Bovine macrophages sense Escherichia coli Shiga toxin 1.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections in cattle are asymptomatic; however, Stx impairs the initiation of an adaptive immune response by targeting bovine peripheral and intraepithelial lymphocytes. As presumptive bovine mucosal macrophages (Mø) are also sensitive to Stx, STEC may even exert immune modulatory effects by acting on steps preceding lymphocyte activation at the Mø level. We therefore studied the expression of the Stx receptor (CD77), cellular phenotype and functions after incubation of primary bovine monocyte-derived Mø with purified Stx1. A significant portion of bovine Mø expressed CD77 on their surface, with the recombinant B-subunit of Stx1 binding to >50% of the cells. Stx1 down-regulated significantly surface expression of CD14, CD172a and co-stimulatory molecules CD80 and CD86 within 4 h of incubation, while MHC-II expression remained unaffected. Furthermore, incubation of Mø with Stx1 increased significantly numbers of transcripts for IL-4, IL-6, IL-10, IFN-γ, TNF-α, IL-8 and GRO-α but not for IL-12, TGF-ß, MCP-1 and RANTES. In the course of bovine STEC infections, Stx1 appears to induce in Mø a mixed response pattern reminiscent of regulatory Mø, which may amplify the direct suppressive effect of the toxin on lymphocytes.

Adherence of Brachyspira hyodysenteriae to porcine intestinal epithelial cells is inhibited by antibodies against outer membrane proteins.

Attachment of Brachyspira hyodysenteriae to intestinal epithelial cell lines and its possible mediation by outer membrane proteins (OMPs) of the spirochete were examined. Different B. hyodysenteriae serotypes were shown to adhere to rat and swine intestinal epithelial cells (IEC-18 and IPEC-J2) in vitro but not to the human rectal tumor cell line (HRT-18). Adherence of strain B204 to IPEC-J2 cells was reduced by rOMP-specific antisera in amounts of 29 % (anti-rBhlp29.7), 59 % (anti-rBhlp16), 70 % (anti-rBhmp39h), and 74 % (anti-rBhmp39h), respectively. By use of pooled antisera against Bhlp16 and Bhmp39f inhibition rates of the other serotypes ranged from 53 to 91 %. In a western blot assay OMPs of all serotypes but one were detected by the respective rOMP antisera. Altogether the results indicated that OMPs of B. hyodysenteriae displayed a serotype overlapping antigenicity and mediated adherence of the spirochetes to animal cell cultures.

Epithelial and mesenchymal cells in the bovine colonic mucosa differ in their responsiveness to Escherichia coli Shiga toxin 1.

Bovine colonic crypt cells express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization of cattle by human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used flow cytometric and real-time PCR analyses of primary cultures of colonic crypt cells to evaluate cell viability, CD77 expression, and gene transcription in the presence and absence of purified Stx1. A subset of cultured epithelial cells had Stx receptors which were located mainly intracellularly, with a perinuclear distribution, and were resistant to Stx1-induced apoptosis and Stx1 effects on chemokine expression patterns. In contrast, a population of vimentin-positive cells, i.e., mesenchymal/nonepithelial cells that had high numbers of Stx receptors on their surface, was depleted from the cultures by Stx1. In situ, CD77(+) cells were located in the lamina propria of the bovine colon by using immunofluorescence staining. A newly established vimentin-positive crypt cell line with high CD77 expression resisted the cytolethal effect of Stx1 but responded to Stx1 with a significant increase in interleukin-8 (IL-8), GRO-alpha, MCP-1, and RANTES mRNA. Combined stimulation with lipopolysaccharide and Stx1 increased IL-10 mRNA. Our results show that bovine colonic crypt cells of epithelial origin are resistant to both the cytotoxic and modulatory effects of Stx1. In contrast, some mucosal mesenchymal cells, preliminarily characterized as mucosal macrophages, are Stx1-responsive cells that may participate in the interaction of STEC with the bovine intestinal mucosa.

Bovine ileal intraepithelial lymphocytes represent target cells for Shiga toxin 1 from Escherichia coli.

The discovery that bovine peripheral lymphocytes are sensitive to Stx1 identified a possible mechanism for the persistence of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) in the bovine reservoir host. If intraepithelial lymphocytes (IEL) are also sensitive to Stx1, the idea that Stx1 affects inflammation in the bovine intestine is highly attractive. To prove this hypothesis, ileal IEL (iIEL) were prepared from adult cattle, characterized by flow cytometry, and subjected to functional assays in the presence and absence of purified Stx1. We found that 14.9% of all iIEL expressed Gb(3)/CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb(3)/CD77(+) cells were activated CD3(+) CD6(+) CD8 alpha(+) T cells, whereas only some CD4(+) T cells and B cells expressed Gb(3)/CD77. However, Stx1 blocked the mitogen-induced transformation to enlarged blast cells within all subpopulations to a similar extent and significantly reduced the percentage of Gb(3)/CD77(+) cells. Although Stx1 did not affect the natural killer cell activity of iIEL, the toxin accelerated the synthesis of interleukin-4 (IL-4) mRNA and reduced the amount of IL-8 mRNA in bovine iIEL cultures. Because the intestinal system comprises a rich network of interactions between different types of cells and any dysfunction may influence the course of intestinal infections, this demonstration that Stx1 can target bovine IEL may be highly relevant for our understanding of the interplay between STEC and its reservoir host.

Verotoxin 1 from Escherichia coli affects Gb3/CD77+ bovine lymphocytes independent of interleukin-2, tumor necrosis factor-alpha, and interferon-alpha.

Verotoxin (VT)-induced immunomodulation has been implicated in the ability of VT-producing Escherichia coli (VTEC) to cause persistent infections in cattle. VT1, also referred to as Shiga toxin 1, is a potent cytotoxin that modulates cytokine secretions and functions. This prompted the current investigation to examine whether the inhibiting effect of VT1 on bovine lymphocytes correlates with the expression of the cellular VT1 receptor Gb3/CD77 or is mediated instead via perturbation of cytokine secretion. Using blood mononuclear cells stimulated by mitogens as a model, VT1 significantly blocked lymphoblast transformation and proliferation in the BoCD8+ T cell and BoCD21+ B cell population. In contrast, VT1 dramatically reduced the number of viable Gb3/CD77+ blast cells within all subpopulations identified (BoCD2+, BoCD4+, BoCD8+, WC1+ [i.e., gammadelta T cells] BoCD21+, and BoCD25+). Similar effects of VT1 were observed when the culture medium was supplemented with selected cytokines: tumor necrosis factor-alpha-sensitizing endothelial cells against VT1, interferon-alpha (IFN-alpha) as bovine IFN-alpha receptors are partially homologous to the B-subunit of VT1, and interleukin-2 that is critical for lymphocyte proliferation in vitro. The addition of these cytokines was neither able to mimic nor to overcome the effects of VT1. Therefore, it is concluded that VT1 directly acts on bovine lymphocytes rather than inducing a cytokine-mediated effect. VT1 considerably affects all main bovine lymphocyte subpopulations, implicating that the immune system is a predominant target for VT1 in cattle.