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Priming of Salmonella enterica serovar typhi-specific CD8(+) T cells by suicide dendritic cell cross-presentation in humans.

Salerno-Goncalves R, Sztein MB - PLoS ONE (2009)

Bottom Line: Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.This study is the first to demonstrate the effect of S.Typhi.

View Article: PubMed Central - PubMed

Affiliation: Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, USA. rmezghan@medicine.umaryland.edu

ABSTRACT

Background: The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8(+) T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-gamma secretion. However, how S. Typhi regulates the development of specific CD8(+) responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8(+) T cells.

Methodology/principal findings: We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-alpha, but low levels of IL-12 p70 and IFN-gamma. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.

Conclusions/significance: This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8(+) cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.

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S. Typhi induces apoptosis in human cells.Blasts or DC were infected with S. Typhi at a MOI of 10∶1 and analyzed for apoptosis 3 hours after infection. DC were also exposed to heated-killed S. Typhi or treated with ZVA-D. Uninfected cells (media) were used as negative controls. Cells treated with staurosporine (8 µg/ml) or 1% formaldehyde were used as positive controls for apoptosis and necrosis respectively. Cell apoptosis was monitored by Annexin V and PI fluorescent dyes and by staining cells for cleaved caspase-3. (A) Blasts from volunteer A00302-5008 were stained with FITC-annexin V and PI or isotype-matched control antibodies and analyzed by flow cytometry. (B) Blasts from volunteers CVD4000#119 and CVD4000#64 and (C) DC from volunteer CVD4000#64 were surface stained for caspase-3 and Salmonella common structural antigens (CSA). Numbers correspond to the percentage of positive cells in the indicated quadrants or regions in each histogram. Arrows in panels B and C depict the expression of caspase 3 on cells expressing, or not, CSA.
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pone-0005879-g003: S. Typhi induces apoptosis in human cells.Blasts or DC were infected with S. Typhi at a MOI of 10∶1 and analyzed for apoptosis 3 hours after infection. DC were also exposed to heated-killed S. Typhi or treated with ZVA-D. Uninfected cells (media) were used as negative controls. Cells treated with staurosporine (8 µg/ml) or 1% formaldehyde were used as positive controls for apoptosis and necrosis respectively. Cell apoptosis was monitored by Annexin V and PI fluorescent dyes and by staining cells for cleaved caspase-3. (A) Blasts from volunteer A00302-5008 were stained with FITC-annexin V and PI or isotype-matched control antibodies and analyzed by flow cytometry. (B) Blasts from volunteers CVD4000#119 and CVD4000#64 and (C) DC from volunteer CVD4000#64 were surface stained for caspase-3 and Salmonella common structural antigens (CSA). Numbers correspond to the percentage of positive cells in the indicated quadrants or regions in each histogram. Arrows in panels B and C depict the expression of caspase 3 on cells expressing, or not, CSA.

Mentions: Based on the above observations, we next investigated the role of apoptosis in the generation of antigens that could then be acquired by uninfected DC. It is known that bacteria-induced apoptotic cells can serve as a reservoir of antigens for cross-presentation by DC [16]. Previous studies have shown that some Salmonella serovars, including S. Typhimurium and S. Typhi, can induce death in infected macrophages by apoptotic mechanisms [33], [34]. Here, cell apoptosis was monitored by Annexin V and PI fluorescent dyes and by staining cells for cleaved caspase-3. Blasts were infected with S. Typhi at MOI of 10∶1 and analyzed for apoptosis after 3 hours of incubation. Uninfected blasts were used as negative controls. Cells treated with staurosporine or formaldehyde were used as positive controls for apoptosis and necrosis, respectively. Cells were stained with Annexin V and PI dyes or anti-caspase-3 antibody as well as isotype-matched control mAbs and analyzed by flow cytometry. We observed that S. Typhi-infected blasts undergo increased apoptosis, as detected by annexin V binding in the absence of PI staining, as compared to uninfected blasts (media) (Fig. 3A). Caspase-3-positive cells were also detected in S. Typhi-infected blasts (Fig. 3B). Both methods of detection identified similar percentages of apoptotic cells in S. Typhi-infected blasts. Similarly, we observed that DC pulsed with live S. Typhi increased apoptosis, as detected by caspase-3 staining (Fig. 3C). Increases, albeit at lower levels, were also apparent when DC were pulsed with heat-inactivated S. Typhi (Fig. 3C). Apoptosis was inhibited ∼30% when the incubation was carried out in the presence of ZVA-D, a powerful, irreversible and cell permeable inhibitor for caspases (Fig. 3C). Together these results further support the possibility that Salmonella-induced apoptosis is responsible for loading uninfected DC with S. Typhi antigen.


Priming of Salmonella enterica serovar typhi-specific CD8(+) T cells by suicide dendritic cell cross-presentation in humans.

Salerno-Goncalves R, Sztein MB - PLoS ONE (2009)

S. Typhi induces apoptosis in human cells.Blasts or DC were infected with S. Typhi at a MOI of 10∶1 and analyzed for apoptosis 3 hours after infection. DC were also exposed to heated-killed S. Typhi or treated with ZVA-D. Uninfected cells (media) were used as negative controls. Cells treated with staurosporine (8 µg/ml) or 1% formaldehyde were used as positive controls for apoptosis and necrosis respectively. Cell apoptosis was monitored by Annexin V and PI fluorescent dyes and by staining cells for cleaved caspase-3. (A) Blasts from volunteer A00302-5008 were stained with FITC-annexin V and PI or isotype-matched control antibodies and analyzed by flow cytometry. (B) Blasts from volunteers CVD4000#119 and CVD4000#64 and (C) DC from volunteer CVD4000#64 were surface stained for caspase-3 and Salmonella common structural antigens (CSA). Numbers correspond to the percentage of positive cells in the indicated quadrants or regions in each histogram. Arrows in panels B and C depict the expression of caspase 3 on cells expressing, or not, CSA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691582&req=5

pone-0005879-g003: S. Typhi induces apoptosis in human cells.Blasts or DC were infected with S. Typhi at a MOI of 10∶1 and analyzed for apoptosis 3 hours after infection. DC were also exposed to heated-killed S. Typhi or treated with ZVA-D. Uninfected cells (media) were used as negative controls. Cells treated with staurosporine (8 µg/ml) or 1% formaldehyde were used as positive controls for apoptosis and necrosis respectively. Cell apoptosis was monitored by Annexin V and PI fluorescent dyes and by staining cells for cleaved caspase-3. (A) Blasts from volunteer A00302-5008 were stained with FITC-annexin V and PI or isotype-matched control antibodies and analyzed by flow cytometry. (B) Blasts from volunteers CVD4000#119 and CVD4000#64 and (C) DC from volunteer CVD4000#64 were surface stained for caspase-3 and Salmonella common structural antigens (CSA). Numbers correspond to the percentage of positive cells in the indicated quadrants or regions in each histogram. Arrows in panels B and C depict the expression of caspase 3 on cells expressing, or not, CSA.
Mentions: Based on the above observations, we next investigated the role of apoptosis in the generation of antigens that could then be acquired by uninfected DC. It is known that bacteria-induced apoptotic cells can serve as a reservoir of antigens for cross-presentation by DC [16]. Previous studies have shown that some Salmonella serovars, including S. Typhimurium and S. Typhi, can induce death in infected macrophages by apoptotic mechanisms [33], [34]. Here, cell apoptosis was monitored by Annexin V and PI fluorescent dyes and by staining cells for cleaved caspase-3. Blasts were infected with S. Typhi at MOI of 10∶1 and analyzed for apoptosis after 3 hours of incubation. Uninfected blasts were used as negative controls. Cells treated with staurosporine or formaldehyde were used as positive controls for apoptosis and necrosis, respectively. Cells were stained with Annexin V and PI dyes or anti-caspase-3 antibody as well as isotype-matched control mAbs and analyzed by flow cytometry. We observed that S. Typhi-infected blasts undergo increased apoptosis, as detected by annexin V binding in the absence of PI staining, as compared to uninfected blasts (media) (Fig. 3A). Caspase-3-positive cells were also detected in S. Typhi-infected blasts (Fig. 3B). Both methods of detection identified similar percentages of apoptotic cells in S. Typhi-infected blasts. Similarly, we observed that DC pulsed with live S. Typhi increased apoptosis, as detected by caspase-3 staining (Fig. 3C). Increases, albeit at lower levels, were also apparent when DC were pulsed with heat-inactivated S. Typhi (Fig. 3C). Apoptosis was inhibited ∼30% when the incubation was carried out in the presence of ZVA-D, a powerful, irreversible and cell permeable inhibitor for caspases (Fig. 3C). Together these results further support the possibility that Salmonella-induced apoptosis is responsible for loading uninfected DC with S. Typhi antigen.

Bottom Line: Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.This study is the first to demonstrate the effect of S.Typhi.

View Article: PubMed Central - PubMed

Affiliation: Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, USA. rmezghan@medicine.umaryland.edu

ABSTRACT

Background: The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8(+) T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-gamma secretion. However, how S. Typhi regulates the development of specific CD8(+) responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8(+) T cells.

Methodology/principal findings: We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-alpha, but low levels of IL-12 p70 and IFN-gamma. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.

Conclusions/significance: This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8(+) cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.

Show MeSH
Related in: MedlinePlus