Lactate boosts TLR4 signaling and NF-kappaB pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation

Abstract

It has been shown that lactate induces insulin resistance. However, the underlying mechanisms have not been well understood. Based on our observation that lactate augments LPS-stimulated inflammatory gene expression, we proposed that lactate may enhance TLR4 signaling in macrophages, which has been shown to play an important role in insulin resistance in adipocytes. In this study, we demonstrated that lactate stimulated MD-2, a coreceptor for TLR4 signaling activation, NF-kappaB transcriptional activity, and the expression of inflammatory genes in human U937 histiocytes (resident macrophages). Similar enhancement of the inflammatory gene expression by lactate was also observed in human monocyte-derived macrophages. The essential role of MD-2 in lactate-augmented TLR4 signaling was confirmed by observation that the suppression of MD-2 expression by small interfering RNA led to significant inhibition of inflammatory gene expression. To further elucidate how lactate treatment enhances TLR4 activation, we showed that the augmentation of inflammatory gene expression by lactate was abrogated by antioxidant treatment, suggesting a critical role of reactive oxygen species in the enhancement of TLR4 activation by lactate. Finally, we showed that alpha-cyano-4-hydroxycinnamic acid, a classic inhibitor for monocarboxylate transporters, blocked lactate-augmented inflammatory gene expression and nuclear NF-kappaB activity, indicating that lactate transport through monocarboxylate transporters is required for lactate-enhanced TLR4 activation. Collectively, this study documents that lactate boosts TLR4 activation and NF-kappaB-dependent inflammatory gene expression via monocarboxylate transporters and MD-2 up-regulation.

FIGURE 1

The effect of lactate on LPS-stimulated inflammatory gene expression by U937 histiocytes. U937 cells were pretreated with 20 mM of sodium lactate for 24 h and then treated with 20 mM of sodium lactate and 100 ng/ml LPS in fresh medium for another 24 h. After the treatment, RNA was isolated and converted to cDNA that was subjected to real-time PCR to quantify IL-1β (A), IL-6 (B), CSF2 (C), CXCL10 (D), and IL-8 (E). F, U937 cells were pretreated with 0, 5, 10, and 20 mM of sodium lactate for 24 h and then treated with 100 ng/ml LPS in the presence of same concentration of sodium lactate for 24 h. The IL-6 released by cells into culture medium was quantified using ELISA. The data (mean ± SD) presented are from one of three independent experiments with similar results.

FIGURE 2

The effect of lactate on LPS-stimulated inflammatory gene expression by HMDMs. HMDMs were pretreated with 20 mM of sodium lactate for 24 h and then treated with 20 mM of sodium lactate and 100 ng/ml LPS in fresh medium for another 24 h. After the treatment, RNA was isolated and converted to cDNA that was subjected to real-time PCR to quantify IL-1β (A), IL-6 (B), CXCL10 (C), and IL-8 (D). The data (mean ± SD) presented are from one of three independent experiments with similar results.

FIGURE 3

The effect of lactate, LPS, or lactate plus LPS on cellular IkBα and IkBβ levels. U937 cells were treated with 20 mM of sodium lactate, 100 ng/ml LPS, or both for 2 and 4 h. After the treatment, cells were lysed and cellular IκBα and IκBβ levels were determined using immunoblotting as described in Materials and Methods. The blot presented is from one of two independent experiments with similar results.

FIGURE 4

NF-κB transcriptional activity in U937 cells treated with lactate, LPS, or both. U937 cells were transfected with NF-κB or CREB promoter-luciferase reporter construct as described in Materials and Methods. After the transfection, cells were treated with 20 mM of sodium lactate, 100 ng/ml LPS, or both for 24 h and the cellular luciferase activity was then determined. The data presented are from one of three independent experiments with similar results.

FIGURE 5

The effect of lactate, LPS, or lactate plus LPS on MD-2 mRNA (A) and protein (B) expression. U937 cells were treated with 20 mM of sodium lactate, 100 ng/ml LPS, or both for 24 h. After the treatment, RNA and protein were isolated and the MD-2 mRNA and protein were determined using real-time PCR and immunoblotting, respectively, as described in Materials and Methods. TLR4 protein was also detected using immunoblotting.

FIGURE 6

Inhibition of lactate-augmented IL-6 secretion by suppression of MD-2 expression using siRNA. A, U937 macrophages were transfected with MD-2 siRNA or control siRNA using Lipofectamine 2000 as the transfection reagent for 24–36 h, and the expression of MD-2 was then determined using immunoblotting. B, After the transfection, the cells were pretreated with 20 mM of sodium lactate for 24 h and medium was changed. The cells were treated with 20 mM of sodium lactate in the presence or absence of 100 ng/ml LPS for another 24 h. The conditioned medium was collected for quantification of IL-6 using ELISA.

FIGURE 7

The effect of antioxidants on lactate-augmented IL-6 gene expression. U937 cells were treated with 20 mM of sodium lactate and 100 ng/ml LPS in the presence or absence of different concentrations of NAC (A) or DMSO (B) for 24 h. After the treatment, culture medium was collected for ELISA to quantify IL-6. The data (mean ± SD) presented are from one of four independent experiments with similar results.

FIGURE 8

The effect of MCT inhibitor on the synergistic effect of lactate and LPS on IL-6 secretion. U937 cells were treated with 20 mM of sodium lactate, 100 ng/ml LPS, or both in the presence or absence of 2 mM of MCT inhibitor α-CHCA for 24 h, and IL-6 released into medium was quantified using ELISA. The data (mean ± SD) presented are from one of three independent experiments with similar results.

FIGURE 9

The effect of MCT inhibitor on NF-κB transcriptional activity. U937 cells were transfected with NF-κB promoter-luciferase reporter construct as described in Materials and Methods. After the transfection, U937 cells were treated with 20 mM of sodium lactate, 100 ng/ml LPS, or both in the presence or absence of 2 mM of MCT inhibitor α-CHCA for 24 h and the cellular luciferase activity was then determined. The data (mean ± SD) presented are from one of three independent experiments with similar results.

FIGURE 10

Inhibition of lactate-augmented MD-2 expression by α-CHCA and NAC. U937 macrophages were treated with 20 mM of sodium lactate, 100 ng/ml LPS, or both in the presence or absence of 2 mM of α-CHCA or 5 mM of NAC for 24 h. After the treatment, RNA was isolated from the cells and reversely transcribed into cDNA, which was used for real-time PCR to quantify MD-2 mRNA expression. The data (mean ± SD) presented are from one of two independent experiments with similar results.