alone and the TNF-α pretreatment group, although we noted a sparing of renal function when TNF-α was administered before toxin. Compared to that of treatment with Stx1 alone, treatment with TNF-α after toxin altered the renal cytokine profile so that the expression of proinflammatory cytokines TNF-α and interleukin-1β (IL-1β) increased, and the expression of the anti-inflammatory cytokine IL-10 decreased. Increased lethality in mice treated with Stx1 followed by TNF-α was associated with higher numbers of dUTP-biotin nick end labeling-positive renal tubule cells, suggesting that increased lethality involved enhanced apoptosis. These data suggest that the early administration of TNF-α is a candidate interventional strategy blocking disease progression, while TNF-α production after intoxication exacerbates disease.Shiga toxins are a family of genetically and functionally related cytotoxic proteins expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Antigenic similarity to Shiga toxin expressed by S. dysenteriae serotype 1 is used to define Shiga toxin type 1 (Stx1) and type 2 (Stx2) expressed by Shiga toxin-producing E. coli (STEC) (44). Shiga toxins consist of a single A subunit in noncovalent association with a pentamer of B subunits. B subunits mediate binding to the neutral glycolipid receptor globotriaosylceramide (Gb₃), while the A subunit possesses an N-glycosidase activity (38). Following toxin internalization and routing to the endoplasmic reticulum (ER), a fragment of the toxin A subunit generated by furin or a furin-like protease is translocated across the ER membrane and mediates the cleavage of a single adenine residue (A4256 in the rat) from the 28S rRNA component of ribosomes (39). Stx-induced depurination leads to the disruption of elongation factor-dependent aminoacyl-tRNA binding to nascent polypeptides (30). Thus, Shiga toxins are potent protein synthesis inhibitors, with 50% cytotoxic doses measured in pg/ml amounts for many cell types in vitro. Shiga toxins also activate the ribotoxic and ER stress pathways, which are important in the activation of proinflammatory cytokine/chemokine production and apoptosis (6, 22, 41).The ingestion of small quantities of Stx-producing bacteria may lead to the development of bloody diarrhea with progression to acute renal failure, designated diarrhea-associated hemolytic uremic syndrome (D+HUS) (33). Epidemiologic studies have shown that the ingestion of STEC strains expressing Stx2 alone or Stx1 and Stx2 are more likely to progress to life-threatening extraintestinal complications (3, 17, 31). D+HUS is a leading cause of pediatric acute renal failure. D+HUS is characterized by rapid-onset oligouria or anuria, azotemia, microangiopathic hemolytic anemia with schistocytosis, and thrombocytopenia (33, 47). The histopathological examination of D+HUS renal tissues showed that glomerular microvascular endothelial cells were frequently swollen and detached from the basement membrane, and glomerular capillary lumina may be occluded with fibrin-rich microthrombi (21, 36).Numerous animal models have been employed to characterize the role of Stx1 and Stx2 in pathogenesis. Studies utilizing nonhuman primates showed that Shiga toxins are essential virulence determinants in the development of microangiopathic lesions. Fontaine et al. (9) showed that macaque monkeys fed toxigenic strains of S. dysenteriae developed colonic microvascular lesions, while baboons given purified intravenous Stx1 developed acute renal failure (48). The bolus intravenous administration of Stx1 or Stx2 into baboons revealed that the animals were more sensitive to Stx2, although the mean times to death were prolonged in Stx2-treated animals compared to that with Stx1 treatment. Both toxins mediated hematologic changes such as thrombocytopenia and schistocytosis, and both toxins produced renal pathology, but with different presentations. Renal damage caused by Stx1 was characterized by moderate congestion at the cortico-medullary junction, while Stx2-treated animals showed severe medullary congestion with cortical ischemia (42). Mice fed Stx2-producing E. coli or given a single bolus injection of purified Shiga toxins died without the development of glomerular thrombotic microangiopathy (50, 54). However, the administration of multiple low doses of Stx2 allowed the animals to survive initial toxin challenge and develop glomerular lesions characteristic of HUS in humans (40). In addition to the toxins, host response factors may contribute to D+HUS pathogenesis. Prodromal hemorrhagic colitis may alter normal colonic barrier function, and patients with D+HUS may be endotoxemic or show evidence of elevated antibody titers against lipopolysaccharides (LPS) expressed by Stx-producing E. coli (2, 10, 26). LPS elicit the expression of a broad array of pro- and anti-inflammatory cytokines and chemokines (45). In accordance with this, D+HUS patients frequently have increased serum or urinary proinflammatory cytokine and chemokine levels (15, 23). Studies using small-animal models support the hypothesis that additional bacterial and host response factors facilitate the development of renal disease. Keepers et al. (19) demonstrated that the coadministration of Stx2 and LPS to C57BL/6 mice did not produce major changes in lethality but resulted in pathophysiological changes more consistent with disease in humans: intraglomerular platelet and fibrin deposition, decreased renal function, neutrophilia, and lymphocytopenia. Barrett et al. (1) showed that the timing of toxin and LPS challenges were critical in disease outcome. LPS enhanced the lethal effects of purified Stx2 when administered to rabbits or mice after toxin challenge, whereas LPS protected the animals from Stx2 toxicity when administered before the toxin. Palermo et al. (32) showed that the LPS-induced modulation of Stx2 lethality was cytokine time and dose dependent. Mice given low doses of TNF-α or IL-1β 1 h before Stx2 treatment showed increased lethality when treated with Stx2, while mice given higher doses of IL-1β (sufficient to elicit corticosteroid production) were protected from Stx2 lethality.The proinflammatory cytokines TNF-α and IL-1β sensitize vascular endothelial cells to the cytotoxic action of Shiga toxins in vitro (24, 34, 53) through a mechanism involving the increased expression of genes involved in the biosynthesis of Gb₃ (43). Murine and human macrophages or macrophage-like cell lines express proinflammatory cytokines and chemokines when treated with purified Shiga toxins (12, 35, 51). Keepers et al. (18) showed a marked monocytic cell infiltrate into the kidneys of mice given Stx2 and LPS. Collectively, these data suggest that the innate immune response to Shiga toxins, in the presence or absence of LPS, alters the outcome of renal disease. In the present study, we have examined the role of a single proinflammatory cytokine, TNF-α, in pathogenesis using the mouse model of Stx-induced renal damage. Our data suggest that the timing of TNF-α production affects the outcome of disease in that the presence of elevated TNF-α levels prior to toxin challenge protects animals from disease, while high TNF-α levels occurring after toxin administration result in accelerated lethality.