Yersinia enterocolitica induces apoptosis and inhibits surface molecule expression and cytokine production in murine dendritic cells

Abstract

Yersinia enterocolitica evades innate immunity by expression of a variety of pathogenicity factors. Therefore, adaptive immunity including CD4(+) T cells plays an important role in defense against Y. enterocolitica. We investigated whether Y. enterocolitica might target dendritic cells (DC) involved in adaptive T-cell responses. For this purpose, murine DC were infected with Y. enterocolitica wild-type and mutant strains prior to incubation with ovalbumin (OVA) as antigen and 5-(6)-carboxyfluorescein diacetate N-succinimidyl ester-labeled OVA-specific T cells from DO11.10 mice. While T-cell proliferation was partially affected by infection of DC with plasmid-cured and YopP-deficient Yersinia mutant strains, no T-cell proliferation occurred after infection of DC with wild-type Y. enterocolitica. Infection of DC with Y. enterocolitica wild type resulted in decreased up-regulation of major histocompatibility complex class II, CD54 (intercellular adhesion molecule 1), CD 80, and CD86 expression. Experiments with plasmid-cured Y. enterocolitica or a YopP-deficient mutant strain revealed that YopP accounts for inhibition of surface molecule expression. Wild-type Y. enterocolitica suppressed the release of KC, tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-12 by DC, while infection of DC with plasmid-cured Y. enterocolitica or with the YopP-deficient mutant resulted in the production of these cytokines. Moreover, infection with wild-type Y. enterocolitica induced apoptosis in DC mediated by YopP. Apoptosis occurred despite translocation of NF-kappaB to the nucleus, as demonstrated by electromobility shift assays. Together, these data demonstrate that Y. enterocolitica targets functions of murine DC that are required for T-cell activation. This might contribute to evasion of adaptive immune responses by Y. enterocolitica.

FIG. 1.

Proliferation of T cells after coculture with DC infected with Y. enterocolitica. DC were incubated with various strains of Y. enterocolitica, wild type (WA-pYV⁺), plasmid cured (WA-pYV⁻), YopP deficient (pYV⁺YopP⁻), and heat-killed wild type (WA-pYV⁺ heat killed), at an MOI of 10. After 1 h, gentamicin (100 μg/ml) and OVA (100 μg/ml) were added. DC were cocultured with CFSE-labeled OVA-specific T-cell receptor transgenic T cells from DO11.10 mice at a ratio of 4 (one DC to four T cells). T-cell proliferation was analyzed 4 days later by flow cytometry by gating on propidium iodide-negative cells (inserts). Results are representative of seven experiments.

FIG. 2.

Death of DC after infection with Y. enterocolitica. (A and B) DC were left untreated or incubated with staurosporine or wild-type Y. enterocolitica (WA-pYV⁺; MOI, 10). Four hours postinfection, DC were stained with TMRE and propidium iodide (PI) (A) or with FITC-conjugated Annexin-V and PI (B). Numbers indicate percentages of cells in each quadrant. Results are representative of 16 (A) and 9 (B) experiments. (C) Electron microscopy analysis of DC incubated with medium demonstrated normal morphology of the cells, while treatment with staurosporine or infection with wild-type Y. enterocolitica (4 h) induced numerous apoptotic bodies containing cytoplasmic and chromatin remnants (arrows).

FIG. 3.

Death of DC after infection with Y. enterocolitica. DC were left untreated or incubated with staurosporine or different strains of Y. enterocolitica: wild type (WA-pYV⁺), plasmid cured (WA-pYV⁻), YopP deficient (pYV⁺YopP⁻), and heat-killed wild type (WA-pYV⁺ heat killed). Four hours postinfection DC were stained with TMRE and propidium iodide (PI), and 24 h postinfection the Nicoletti procedure was performed. Results are representative of three experiments.

FIG. 4.

Expression of MHC class II and costimulatory molecules by DC after infection with Y. enterocolitica. (A) DC were incubated with various strains of Y. enterocolitica: wild type (WA-pYV⁺), plasmid cured (WA-pYV⁻), YopP deficient (pYV⁺YopP⁻), and heat-killed wild type (WA-pYV⁺ heat killed). Three hours after starting infection DC were stained (black lines) for MHC class II, CD86, CD54 (ICAM-1), and CD80 and analyzed by flow cytometry. By gating out propidium iodide-positive cells, 10,000 live cells were examined in each measurement. Gray lines, isotype-matched immunoglobulin G controls. The differences between the means of monoclonal antibodies to isotype controls are indicated in the top corner of each events graph. (B) Means of four independent experiments are shown. Numbers above graphs indicate P values.

FIG. 4.

Expression of MHC class II and costimulatory molecules by DC after infection with Y. enterocolitica. (A) DC were incubated with various strains of Y. enterocolitica: wild type (WA-pYV⁺), plasmid cured (WA-pYV⁻), YopP deficient (pYV⁺YopP⁻), and heat-killed wild type (WA-pYV⁺ heat killed). Three hours after starting infection DC were stained (black lines) for MHC class II, CD86, CD54 (ICAM-1), and CD80 and analyzed by flow cytometry. By gating out propidium iodide-positive cells, 10,000 live cells were examined in each measurement. Gray lines, isotype-matched immunoglobulin G controls. The differences between the means of monoclonal antibodies to isotype controls are indicated in the top corner of each events graph. (B) Means of four independent experiments are shown. Numbers above graphs indicate P values.

FIG. 5.

Production of IL-12 (A), IL-10 (B), KC (C), and TNF-α (D) by DC after infection with Y. enterocolitica. DC from BALB/c mice were incubated with LPS or with various strains of Y. enterocolitica: wild type (WA-pYV⁺), plasmid cured (WA-pYV⁻), YopP deficient (pYV⁺ YopP⁻), and heat-killed wild type (WA-pYV⁺ heat killed). Gentamicin (100 μg/ml) was added 1 h after infection. Three hours (KC) or 23 h (IL-12, IL-10, and TNF-α) later, supernatants were harvested and analyzed by ELISA. Data represent the means of results from three independent experiments.

FIG. 6.

Y. enterocolitica-induced activation of NF-κB in DC. DC were incubated with LPS or with various strains of Y. enterocolitica: wild type (WA-pYV⁺), plasmid cured (WA-pYV⁻), YopP deficient (pYV⁺YopP⁻), and heat-killed wild type (WA-pYV⁺ heat killed). After 30, 60, and 90 min, nuclear extracts were prepared and analyzed in gel shift experiments with ³²P-labeled probes for NF-κB. (A) Time course of Yersinia-induced NF-κB activation. (B) Supershift analyses were performed by incubating nuclear extracts with antibodies against NF-κB subunits p50 or p65 and for competition experiments with a 100-fold excess of unlabeled NF-κB probe. NF-κBc, consensus sequence; NF-κBm, mutated sequences. Results are representative of three experiments.