Figure 1.
Flagellin is taken up by rat IEC monolayers.
A. Control IEC-18 cells that were not exposed to CBir1 flagellin. B and C. Following a 2 h incubation, CBir1 was internalized by IEC-18 cells as detected flagellin labeled with Alexa-488 (green) and biotin. E represents merged images of B-D showing the dual stained flagellin as visualized by fluorescent microscopy. Cell nuclei were stained with DAPI (blue).
Figure 2.
Flagellin does not interfere with intestinal barrier integrity.
TER measurements were taken every hour for 4 h in the AP and BL chambers of polarized Caco-2BBe (A) and T-84 (B) cells exposed to flagellin that showed no significant change. C and D. Examination the tight junction protein, ZO-1 by confocal microscopy, in polarized Caco-2BBe cells. The appearance of the junctions stained with anti-ZO-1 antibody and appropriate secondary antibody (green) did not change following exposure of flagellin to the AP surface. Cell nuclei with stained with DAPI (blue). The confocal images shown are representative of 2 experiments.
Figure 3.
Flagellin monomers were internalized by polarized IEC.
Note: White arrows in A-D indicate cluster of flagellin monomers. A. GFP-labeled flagellin monomers (green) were internalized by Caco-2BBe cells at 30 min post-AP exposure and visualized via confocal microscopy. The cell body was visualized by staining for F-actin using phalloidin (red). Accumulated flagellin was detected inside the cells as shown in the mid z-sections taken by confocal microscopy. A transverse image reconstruction from optical z-stacks is shown below and shows flagellin below the cell surface. B. GFP-labeled flagellin was added to the AP surface of polarized T-84 cells and 30 min post-exposure flagellin clusters could be detected within the cell body from mid z-sections taken by confocal microscopy using a 60x objective lens. Cell nuclei were stained by DAPI (blue). C. Caco-2 BBe cells internalized GFP-labeled flagellin (green) which was also immunostained using a rhodamine rabbit anti-flagellin antibody (red). Co-localization GFP and rhodamine signals is seen in the merged images and in the transverse image reconstruction from z-stacks taken by confocal microscopy (at right). D. Flagellin purified from S. Typhimurium was internalized by Caco-2BBe cells. Intracellular staining of purified S. Typhimurium flagellin were visualized, via the middle z-section taken by confocal microscopy after 30 min of apically applied flagellin using a rabbit anti-flagellin antibody followed by a secondary anti-rabbit antibody conjugated to rhodamine (red). Cell nuclei were stained with DAPI (blue) E. Optical z-sections showing apical and basolateral surfaces as well as the middle plane through the cells together with a transverse image reconstructed from z-stacks showing lack of uptake of latex beads (MW 40,000; green) by Caco-2BBe cells after a 30 min incubation. F-actin was stained using phalloidin (red) and cell nuclei were stained with DAPI (blue). Images were acquired using a 60X objective lens. The confocal images shown are representative of 1 of 4 experiments producing similar results.
Figure 4.
Flagellin was not internalized by siTLR5 Caco-2BBe cells or IEC in which TLR5 was blocked.
A. Immunoblotting for TLR5 via Western blot showed that TLR5 expression is dramatically reduced in siTLR5 Caco-2BBe cells compared to wild type Caco-2BBe cells. Further, wild type Caco-2BBe cells infected with S. Typhimurium (MOI 500) did not change the expression of TLR5 2 h post-infection. B. Transverse image of polarized siTLR5 Caco-2BBe cells constructed from optical z-stacks taken by confocal microscopy, showing flagellin (green) was localized to the AP surface at thirty minutes post-exposure. Cell nuclei were stained with DAPI (blue). C. The majority of flagellin remained on the apical surface of siTLR5 Caco-2BBe cells and wild type Caco-2BBe cells pre-treated with TLR5 blocker antibody following 1 h incubation with 5 µg of flagellin. Whereas, the predominant quantity of flagellin was found in the cell layer of wild type Caco-2BBe controls. Flagellin was not detected in the BL supernatants of any of the tested cell samples. These results represent 1 of 3 repeated experiments with similar results.
Figure 5.
Flagellin co-localizes with endosomal and lysosomal markers.
A. Following AP exposure to Caco-2BBe cells, GFP-labeled flagellin (green) was internalized and, within 15 min, co-localized with an antibody that recognized an early endosomal surface marker (EEA1) and secondary antibody (red) as shown in the middle z-sections by confocal microscopy. Lower right panel shows merged images and indicates co-localization of flagellin with EEA1.Cell nuclei were stained with DAPI (blue). B. Within 1 h flagellin (green) co-localized with the lysosome marker LAMP-1 (red). The confocal images shown are representative of 3 independent experiments. C–F. 6HIS-Flagellin was added to the AP or BL side of polarized Caco-2BBE cells at 19°C allowing traffic up to (but not beyond) the recycling endosome. In some experiments biotinylated transferrin also was added to the apical surface as a marker for the endosomal pathway. Cells then were either fractionated (see methods) without allowing further traffic or warmed to 37°C for the times indicated prior to fractionation. NOTE: White arrows indicate flagellin protein in A and B. C. Intracellular localization of internalized flagellin protein (normalized for total cell-associated protein) following incubation at 19°C. D. Western blots of endosomal fractionation as described in C and in the methods. E. Intracellular localization of 6HIS-Flagellin when internalization of protein at 19°C was followed by incubation at 37°C allowing traffic beyond the recycling endosome. F-G. Western blots of fractionation as described in E and in the methods.
Figure 6.
IEC AP or BL exposure to flagellin induces IL-8 secretion.
Purified Salmonella flagellin or D3 recombinant flagellin was added to the AP or BL chamber of Caco-2BBe (A) or T-84 (B) cells grown on transwell filters. Supernatants were collected at 6 h post-exposure and analyzed for IL-8 secretion. Both cell lines (A and B) secreted IL-8 regardless of AP or BL stimulation with full-length flagellin compared to the D3 protein and un-stimulated cells. However, higher levels of IL-8 were detected in the BL supernatant compared to the AP supernatant. Further, Caco-2BBe (C) and T-84 (D) cells were apically stimulated with various types of flagellin proteins. Un-stimulated cells served as negative controls. S. dublin flagellin, ECO83:HI flagellin, E. coli K12, ND1/2 ECH CD2/1 flagellin induced higher IL-8 secretion from the BL surface of both cells types compared to D3 truncated flagellin or un-stimulated controls. Data is expressed as mean ± SD. *p<0.05 vs. all groups.
Figure 7.
Immune cell migration through IEC in response to luminal exposure to flagellin.
Polarized Caco-2BBe and T-84 cells were stimulated with flagellin proteins 2 h prior to the addition of human DCs (A and B) or neutrophils (C and D) to the BL surface of IEC. Supernatants were collected 4 h post-migration and immune cells were counted to determine migration from the BL to the AP surface of IEC in response to flagellins. Data is expressed as mean ± SD. In Figure 2A, *p<0.01 vs. all groups except D3 flagellin and **p<0.05 vs. all groups. In Figure 2B, *p<0.01 vs. all groups except D3 flagellin and **p<0.05 vs. all groups excluding controls. Figure 2C, p<0.05 vs. S. dublin and ECO83:H1 flagellin. Figure 2D, *p<0.05 vs. all groups except D3 flagellin and **p<0.05 vs. D3 flagellin.
Figure 8.
IL-8 secretion decreased from TLR5 silenced (siRNA) Caco-2BBe cells exposed to flagellin.
A. S. dublin flagellin was added to the AP or BL surface of polarized Caco-2BBe cells where TLR5 was knocked down using siRNA. Parallel wild type Caco-2BBe cells were treated as above. Six hours post-flagellin exposure, AP and BL supernatants were collected to quantitate IL-8 secretion. IL-8 secretion was significantly decreased from siRNA Caco-2BBe cells stimulated with S. dublin flagellin regardless of AP or BL exposure compared to wild type Caco-2BBe cells. B. IL-8 secretion was significantly decreased in siTLR5 Caco-2BBe cells apically exposed to flagellin from S. dublin, ECO83, E. coli K12, and ND1/2ECHCD2/1 compared to wild type cells. Data is expressed as mean ± SD. *p<0.05 vs. all groups.