Gabriel Cabrera

Protection of mice against Shiga toxin 2 (Stx2)-associated damage by maternal immunization with a Brucella lumazine synthase-Stx2 B subunit chimera.

ABSTRACT Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.

Early Increases in Superantigen-Specific Foxp3+ Regulatory T Cells during Mouse Mammary Tumor Virus Infection

ABSTRACT Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3+ regulatory T cells (Treg) in Peyers patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4+ CD25+ T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25+ cells at different stages of the infection. When CD25+ cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3− T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that Treg cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25+ cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages Treg cells may favor viral persistence. Thus, our results indicated that Treg cells play an important and complex role during MMTV infection.

Shiga toxin-producing Escherichia coli O157 : H7 shows an increased pathogenicity in mice after the passage through the gastrointestinal tract of the same host.

Haemolytic uraemic syndrome (HUS) is a rare but life-threatening complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, characterized by acute renal failure, thrombocytopenia and haemolytic anaemia. Although the main infection route is the consumption of contaminated food or water, person-to-person transmission has been suggested in several situations. Moreover, epidemiological data indicate that the horizontal transmission of several pathogens, including STEC, among individuals of the same species requires significantly lower doses than those used in animal models infected with laboratory-cultured bacteria. Thus, the aim of this study was to evaluate whether the passage of a clinically isolated STEC strain through the gastrointestinal tract of mice affects its pathogenicity in mice. To test this, weaned mice were orally inoculated by gavage with either an E. coli O157:H7 isolate from an HUS patient, or the same strain recovered from stools after one or two successive passages through the gastrointestinal tract of the mice. We show that stool-recovered strains are able to induce a more generalized and persistent colonization than the parent strain. Furthermore, a 10(4)-fold-reduced inoculum of the stool-recovered strains still causes gut colonization and mouse mortality, which are not observed with the parent strain. These results indicate an increased pathogenicity in stool-recovered strains that may be associated with an increased ability to colonize the mouse intestine.

B-cell lymphopoiesis is regulated by cathepsin L.

Cathepsin L (CTSL) is a ubiquitously expressed lysosomal cysteine peptidase with diverse and highly specific functions. The involvement of CTSL in thymic CD4+ T-cell positive selection has been well documented. Using CTSLnkt/nkt mice that lack CTSL activity, we have previously demonstrated that the absence of CTSL activity affects the homeostasis of the T-cell pool by decreasing CD4+ cell thymic production and increasing CD8+ thymocyte production. Herein we investigated the influence of CTSL activity on the homeostasis of peripheral B-cell populations and bone marrow (BM) B-cell maturation. B-cell numbers were increased in lymph nodes (LN), spleen and blood from CTSLnkt/nkt mice. Increases in splenic B-cell numbers were restricted to transitional T1 and T2 cells and to the marginal zone (MZ) cell subpopulation. No alterations in the proliferative or apoptosis levels were detected in peripheral B-cell populations from CTSLnkt/nkt mice. In the BM, the percentage and the absolute number of pre-pro-B, pro-B, pre-B, immature and mature B cells were not altered. However, in vitro and in vivo experiments showed that BM B-cell production was markedly increased in CTSLnkt/nkt mice. Besides, BM B-cell emigration to the spleen was increased in CTSLnkt/nkt mice. Colony-forming unit pre-B (CFU pre-B) assays in the presence of BM stromal cells (SC) and reciprocal BM chimeras revealed that both BM B-cell precursors and SC would contribute to sustain the increased B-cell hematopoiesis in CTSLnkt/nkt mice. Overall, our data clearly demonstrate that CTSL negatively regulates BM B-cell production and output therefore influencing the homeostasis of peripheral B cells.

Lymphadenectomy exacerbates tumor growth while lymphadenectomy plus the adoptive transfer of autologous cytotoxic cells and low-dose cyclophosphamide induces regression of an established murine fibrosarcoma

Tumor-draining lymph node (TDLN) ablation is routinely performed in the management of cancer; nevertheless, its usefulness is at present a matter of debate. TDLN are central sites where T cell priming to tumor antigens and onset of the antitumor immune response occur. However, tumor-induced immunosuppression has been demonstrated at TDLN, leading to downregulation of antitumor reaction and tolerance induction. Tolerance in turn is a main impairment for immunotherapy trials. We used a murine immunogenic fibrosarcoma that evolves to a tolerogenic state, to study the cellular and molecular mechanisms underlying tolerance induction at the level of TDLN and to design an appropriate immunotherapy. We determined that following a transient activation, the established tumor induces signs of immunosuppression at TDLN that coexist with local and systemic evidences of antitumor response. Therefore, we evaluated the feasibility of removing TDLN in order to eliminate a focus of immunosuppression and favor tumor rejection; but instead, a marked exacerbation of tumor growth was induced. Combining TDLN ablation with the in vivo depletion of regulatory cells by low-dose cyclophosphamide and the restoring of the TDLN-derived cells into the donor mouse by adoptive transference, resulted in lowered tumor growth, enhanced survival and a considerable degree of tumor regression. Our results demonstrate that important antitumor elements can be eliminated by lymphadenectomy and proved that the concurrent administration of low-dose chemotherapy along with the reinoculation of autologous cytotoxic cells provides protection. We suggest that this protocol may be useful, especially in the cases where lymphadenectomy is mandatory.

Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells.

Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

Leukotriene C4 increases the susceptibility of adult mice to Shiga toxin-producing Escherichia coli infection.

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that causes hemorrhagic colitis. Under some circumstances, Shiga toxin (Stx) produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome (HUS). Despite STEC human infection is characterized by acute inflammation of the colonic mucosa, little is known regarding the role of proinflammatory mediators like cysteine leukotrienes (cysLTs) in this pathology. Thus, the aim of this work was to analyze whether leukotriene C4 (LTC4) influences STEC pathogenesis in mice. We report that exogenous LTC4 pretreatment severely affected the outcome of STEC gastrointestinal infection. LTC4-pretreated (LTC4+) and STEC-infected (STEC+) mice showed an increased intestinal damage by histological studies, and a decreased survival compared to LTC4-non-pretreated (LTC4-) and STEC+ mice. LTC4+/STEC+ mice that died after the infection displayed neutrophilia and high urea levels, indicating that the cause of death was related to Stx2-toxicity. Despite the differences observed in the survival between LTC4+ and LTC4- mice after STEC infection, both groups showed the same survival after Stx2-intravenous inoculation. In addition, LTC4 pretreatment increased the permeability of mucosal intestinal barrier, as assessed by FITC-dextran absorption experiments. Altogether these results suggest that LTC4 detrimental effect on STEC infection is related to the increased passage of pathogenic factors to the bloodstream. Finally, we showed that STEC infection per se increases the endogenous LTC4 levels in the gut, suggesting that this inflammatory mediator plays a role in the pathogenicity of STEC infection in mice, mainly by disrupting the mucosal epithelial barrier.

Retinoid Levels Influence Enterohemorrhagic Escherichia coli Infection and Shiga Toxin 2 Susceptibility in Mice

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that produces Shiga toxin (Stx) and causes hemorrhagic colitis. Under some circumstances, Stx produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome. Although retinoids like vitamin A (VA) and retinoic acid (RA) are beneficial to gut integrity and the immune system, the effect of VA supplementation on gastrointestinal infections of different etiologies has been controversial. Thus, the aim of this work was to study the influence of different VA status on the outcome of an EHEC intestinal infection in mice. We report that VA deficiency worsened the intestinal damage during EHEC infection but simultaneously improved survival. Since death is associated mainly with Stx toxicity, Stx was intravenously inoculated to analyze whether retinoid levels affect Stx susceptibility. Interestingly, while VA-deficient (VA-D) mice were resistant to a lethal dose of Stx2, RA-supplemented mice were more susceptible to it. Given that peripheral blood polymorphonuclear cells (PMNs) are known to potentiate Stx2 toxicity, we studied the influence of retinoid levels on the absolute number and function of PMNs. We found that VA-D mice had decreased PMN numbers and a diminished capacity to produce reactive oxygen species, while RA supplementation had the opposite effect. These results are in line with the well-known function of retinoids in maintaining the homeostasis of the gut but support the idea that they have a proinflammatory effect by acting, in part, on the PMN population.

Oral administration of Shiga toxin-producing Escherichia coli induces intestinal and systemic specific immune response in mice

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer’s patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare.

Increases in IgA+ B cells in Peyer's patches during milk-borne mouse mammary tumor virus infection are influenced by Toll-like receptor 4 and are completely dependent on the superantigen response.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyers patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.