TLR4-initiated and cAMP-mediated abrogation of bacterial invasion of the bladder - PubMed (original) (raw)

TLR4-initiated and cAMP-mediated abrogation of bacterial invasion of the bladder

Jeongmin Song et al. Cell Host Microbe. 2007.

Abstract

The remarkable resistance of the urinary tract to infection has been attributed to its physical properties and the innate immune responses triggered by pattern recognition receptors lining the tract. We report a distinct TLR4 mediated mechanism in bladder epithelial cells (BECs) that abrogates bacterial invasion, a necessary step for successful infection. Compared to controls, uropathogenic type 1 fimbriated Escherichia coli and Klebsiella pneumoniae invaded BECs of TLR4 mutant mice in 10-fold or greater numbers. TLR4 mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Artificially increasing intracellular cAMP levels in BECs of TLR4 mutant mice restored resistance to type 1 fimbriated bacterial invasion. This finding reveals a novel function for TLR4 and another facet of bladder innate defense.

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Figures

Figure 1

Figure 1. Increased invasion of mouse bladders by Gram negative bacteria when TLR4 signaling is abrogated

A, TLR4 mutant C3H/HeJ (HeJ) and isogenic control C3H/HeN (HeN) mice were inoculated transurethrally with 1×108 of E. coli CI5 (A). Bacterial invasion after 1 hr was assessed by gentamicin protection assays followed by colony counts of bladder homogenates. B–C, Fluorescent images of bladder sections of HeN (B) and HeJ (C) mice 1 hr following instillation of 1×108 E. coli CI5. Green, E. coli; Red, Wheat Germ Agglutinin (WGA). D–F, TLR4 immunostaining images of bladder sections of HeN mice before (D and E) and 1 hr after (F) instillation of 1×108 E. coli CI5. E and F were stained with TLR4-specific antibody whereas D was stained with isotype control antibody. G–H, TLR4 mutant HeJ and control HeN mice were inoculated transurethrally with 1×108 of type 1 fimbriated K. pneumoniae 1236 (G), or S. aureus 54 (H). Bacterial invasion after 1 hr was assessed as before. I, TLR4 mutant HeJ and control HeN mice were inoculated transurethrally with 1×108 of type 1 fimbriated msbB mutant MLK1067 (msbB-) or corresponding wild-type (WT) E. coli W3110. Bacterial invasion after 1 hr was assessed as before. In A, G, H, and I, **P <0.0001; * P<0.01, relative to values of HeN; #P<0.05, relative to values of wild-type E. coli W3110 infected HeN. Bars represent the mean + S. D.. In B–F, magnification was 200×. *L stands for lumen.

Figure 2

Figure 2. TLR4-LPS signaling abrogates in vitro invasion of BECs by type 1 fimbriated Enterobacteria

A, Bacterial invasion following exposure of BECs to wild-type E. coli W3110 (WT) or msbB mutant E. coli MLK1067 (msbB-). **P< 0.001, relative to values of wild-type E. coli infected BECs. B, RT-PCR of control-transfected BECs (Ctrl) and TLR4 knockdown BECs (TLR4 KD). Glyseraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as a loading control. C–D, Bacterial invasion (C) or bacterial adherence (D) following exposure of non-transfected BECs (NT), control-transfected BECs (Ctrl), and TLR4 KD BECs to type 1 fimbriated E. coli ORN103(pSH2). *P< 0.03, relative to control values. Bars represent the mean + S. D. in A, C and D.

Figure 3

Figure 3. TLR4 KD BECs exhibit an enhanced Rac-1 response to E. coli which correlates with enhanced bacterial invasion

A, Invasion of control-transfected BECs, BECs overexpressing constritutively active Rac1, or BECs with dominant-negative Rac1 by type 1 fimbriated E. coli ORN103(pSH2). **P< 0.01; *P<0.05, relative to control values. Bars represent the mean + S. D.. B, GTP-bound Rac1 levels of non-transfected (NT), control-transfected (Ctrl), and TLR4 KD BECs before (0) and 30 min after (30) exposure to E. coli. An actin-specific Western blot was used as a loading control.

Figure 4

Figure 4. TLR4 KD BECs evoke a reduced cAMP response to E. coli which correlates with increased bacterial invasion

A, Intracellular cAMP levels of control-transfected BECs (Ctrl) and TLR4 KD BECs after exposure to type 1 fimbriated E. coli ORN103(pSH2) or E. coli LPS. *P<0.05 relative to E. coli (EC) or LPS treated control. B, E. coli invasion of non-transfected (NT), control-transfected (Ctrl), and TLR4 KD BECs left untreated (UT) or following treatment with 50 μM forskolin (Fsk) or 1 mM dibutryl cAMP (dbcAMP). *P<0.001 relative to values of respective UT BECs. C, GTP-bound Rac1 levels in untreated (UT) and forskolin-treated (Fsk) BECs before (0) or after 30 min (30) exposure to E. coli. An actin-specific Western blot was used as a loading control. D, E. coli invasion of untreated (UT) BECs or BECs following 30 min treatment with 0.2 μM PKA inhibitor peptide (PKI), 1 mM dibutyryl cAMP (dbcAMP), 1 mM 6-Bnz-cAMP, or 1 mM 8-CPT-2-cAMP. **P< 0.001; *P< 0.01, relative to UT values. Bars represent the mean + S. D. in A, B, and D.

Figure 5

Figure 5. The BEC cAMP response to E. coli and its subsequent effect on bacterial invasion is mediated by Adenylyl cyclase 3

A, RT-PCR of control-transfected BECs (Ctrl) and AC-3, -4, -6, or -7 KD BECs. GAPDH-specific RT-PCR was used as a loading control. B, Intracellular cAMP levels in non-transfected BECs (NT), control-transfected BECs (Ctrl), or AC-3, -4, -6 or -7 KD BECs before (UT) or after exposure to E. coli ORN103(pSH2) (EC). *P<0.005 and **P<0.02 relative to respective UT values. C, E. coli invasion of control-transfected BECs (Ctrl), or AC-3, -4, -6, or -7 KD BECs. **P<0.0001 relative to control values. Bars represent the mean + S. D. in B and C. D, Active-Rac1 levels of control and AC3 KD BECs before and at 15 or 30 min after exposure to E. coli. An actin-specific Western blot was used as a loading control.

Figure 6

Figure 6. Reduced intracellular levels of cAMP in BECs of TLR4 mutant mice and use of Fsk to enhance resistance of BECs to invasion by type 1 fimbriated Enterobacteria

A, Intracellular cAMP in superficial BECs obtained from control (HeN), TLR4 mutant (HeJ) mice, and TLR4 mutant mice treated with Fsk (HeJ+Fsk) (n=3–5).**P<0.01, relative to HeN values as well as relative to HeJ+Fsk values. B–D, Invasion of bladders of control (HeN) mice, TLR4 mutant (HeJ) mice and TLR4 mutant (HeJ) mice pretreated with Fsk by 1×108 of E. coli CI5 (B), K. pneumoniae 1236 (C), or S. aureus 54 (D). When indicated, the mice were pretreated for 1 h with forskolin via intravesicular catheter instillation and intraperitoneal (IP) injection. Bars represent the mean + S. D. in A–D. ##P<0.001; #P<0.01, relative to HeN values. ** P<0.01; * P<0.03, relative to HeJ values.

Figure 7

Figure 7. Diagrammatic depiction of the parallel signaling reactions occurring in BECs during infection by type 1 fimbriated enterobacteria

Highlighted in the figure is the TLR4 initiated and AC-3, cAMP, and PKA dependent signaling pathway that dissects with and abrogates the lipid raft mediated endocytic pathway.

References

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