Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections

Abstract

Background: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC).

Methodology/principal findings: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kappaB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kappaB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kappaB dependent pathways in a concentration- and time-dependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kappaB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted.

Conclusions/significance: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.

Figure 1. Palmitate effects on IL-6 and MCP-1 mRNA and protein levels.

Time- and concentration- dependent regulation of IL-6 and MCP-1 protein (A, C) and mRNA (B, D). IL-6 protein content (A) was measured in cytosol (white bars, ng/µg) and supernatant (black bars, ng/ml), while MCP-1 protein was measured exclusively in supernatant (C, ng/ml). All bars indicate difference to medium control. For each measurement a medium treated control was used. Data are shown as mean and SEM. Significant differences are indicated by lines. mRNA (B, D) was normalized to natural logarithm LN.

Figure 2. LPS effects on IL-6 and MCP-1 mRNA and protein levels.

Time- and concentration- dependent regulation of IL-6 and MCP-1 protein (A, C) and mRNA (B, D). IL-6 protein content (A) was measured in cytosol (white bars, ng/µg) and supernatant (black bars, ng/ml), while MCP-1 protein was measured exclusively in supernatant (C, ng/ml). All bars indicate difference to medium control. For each measurement a medium treated control was used. Data are shown as mean and SEM. Significant differences are indicated by lines. Post-hoc Bonferroni test was used after ANOVA. Significance level was p<0.05. mRNA (B, D) was normalized to natural logarithm LN.

Figure 3. Palmitate effects on the expression of IL-6 receptor subunit gp80, gp80_soluble, gp130 and pSTAT3.

(A) Membrane gp80 receptor mRNA (white bars), soluble gp80 mRNA (black bars). (B) Protein expression of gp80 (white bars) and pSTAT3 (black bars) was measured by ELISA, and membrane gp130 receptor was analysed by Western blot. (C) Gp130 mRNA (white bars), STAT3 mRNA (black bars). Data are shown as mean and SEM. mRNA was normalized to natural logarithm LN.

Figure 4. LPS effects on the expression of IL-6 receptor subunit gp80, gp80_soluble, gp130 and pSTAT3.

(A) Membrane gp80 receptor mRNA (white bars), soluble gp80 mRNA (black bars). (B) Protein expression of gp80 (white bars) and pSTAT3 (black bars) was measured by ELISA, and membrane gp130 receptor was analysed by Western blotting. (C) Gp130 mRNA (white bars), STAT3 mRNA (black bars). Data are shown as mean and SEM. mRNA was normalized to natural logarithm LN.

Figure 5. Confocal immunofluorescence of cultures treated for 48 hrs with 1 µg/ml LPS or 0.25 mM palmitate.

(A1–C1) Medium control; (A2–C2) LPS treated; (A3–C3) palmitate treated. Cells were double labelled for pSTAT3 (green) and SOCS3 (orange). Monoclonal SHP2 (red) antibody was used in single labelling experiments. Nuclei were stained with DAPI (blue) recorded with standard fluorescence and merged into the confocal images. The scale bar in B3 applies to all images except the insets, which have been enlarged two times.

Figure 6. Palmitate and LPS effects on SOCS3 and SHP2.

Time- and concentration- dependent palmitate effects on SOCS3 mRNA (A), SHP2 mRNA (C), SOCS3 protein expression (B), and SHP2 protein expression (D). LPS effects on SOCS3 mRNA (E), SHP2 mRNA (F), SOCS3 protein expression (G), and SHP2 protein expression (H). Protein content was analysed in the cytosol using Western blotting analysis. All bars indicate difference to medium control. For each measurement a medium treated control was used. Data are shown as mean and SEM. Significant differences are indicated by lines. mRNA was normalized to natural logarithm LN.

Figure 7. Gene regulation and pathway analysis.

Time- and concentration-dependent palmitate and LPS effects on pNF-κB protein expression (A, B). Protein content of pNF-κB was measured in cytosol (A, black bars) and is indicated as difference to medium control (AU in pg/mg) by ELISA. All bars indicate difference to medium control (A, B). For each measurement a medium treated control was used. Confocal double immunofluorescence images demonstrate nuclear translocation of NF-κB (green) and IL-6 (red). Nuclei are depicted by DAPI (blue). Dot blot analysis of phosphorylated NF-κB are shown as ratio of nuclear pNF-κB protein content to cytosolic pNF-κB protein content related to total protein content. Cells were stimulated for 48 h either with 0.25 mM palmitate or with 1 µg/ml LPS (C). Pathway analysis of palmitate induced IL-6 (D) and MCP-1 (E) regulation. Protein content was measured by ELISA in supernatants (ng/ml). Cells were stimulated with palmitate (0.5 mM) and inhibitor of proteasomal degradation of NF-κB (MG132, 40 µM) or MEK1 (PD98059, 20 µM) for 48 hrs. Medium control is indicated as black bar (D, E). Data are shown as mean and SEM. Significant differences are indicated by lines.