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. 2011 Feb 28:12:18.
doi: 10.1186/1471-2172-12-18.

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Humberto M Garay-Malpartida  1 , Roberta F Mourão, Marluce Mantovani, Icaro A Santos, Mari C Sogayar, Anna C Goldberg
Affiliations

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Humberto M Garay-Malpartida et al. BMC Immunol. .

Abstract

Background: Toll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic β-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic β-cells is still to be clearly established. We investigated whether TLR4 is present in β-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis.

Results: CD11b positive macrophages were practically absent from isolated human islets obtained from non-diabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to β-cells. A significant loss of cell viability was observed in these β-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in β-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content.

Conclusions: Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic β-cells.

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Figures

Figure 1
Figure 1
Flow cytometric analysis of TLR4 expression in human islet cells. Free-floating 8-12 day islet cultures (500-1000 IEQ) were dissociated into single-cell suspensions and assessed by flow cytometry. A representative image from three independent experiments is shown. (a) Histogram compares total TLR4-positive cells (gray) with isotype-matched goat anti-rabbit IgG control for TLR4 staining (no color). (b) The right upper quadrant of the dot plot shows the TLR4 positive β-cell population, double-stained for Newport Green (NG) and TLR4-APC conjugate, (c) The right upper quadrant of the dot plot shows TLR4-positive macrophage-derived cells (non β-cells) double stained with TLR4-APC conjugate and CD11b-PE conjugate, (d) Tetramethylrhodamine (TMRE) and NG double staining marks viable β-cells.
Figure 2
Figure 2
LPS-induced TLR4 expression in β-cells. 5 × 105 human β-cells from adherent cultures were treated with LPS (5 and 50 ng/mL) and analyzed after 48 h. Expression of (a) TLR4 and (b) CD14 mRNA is shown as mean ± SD (3 independent experiments) of relative fold increase in gene expression at 48 h, compared to control (untreated) cells. Statistically significant differences are shown as (*) p < 0.05 and (**) p < 0.005.
Figure 3
Figure 3
Long-term exposure to LPS induces a loss of β-cell viability. Under the same conditions described in the legend to Figure 2, cell viability was analyzed by flow cytometry using TMRE staining. (a) Histogram showing the percentage of TMRE-positive cells upon treatment with 50 ng/mL LPS (red line), untreated control cells (green line) and isotype-matched control for TLR4 staining (dotted black line). (b) Values represent the relative percentage of TMRE-positive β-cells. Data are expressed as mean ± SD for β-cell cultures derived from different pancreas donors (n = 7), incubated with or without LPS 50 ng/mL for 48 h and analyzed in triplicate. Statistically significant differences are indicated as (**) p < 0.005, compared to non-treated control cells.
Figure 4
Figure 4
Effects of LPS treatment on insulin synthesis and secretion by β-cells. After 48 h of LPS stimulation, the effects on insulin homeostasis were measured by flow cytometry. (a) Representative histogram of percent NG-positive cells after treatment with LPS (red line), untreated or control cells (green line) and isotype-matched controls for the TLR4 staining (dotted black line). Data represent relative percentage of (b) NG-positive cells compared to control cells, expressed as mean ± SD for several β-cell cultures derived from different pancreas donors (n = 7), and analyzed in triplicate. (c) Insulin mRNA expression is shown as mean ± SD (3 independent experiments) of relative fold increase compared to control (untreated) cells. (d) Insulin secretion measured by chemiluminescence in β-cell cultures isolated from two different pancreata expressed as relative percentage of insulin secretion observed in control cells, and analyzed in triplicate (mean ± SD). Statistically significant differences are indicated as (**) p < 0.005, as compared to non-treated control cells.
Figure 5
Figure 5
Effects of LPS on TLR4 expression, cell viability and insulin synthesis in murine insulinoma cells. 5 × 105 MIN-6 cells treated with LPS (50 ng/mL) were analyzed after 48 h for gene expression, cell viability and insulin production. (a) TLR4 and CD14 mRNA expression is shown as mean ± SD (n = 3) of relative fold increase compared to control (untreated) cells. Representative flow cytometry histogram of (b) percent TMRE-positive or (d) percent NG-positive cells treated with LPS (red line), untreated or control cells (green line) and isotype-matched control for TLR4 staining (dotted black line). Relative percentage of (c) TMRE positive or (e) NG positive cells expressed as mean ± SD (n = 3). In all cases, statistically significant differences are indicated as (*) p < 0.05 and (**) p < 0.005, compared to non-treated control cells.
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