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In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

Fiorentino M, Lammers KM, Levine MM, Sztein MB, Fasano A - Front Immunol (2013)

Bottom Line: Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure.We conclude that wild-type S.Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Biology Research Center, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

No MeSH data available.


Related in: MedlinePlus

S. Typhi attenuated strains induce IL-8 secretion by Caco2 cells. (A) IL-8 secreted by Caco2 cells infected with vaccine candidates CVD 908-htrA and CVD 909 applied at MOIs of 4000:1 and 400:1, HK S. Typhi and conditioned media (22 h post-infection). (B) IL-8 released by Caco2 cells infected with the aro mutants at a MOI of 40:1at 22 h post-infection; (C) IL-8 secretion measured at 4 h post-infection at a MOI of 400:1 (statistical analysis between the apical and basolateral compartments for the same strain); (D) IL-8 measured at 22 h after infection with the vaccine strain Ty21a applied at MOIs of 4000 and 400. Wild-type S. Typhi served as positive control. Values shown represent the mean ± SEM of three independent assays. #p < 0.05 over uninfected; ***p < 0.001, **p < 0.01, *p < 0.05 for comparisons between wild-type S. Typhi and mutant strains applied at the same titer (ANOVA).
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Figure 9: S. Typhi attenuated strains induce IL-8 secretion by Caco2 cells. (A) IL-8 secreted by Caco2 cells infected with vaccine candidates CVD 908-htrA and CVD 909 applied at MOIs of 4000:1 and 400:1, HK S. Typhi and conditioned media (22 h post-infection). (B) IL-8 released by Caco2 cells infected with the aro mutants at a MOI of 40:1at 22 h post-infection; (C) IL-8 secretion measured at 4 h post-infection at a MOI of 400:1 (statistical analysis between the apical and basolateral compartments for the same strain); (D) IL-8 measured at 22 h after infection with the vaccine strain Ty21a applied at MOIs of 4000 and 400. Wild-type S. Typhi served as positive control. Values shown represent the mean ± SEM of three independent assays. #p < 0.05 over uninfected; ***p < 0.001, **p < 0.01, *p < 0.05 for comparisons between wild-type S. Typhi and mutant strains applied at the same titer (ANOVA).

Mentions: Supernatants from both the monolayer apical and basolateral compartments were evaluated by Luminex assay for the detection of the pro-inflammatory cytokines IL-1β, IL-8, IL-6, TNF-α, IL-17, and IFN-γ. Moreover, we assayed supernatants for the measurement of IL-10 and TGF-β. Media collected from Ty21a infected cells were assessed for levels of IL-8 and IL-6 only. Cytokines were measured at 4 and 22 h post-infection. The only cytokines detected in these studies were IL-8 and IL-6. Levels of all other cytokines were very low or below detection. Figure 9A shows the results for IL-8 in media collected from the apical and basolateral sides of uninfected, live wild-type S. Typhi as positive control, filtrate, heat-inactivated S. Typhi, and S. Typhi aro mutants-infected Caco2 monolayers at 22 h post-infection. As expected, we detected a significant cytokine secretion in the basolateral compartment but interestingly a remarkable release of IL-8 was also measured on the apical side. IL-8 secretion induced by wild-type S. Typhi was significantly higher than that observed in uninfected cells on both sides and at all bacterial loads applied except for CVD909, apical secretion (Figure 9A). The largest IL-8 secretion was measured at a MOI of 400:1 for which we detected amounts of 2239 ± 573 and 838 ± 197 pg/ml compared to the uninfected monolayer, on the basolateral and apical sides, respectively. Even at the lowest MOI of 40:1 the fold increase was highly significant compared to uninfected, both on the basolateral (918 ± 163) and apical (340 ± 585; Figure 9B) side. IL-8 secretion in the basolateral side induced by filtered and heat-inactivated wild-type bacteria (Figure 9A) is remarkably lower than live, wild-type S. Typhi, suggesting that a physical interaction with the live pathogen is needed to trigger a strong cytokine response. S. Typhi aro mutants elicit remarkable levels of cytokine secretion, although the overall IL-8 amounts are lower than those secreted by wild-type bacteria-infected cells, in both the apical and basolateral compartments (Figures 9A,B). Similar to the wild-type strain, we measured the highest secretion of IL-8 at a MOI of 400:1 CFU/cell for CVD 908-htrA: 336.5 ± 66.5 and 832 ± 175 pg/ml in the apical and basolateral sides, respectively. IL-8 secretion elicited by the mutant strain CVD 909 was higher than untreated cells at all MOIs, with the greatest difference being observed at the MOI of 4000:1, with IL-8 levels 210 ± 40.1 and 814 ± 179 pg/ml compared to 7.49 ± 2.03 and 15.0 ± 2.94 pg/ml of uninfected cells, in the apical and basolateral compartments, respectively.


In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

Fiorentino M, Lammers KM, Levine MM, Sztein MB, Fasano A - Front Immunol (2013)

S. Typhi attenuated strains induce IL-8 secretion by Caco2 cells. (A) IL-8 secreted by Caco2 cells infected with vaccine candidates CVD 908-htrA and CVD 909 applied at MOIs of 4000:1 and 400:1, HK S. Typhi and conditioned media (22 h post-infection). (B) IL-8 released by Caco2 cells infected with the aro mutants at a MOI of 40:1at 22 h post-infection; (C) IL-8 secretion measured at 4 h post-infection at a MOI of 400:1 (statistical analysis between the apical and basolateral compartments for the same strain); (D) IL-8 measured at 22 h after infection with the vaccine strain Ty21a applied at MOIs of 4000 and 400. Wild-type S. Typhi served as positive control. Values shown represent the mean ± SEM of three independent assays. #p < 0.05 over uninfected; ***p < 0.001, **p < 0.01, *p < 0.05 for comparisons between wild-type S. Typhi and mutant strains applied at the same titer (ANOVA).
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Figure 9: S. Typhi attenuated strains induce IL-8 secretion by Caco2 cells. (A) IL-8 secreted by Caco2 cells infected with vaccine candidates CVD 908-htrA and CVD 909 applied at MOIs of 4000:1 and 400:1, HK S. Typhi and conditioned media (22 h post-infection). (B) IL-8 released by Caco2 cells infected with the aro mutants at a MOI of 40:1at 22 h post-infection; (C) IL-8 secretion measured at 4 h post-infection at a MOI of 400:1 (statistical analysis between the apical and basolateral compartments for the same strain); (D) IL-8 measured at 22 h after infection with the vaccine strain Ty21a applied at MOIs of 4000 and 400. Wild-type S. Typhi served as positive control. Values shown represent the mean ± SEM of three independent assays. #p < 0.05 over uninfected; ***p < 0.001, **p < 0.01, *p < 0.05 for comparisons between wild-type S. Typhi and mutant strains applied at the same titer (ANOVA).
Mentions: Supernatants from both the monolayer apical and basolateral compartments were evaluated by Luminex assay for the detection of the pro-inflammatory cytokines IL-1β, IL-8, IL-6, TNF-α, IL-17, and IFN-γ. Moreover, we assayed supernatants for the measurement of IL-10 and TGF-β. Media collected from Ty21a infected cells were assessed for levels of IL-8 and IL-6 only. Cytokines were measured at 4 and 22 h post-infection. The only cytokines detected in these studies were IL-8 and IL-6. Levels of all other cytokines were very low or below detection. Figure 9A shows the results for IL-8 in media collected from the apical and basolateral sides of uninfected, live wild-type S. Typhi as positive control, filtrate, heat-inactivated S. Typhi, and S. Typhi aro mutants-infected Caco2 monolayers at 22 h post-infection. As expected, we detected a significant cytokine secretion in the basolateral compartment but interestingly a remarkable release of IL-8 was also measured on the apical side. IL-8 secretion induced by wild-type S. Typhi was significantly higher than that observed in uninfected cells on both sides and at all bacterial loads applied except for CVD909, apical secretion (Figure 9A). The largest IL-8 secretion was measured at a MOI of 400:1 for which we detected amounts of 2239 ± 573 and 838 ± 197 pg/ml compared to the uninfected monolayer, on the basolateral and apical sides, respectively. Even at the lowest MOI of 40:1 the fold increase was highly significant compared to uninfected, both on the basolateral (918 ± 163) and apical (340 ± 585; Figure 9B) side. IL-8 secretion in the basolateral side induced by filtered and heat-inactivated wild-type bacteria (Figure 9A) is remarkably lower than live, wild-type S. Typhi, suggesting that a physical interaction with the live pathogen is needed to trigger a strong cytokine response. S. Typhi aro mutants elicit remarkable levels of cytokine secretion, although the overall IL-8 amounts are lower than those secreted by wild-type bacteria-infected cells, in both the apical and basolateral compartments (Figures 9A,B). Similar to the wild-type strain, we measured the highest secretion of IL-8 at a MOI of 400:1 CFU/cell for CVD 908-htrA: 336.5 ± 66.5 and 832 ± 175 pg/ml in the apical and basolateral sides, respectively. IL-8 secretion elicited by the mutant strain CVD 909 was higher than untreated cells at all MOIs, with the greatest difference being observed at the MOI of 4000:1, with IL-8 levels 210 ± 40.1 and 814 ± 179 pg/ml compared to 7.49 ± 2.03 and 15.0 ± 2.94 pg/ml of uninfected cells, in the apical and basolateral compartments, respectively.

Bottom Line: Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure.We conclude that wild-type S.Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Biology Research Center, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

No MeSH data available.


Related in: MedlinePlus