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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

Fluorescence microscopy of Caco2 monolayers labeled with ZO-1 or fluorescein-phalloidin (actin) show disruption of the tight-junction complex and the cytoskeleton at 4 h post-infection. Caco2 polarized monolayers were infected with wild-type S. Typhi (MOI 400:1) for 4 h, washed with PBS, fixed and stained along with uninfected controls. (A) ZO-1 staining in uninfected monolayers. Note the characteristic chicken wire patterning. (B) Disrupted ZO-1organization in wild-type S. Typhi -infected monolayers. Areas of cell-cell detachment are marked by arrows. (C) Actin staining of the cytoskeleton in uninfected controls. (D) Actin fibers stained in the S. Typhi -infected cells. (E) Merge of (A,C). (F) Merge of (B,D). (G) ZO-1 distribution in CVD 909 infected monolayers. (H) ZO-1 (red), actin (green) and DAPI for the nuclei (blu) merged staining of CVD 909 infected cells. Bar, 25 μm.
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Figure 6: Fluorescence microscopy of Caco2 monolayers labeled with ZO-1 or fluorescein-phalloidin (actin) show disruption of the tight-junction complex and the cytoskeleton at 4 h post-infection. Caco2 polarized monolayers were infected with wild-type S. Typhi (MOI 400:1) for 4 h, washed with PBS, fixed and stained along with uninfected controls. (A) ZO-1 staining in uninfected monolayers. Note the characteristic chicken wire patterning. (B) Disrupted ZO-1organization in wild-type S. Typhi -infected monolayers. Areas of cell-cell detachment are marked by arrows. (C) Actin staining of the cytoskeleton in uninfected controls. (D) Actin fibers stained in the S. Typhi -infected cells. (E) Merge of (A,C). (F) Merge of (B,D). (G) ZO-1 distribution in CVD 909 infected monolayers. (H) ZO-1 (red), actin (green) and DAPI for the nuclei (blu) merged staining of CVD 909 infected cells. Bar, 25 μm.

Mentions: As shown in Figure 6A, in uninfected monolayers ZO-1 is localized at the cell-cell boundary in a typical chicken wire-like pattern throughout the monolayer, indicating intact tight-junction complexes. In contrast, following infection with wild-type S. Typhi for 4 h (Figure 6B; MOI 400:1), ZO-1 distribution appears altered: while still observing the protein at the cell boundaries, ZO-1 also appeared clustered into the cytoplasm and co-localized with aggregates of actin fibers of the disrupted cytoskeleton (Figures 6D,F). In contrast, as expected, in uninfected monolayers the actin cytoskeleton was organized in a network of filaments normally distributed beneath the plasma membrane and throughout the cytoplasm (Figures 6C,E). As a plausible effect of this tight-junction and cytoskeletal rearrangement we observed the detachment of adjacent cells from each other (Figure 6B, arrows). Similar to ZO-1, in infected monolayers we observed the clustering of claudin-1into the cytoplasm around the nucleus and the detachment of adjacent cells (data not shown). Consistent with TEER data, in monolayers infected with CVD 909 we did not observe major alterations of ZO-1 localization. As shown in Figures 6G,H, ZO-1 is localized at the cell-cell boundary with minimal internalization and also minimal alteration of the actin cytoskeleton. At 22 h post-infection, we still observed a severely altered distribution of ZO-1 in wild-type S. Typhi treated cells (Figure 7D) whereas the effect appears largely attenuated in Ty21a infected cells, for which only a few areas of cell–cell detachment and some ZO-1 internalization were detected (Figure 7C, arrows). No effect on the distribution of ZO-1 was observed with CVD 909 (Figure 7B) or CVD 908 (not shown). These results strongly support the notion that changes in epithelial permeability induced by bacterial infection are caused by alterations in the paracellular pathway due to disruption and/or modulation of the sealing function of tight junctions.


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)

Fluorescence microscopy of Caco2 monolayers labeled with ZO-1 or fluorescein-phalloidin (actin) show disruption of the tight-junction complex and the cytoskeleton at 4 h post-infection. Caco2 polarized monolayers were infected with wild-type S. Typhi (MOI 400:1) for 4 h, washed with PBS, fixed and stained along with uninfected controls. (A) ZO-1 staining in uninfected monolayers. Note the characteristic chicken wire patterning. (B) Disrupted ZO-1organization in wild-type S. Typhi -infected monolayers. Areas of cell-cell detachment are marked by arrows. (C) Actin staining of the cytoskeleton in uninfected controls. (D) Actin fibers stained in the S. Typhi -infected cells. (E) Merge of (A,C). (F) Merge of (B,D). (G) ZO-1 distribution in CVD 909 infected monolayers. (H) ZO-1 (red), actin (green) and DAPI for the nuclei (blu) merged staining of CVD 909 infected cells. Bar, 25 μm.
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Figure 6: Fluorescence microscopy of Caco2 monolayers labeled with ZO-1 or fluorescein-phalloidin (actin) show disruption of the tight-junction complex and the cytoskeleton at 4 h post-infection. Caco2 polarized monolayers were infected with wild-type S. Typhi (MOI 400:1) for 4 h, washed with PBS, fixed and stained along with uninfected controls. (A) ZO-1 staining in uninfected monolayers. Note the characteristic chicken wire patterning. (B) Disrupted ZO-1organization in wild-type S. Typhi -infected monolayers. Areas of cell-cell detachment are marked by arrows. (C) Actin staining of the cytoskeleton in uninfected controls. (D) Actin fibers stained in the S. Typhi -infected cells. (E) Merge of (A,C). (F) Merge of (B,D). (G) ZO-1 distribution in CVD 909 infected monolayers. (H) ZO-1 (red), actin (green) and DAPI for the nuclei (blu) merged staining of CVD 909 infected cells. Bar, 25 μm.
Mentions: As shown in Figure 6A, in uninfected monolayers ZO-1 is localized at the cell-cell boundary in a typical chicken wire-like pattern throughout the monolayer, indicating intact tight-junction complexes. In contrast, following infection with wild-type S. Typhi for 4 h (Figure 6B; MOI 400:1), ZO-1 distribution appears altered: while still observing the protein at the cell boundaries, ZO-1 also appeared clustered into the cytoplasm and co-localized with aggregates of actin fibers of the disrupted cytoskeleton (Figures 6D,F). In contrast, as expected, in uninfected monolayers the actin cytoskeleton was organized in a network of filaments normally distributed beneath the plasma membrane and throughout the cytoplasm (Figures 6C,E). As a plausible effect of this tight-junction and cytoskeletal rearrangement we observed the detachment of adjacent cells from each other (Figure 6B, arrows). Similar to ZO-1, in infected monolayers we observed the clustering of claudin-1into the cytoplasm around the nucleus and the detachment of adjacent cells (data not shown). Consistent with TEER data, in monolayers infected with CVD 909 we did not observe major alterations of ZO-1 localization. As shown in Figures 6G,H, ZO-1 is localized at the cell-cell boundary with minimal internalization and also minimal alteration of the actin cytoskeleton. At 22 h post-infection, we still observed a severely altered distribution of ZO-1 in wild-type S. Typhi treated cells (Figure 7D) whereas the effect appears largely attenuated in Ty21a infected cells, for which only a few areas of cell–cell detachment and some ZO-1 internalization were detected (Figure 7C, arrows). No effect on the distribution of ZO-1 was observed with CVD 909 (Figure 7B) or CVD 908 (not shown). These results strongly support the notion that changes in epithelial permeability induced by bacterial infection are caused by alterations in the paracellular pathway due to disruption and/or modulation of the sealing function of tight junctions.

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