Differential Outcome between BALB/c and C57BL/6 Mice after Escherichia coli O157:H7 Infection Is Associated with a Dissimilar Tolerance Mechanism.

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism.

Absence of interleukin-10 reduces progression of shiga toxin-induced hemolytic uremic syndrome.

Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10-/-) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10-/- mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN increased at 72 h in both mouse strains accompanied by an up-regulation of CD11b in control mice. In parallel, renal PMN were significantly increased only in control mice after toxin. Plasma TNF-α, IL-6 and corticosterone levels were higher increased in IL-10-/- than control mice. Simultaneously renal TNF-α raised constantly but was accompanied by increased TGF-ß levels in IL-10-/- mice. These results demonstrate that the profile of circulating and renal cytokines after Stx2 differed between strains suggesting that balance of these factors could participate in renal protection. We conclude that IL-10 absence has a protective role in an experimental model of HUS by reducing PMN recruitment into kidney and renal damage, and increasing mice survival.

Assessment of interleukin-10 promoter variant (-1082A/G) and cytokine production in patients with hemolytic uremic syndrome.

Introduction: Hemolytic uremic syndrome (HUS) is a condition that results in acute kidney failure mainly in children, which is caused by Shiga toxin-producing Escherichia coli and inflammatory response. Although anti-inflammatory mechanisms are triggered, studies on the implication in HUS are scarce. Interleukin-10 (IL-10) regulates inflammation in vivo, and the interindividual differences in its expression are related to genetic variants. Notably, the single nucleotide polymorphism (SNP) rs1800896 -1082 (A/G), located in the IL-10 promoter, regulates cytokine expression. Methods: Plasma and peripheral blood mononuclear cells (PBMC) were collected from healthy children and HUS patients exhibiting hemolytic anemia, thrombocytopenia, and kidney damage. Monocytes identified as CD14+ cells were analyzed within PBMC by flow cytometry. IL-10 levels were quantified by ELISA, and SNP -1082 (A/G) was analyzed by allele-specific PCR. Results: Circulating IL-10 levels were increased in HUS patients, but PBMC from these patients exhibited a lower capacity to secrete this cytokine compared with those from healthy children. Interestingly, there was a negative association between the circulating levels of IL-10 and inflammatory cytokine IL-8. We observed that circulating IL-10 levels were threefold higher in HUS patients with -1082G allele in comparison to AA genotype. Moreover, there was relative enrichment of GG/AG genotypes in HUS patients with severe kidney failure. Discussion: Our results suggest a possible contribution of SNP -1082 (A/G) to the severity of kidney failure in HUS patients that should be further evaluated in a larger cohort.

Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro.

Enterohemorrhagic Escherichia coli (EHEC) strains are food-borne pathogens that can cause different clinical conditions. Shiga toxin 2a and/or 2c (Stx2)-producing E. coli O157:H7 is the serotype most frequently associated with severe human disease. In this work we analyzed the hypothesis that host cells participate in Stx2 production, cell damage, and inflammation during EHEC infection. With this aim, macrophage-differentiated THP-1 cells and the intestinal epithelial cell line HCT-8 were incubated with E. coli O157:H7. A time course analysis of cellular and bacterial survival, Stx2 production, stx2 transcription, and cytokine secretion were analyzed in both human cell lines. We demonstrated that macrophages are able to internalize and kill EHEC. Simultaneously, Stx2 produced by internalized bacteria played a major role in macrophage death. In contrast, HCT-8 cells were completely resistant to EHEC infection. Besides, macrophages and HCT-8 infected cells produce IL-1ß and IL-8 inflammatory cytokines, respectively. At the same time, bacterial stx2-specific transcripts were detected only in macrophages after EHEC infection. The interplay between bacteria and host cells led to Stx production, triggering of inflammatory response and cell damage, all of which could contribute to a severe outcome after EHEC infections.

Cytokines use different intracellular mechanisms to upregulate the membrane expression of CX3CR1 in human monocytes.

Membrane expression of fractalkine (CX3CL1)-receptor (CX3CR1) is relevant in monocytes (Mo) because CX3CR1-CX3CL1 interactions might participate on both, homeostatic and pathologic conditions. We have previously demonstrated that CX3CR1 levels are decreased during culture and when Mo are differentiated into dendritic cells, but enhanced when differentiated into macrophages. Regarding soluble factors, lipopolysaccharide (LPS) accelerated the loss of CX3CR1, while interleukin (IL)-10 and Interferon-gamma (IFN-γ) prevented it. However, the comprehensive knowledge about the intracellular pathways that underlay the level of CX3CR1 expression in Mo is still incomplete. In the current work, we studied the effect of anti-inflammatory cytokines (IL-4, IL-13, IL-10), alone or together with IFN- γ on CX3CR1 expression. We found that only IL-10 and IFN-γ separately were able to prevent CX3CR1 down-modulation during culture of human Mo. Besides, Mo incubated with IL-10 plus IFN-γ showed the highest CX3CR1 expression by cell, suggesting cooperation between two different mechanism used by both cytokines. By studying intracellular mechanisms triggered by IL-10 and IFN-γ, we demonstrated that they specifically induced PI3K-dependent serine-phosphorylation of signal transducer and activator of transcription (STAT)3 or STAT1, respectively. Moreover, chemical inhibitors of STAT1 or STAT3 abrogated IFN-γ or IL-10 effects on CX3CR1 expression. Strikingly, only IL-10 increased CX3CR1 mRNA level, as consequence of augmenting mRNA stability. CX3CR1 mRNA increase was PI3K-dependent, supporting the causal link between the action of IL-10 at the CX3CR1 transcript and CX3CR1 protein level on Mo. Thus, both cytokines up-regulate CX3CR1 expression on human Mo by different intracellular mechanisms.