TLR4 facilitates translocation of bacteria across renal collecting duct cells

Abstract

Uropathogenic Escherichia coli (UPEC) are the most frequent causes of urinary tract infections and pyelonephritis. Renal medullary collecting duct (MCD) cells are the intrarenal site to which UPEC strains prefer to adhere and initiate an inflammatory response, but the ability of UPEC strains to translocate across impermeant MCD cells has not been demonstrated definitively. Here, several UPEC strains adhered to the apical surface and translocated across confluent murine inner MCD cells grown on filters. UPEC strains expressing cytolytic and vacuolating cytotoxins disrupted the integrity of cell layers, whereas noncytolytic UPEC strains passed through the cell layers without altering tight junctions. Apical-to-basal transcellular translocation was dramatically reduced after extinction of Toll-like receptor 4 (TLR4) and the lipid raft marker caveolin-1 by small interfering RNA. Furthermore, disruption of lipid raft integrity by filipin III and methyl-beta-cyclodextrin significantly reduced both the transcellular translocation of UPEC across murine inner MCD cell layers and the stimulation of proinflammatory mediators. Bacterial translocation was also significantly reduced in primary cultures of TLR4-deficient mouse MCD cells compared with MCD cells from wild-type mice. Benzyl alcohol, an anesthetic that enhances membrane fluidity, favored the recruitment of caveolin-1 in lipid rafts and increased the translocation of UPEC across cultured TLR4-deficient MCD cells. These findings demonstrate that the transcellular translocation of UPEC strains across impermeant layers of MCD cells may occur through lipid rafts via a TLR4-facilitated process.

Figure 1.

Adherence and invasion of renal mpkIMCD cells by uropathogenic E. coli. Confluent mpkIMCD cells were incubated with various 5 × 10⁵ E. coli isolates for 3 h and then processed for adhesion and invasion assays as described in the Concise Methods section. (A) Percentage of uropathogenic CFT073, HT7, and HT91 isolates; nonpathogenic MG1655; and commensal HT11551 adhering to the apical surface of mpkIMCD cells. (B) Gentamicin protection assays showing that CFT073, HT7, and HT91 isolates invade mpkIMCD cells, whereas MG1655 and HT11551 did not (ND, not detected). (C) Percentage of internalized CFT073, HT7, and HT91 isolates in mpkIMCD cells preincubated without or with 25 mg/ml d-mannose. (D) Percentage of internalized HT7 in mpkIMCD cells preincubated with 25 mg/ml d-mannose, 5 mg/ml globotriose, or mannose plus globotriose. Data are means ± SEM; n = 3 to 6 separate experiments for each experimental condition.

Figure 2.

Effects of UPEC on the integrity of confluent renal mpkIMCD cell layers. (A) E. coli isolates (5 × 10⁵ bacteria/well) were added (3 h) to the apical side of confluent mpkIMCD cells grown on 1-μm-pore-size permeable filters, then fixed and processed for indirect immunofluorescence using an anti–ZO-1 antibody (in purple), and then stained with phalloidin (in red) to visualize F-actin. Cells were also stained with LIVE/DEAD fluorescent dye staining (red, dead cells; green, viable cells). CFT073 but not HT7 and HT91 altered the integrity of the cell layer with numerous dead cells (*). Bars = 10 μm. (B) Indirect immunofluorescence of cells grown on glass slide showing actin rearrangements (in red) forming close contacts (arrowhead) with an adherent HT7 bacteria (in green, and merge image in yellow). (C) Transmission electron micrographs of adherent HT7 bacteria to the apical membrane of mpkIMCD cells (left) and internalized bacteria (arrowheads; right). Bars = 1 μm. (D) Western blot analysis of active caspase-3 expression in nonpathogenic and uropathogenic E. coli strains. (E and F) Illustrations (E) and number of caspase-3–positive stained cells (F) incubated with CFT073 and without or with the pan-caspase inhibitor Z-VAD.fmk (20 μM, 30 min). Bars are means ± SEM; n = 3 independent experiments. *P < 0.05 between groups. Bar = 10 μm.

Figure 3.

Differential effects of UPEC on paracellular permeability, bacterial translocation, and expression of tight junction and adhesion proteins in mpkIMCD cells. (A through C) E. coli (5 ± 10⁵ bacteria/well) were added to the apical side of confluent layers of mpkIMCD cells grown on permeable filters. R_T (A), number of bacteria recovered in the basal medium (B), and percentage of bacterial translocation determined as described in the Concise Methods section (C) were measured during a 3-h period. Data are means ± SEM; n = 6 to 9 experiments for each condition tested. *P < 0.05 versus time 0 values. (D) Western blot analyses of expression of claudin-4, intercellular adhesion molecule 1 (ICAM-1), E-cadherin, and the corresponding β-actin in mpkIMCD cells incubated without (None) or with MG1655, CFT073, or HT7 (5 ± 10⁵ bacteria/well) for 3 h. Bars are mean ratio values ± SEM of densitometric analyses of claudin-4, ICAM-1, or E-cadherin over β-actin–labeled bands from three to four separate cultures of mpkIMCD in each group tested. *P < 0.05 versus None values. (E and F) R_T values (E) and bacterial invasion (F) in mpkIMCD cells preincubated (30 min) without or with LY294002 (50 μM) or wortmannin (1 μM) before addition of HT7. The percentage of bacterial invasion was determined using the gentamicin protection assay as described in the Concise Methods section. Data are means ± SEM; n = 4 separate filters for each condition tested. *P < 0.05 versus None values.

Figure 4.

Caveolin-1 requirement for internalization of UPEC in mpkIMCD cells. (A) Western blot analysis of caveolin-1 and β-actin in mpkIMCD cells incubated without (control) or with HT7 (5 ± 10⁵ bacteria/well, 3 h). Bars are the mean ratio values ± SEM of densitometric analyses of caveolin-1 over β-actin–labeled bands from three to four separate cultures. (B) Western blot analysis of caveolin-1 in DRM fractions fractionated by flotation centrifugation gradient in uninfected and HT7-infected mpkIMCD cells. (C) Caveolin-1 mRNA (bottom) and protein (top) expressions in uninfected mpkIMCD cells (lane 1) or cells transfected with a negative control siRNA (lane 2), a caveolin-1 siRNA (lane 3), or with non–reverse-transcribed caveolin-1 siRNA (lane 4). (D) Nontransfected cells (None) and transfected cells with caveolin-1 siRNA or negative control siRNA (control siRNA) were incubated with HT7 (5 ± 10⁵ bacteria/well, 3 h) before invasion assay. Bars (means ± SEM) represent the percentage of internalized bacteria. *P < 0.05 versus None values.

Figure 5.

Intracellular TLR4 requires intact cell trafficking endocytosis pathways and mediates the internalization of UPEC in mpkIMCD cells. (A) FACS analysis for TLR4 in nonpermeabilized and permeabilized cells. Nonbold lines correspond to the isotype control. (B) Double immunofluorescence using antibodies raised against the Golgi apparatus marker p58K (in green) and TLR4 (in red). Nuclei (N) were counterstained with Hoechst 33258 (in blue). (C) tlr4 mRNA and protein expressions in uninfected mpkIMCD cells (lane 1) and cells transfected with a negative control siRNA (lane 2), tlr4 siRNA (lane 3), or non–reverse-transcribed tlr4 siRNA (lane 4). (D) MIP-2 secretion in nontransfected cells and cells transfected with a negative control siRNA or tlr4 siRNA incubated with LPS (10 ng/ml, 6 h). Data are means ± SEM; n = 5 separate cultures in each condition tested. (E) Nontransfected cells and cells transfected with tlr4 siRNA or negative control siRNA were incubated with HT7 before invasion assay as described already. Bars (means ± SEM) represent the percentage of internalized bacteria. *P < 0.05 between groups.

Figure 6.

Effects of cholesterol-affecting drugs and cholesterol repletion on the expression of proinflammatory mediators and translocation of UPEC across renal mpkIMCD cells. The relative mRNA expression levels of proinflammatory mediators quantified by real-time PCR (A), secretion of MIP-2 and TNF-α in cell supernatants (B), R_T (C), and apical-to-basal translocation of HT7 (D) were measured in confluent cultures of mpkIMCD cells grown on filters and preincubated (30 min) without (black bars, ▪) or with 10 mM MβCD (hatched bars, ▴) or 5 μM filipin III (hatched bars, ▾) and then incubated with HT7 (5 × 10⁵ bacteria, 3 h). Sets of MβCD- or filipin III–treated cells incubated with HT7 were rinsed and then further incubated with HT7 in the presence of 100 μM cholesterol for an additional 3 h (hatched gray bars). Data are means ± SEM; n = 5 to 7 separate cultures for each experimental condition. *P < 0.05 versus HT7 values.

Figure 7.

Translocation of UPEC across confluent cultures of Lpsⁿ and Lps^d MCD cells. (A through C) R_T (A), number of HT7 recovered in the basal medium (B), and percentage of apical-to-basal translocation of bacteria (C) measured in confluent cultures of Lpsⁿ and Lps^d MCD grown on permeable filters and incubated on the apical side with HT7 (5 × 10⁵ bacteria/well). (D) Western blot analysis of caveolin-1 in DRM fractions fractionated by flotation centrifugation gradient in uninfected (−HT7) and HT7-infected (+HT7) Lpsⁿ MCD and Lps^d MCD. (E) Western blot analysis of caveolin-1 in flotation centrifugation fractionated DRM fractions of confluent cultures of Lps^d MCD incubated with HT7 and with (+Ba) or without (−Ba) 20 mM benzyl alcohol (Ba) for 4 h. (F and G) Effects of increasing concentrations of Ba on R_T (F) and percentage of apical-to-basal translocation of HT7 (G) in confluent cultures of Lps^d MCD. Data are means ± SEM; n = 6 separate cultures of MCD dissected from the kidneys of two to three mice for each group tested. *P < 0.05 between groups.