Murine RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in complete high glucose Dulbecco's modified Eagle's Medium (DMEM; Welgene, Inc., Daejeon, Korea) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified incubator under conditions of 5% CO₂.

Cell viability was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells were seeded in a 96-well culture plate at a cell density of 2.0×10⁴ cells/well. Then, MTT colorimetric solutions were added to each well, and the absorbance was measured at 570 nm using a microplate spectrophotometer (BioTek instruments, Winooski, VT, USA).

Total RNA was isolated using the easy-spin Total RNA Extraction kit (iNtRON Biotechnology, Sungnam, Korea), according to the manufacturer's procedure. cDNA was synthesized from RNA in a PCR Thermal Cycler (Takara Bio, Inc., Otsu, Japan) using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-qPCR analysis was performed with the SYBR Green Master Mix (Takara Bio, Inc.), using gene-specific primers, in a StepOnelus Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). PCR conditions were initially maintained at 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 45 sec. The level of mRNA expression was normalized to the level of GAPDH expression. The relative value of gene expression was analyzed using the 2^−∆∆Cq method (13). The primer sequences used in this study are presented in Table I.

RAW 264.7 cells were seeded in a 6-well culture plate in the presence and absence of FX11 or LPS. Culture medium was collected and lactate levels were measured by UPLC-tandem mass spectrometry (Triple Quad 5500; Sciex, Framingham, MA, USA), as described previously, with modifications (14).

RAW 264.7 cells were seeded in a 6-well culture plate. The next day, cells were treated with different concentrations of FX11 or LDHA-specific small interfering RNA (siRNA) (50 nM). The cells were harvested by lysis buffer (150 mM Sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5 and 2 mM EDTA) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) to obtain whole cell lysates. Protein concentrations were measured by using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of the proteins were subjected to SDS-PAGE, and then transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk and incubated with specific primary antibodies against p-JNK, JNK, p-ERK, ERK, p-p38, p38, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), HO-1, and LDHA (Cell Signaling Technology, Inc., Danvers, MA, USA), and β-actin (EMD Millipore, Billerica, MA, USA). The blots were then incubated with the horseradish peroxidase (HRP)-linked Goat anti-Rabbit (or Mouse) secondary IgG and IgM (EMD Millipore) at room temperature for 1 h. The signals were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). A chemiluminescence image analyzer (Vilber Lourmat Corporation, Torcy, France) was used for the detection of signals.

The concentrations of nitrite in the supernatant of cell culture media were measured by Griess reagent (Molecular Probes; Thermo Fisher Scientific, Inc.). The cell culture media and Griess reagent were taken in a 96-well plate. This mixture was then incubated for 30 min at room temperature. The nitrite concentrations were measured by measuring the absorbance at 548 nm using a microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA).

The concentrations of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6] in the supernatant of cell culture media were measured using a mouse ELISA kit (Enzo Life Sciences, Ann Arbor, MI, USA), according to the manufacturer's instructions.

LDHA silencing in RAW 264.7 macrophages was performed by transiently transfecting the cells with mouse LDHA-specific siRNAs. LDHA-siRNA duplexes and scrambled siRNA were purchased from OriGene Technologies (Rockville, MD, USA). RAW 264.7 cells were seeded in a 6-well culture plate at a cell density of 4×10⁵ cells/well. The cells were transfected with LDHA-specific siRNA for 48 h using the Lipofectamine 2000 reagent, according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.).

The Affymetrix whole transcript expression array process was performed according to the manufacturer's protocol (Genechip whole transcript plus reagent kit). cDNA was synthesized using the genechip whole transcript amplification kit according to the manufacturer's instructions. The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the Genechip WT terminal labeling kit. Approximately 5.5 µg of the labeled DNA target was hybridized to the Affymetrix genechip mouse 2.0 ST array at 45°C for 16 h. The hybridized arrays were washed and stained on a geneChip fluidics station 450 and scanned on a GCS3000 scanner (Affymetrix; Thermo Fisher Scientific, Inc.). Signal values were determined by using the Affymetrix^® GeneChip^™ command console software.

Raw data were extracted automatically in Affymetrix data extraction protocol using the software provided by Affymetrix genechip Command Console Software (AGCC). After importing the CEL files, the data were summarized and normalized using the robust multi-average (RMA) method implemented in the Affymetrix expression console software (EC). We exported the result with gene level RMA analysis and performed the differentially expressed gene (DEG) analysis. For a DEG set, a hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-Enrichment and functional annotation analysis for significant probe lists was performed using gene ontology (www.geneontology.org/) and KEGG (www.genome.jp/kegg/). All data analysis and visualization of DEGs were performed by using R 3.1.2 (www.r-project.org).

Experiments were performed using whole heparinized blood obtained by venopuncture from healthy donors and from patients with Behçet's disease (Healthy controls, n=4. Behçet's disease, n=6). The present study was conducted in accordance with the provisions of the Declaration of Helsinki for the participation of human subjects in research and was approved by the Institutional Review Board of Ajou University Medical Center (IRB no. BMR-GEN-14-463); written informed consent was obtained from all participants. PBMCs were isolated using histopaque-1077 density-centrifugation and were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% non-essential amino acids (100 mM), and 1% L-glutamine (200 mM). The cultures were maintained at 37°C in a 5% CO₂ atmosphere with 95% humidity.

All the data are expressed as mean ± standard error mean. Statistical differences among groups were calculated using one-way analysis of variance with a Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed with GraphPad prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA).

Lactate is implicated in excessive cell proliferation and autoimmune diseases (6,11). Therefore, we hypothesized that modulation of lactate regulates inflammatory response induced by LPS. We investigated whether the pharmacological inhibition of LDHA affects the transcriptional expression of cytokines. Among various LDH inhibitors, we utilized FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid], a gossypol derivative, which demonstrates a higher selectivity for the forward reaction of LDHA, than for the reverse reaction of LDHB (Ki=0.05 µM for LDHA, Ki=20 µM for LDHB) (15,16). First, we measured the cell viability, to determine a tolerable dose range of FX11 for the study. When RAW 264.7 cells were treated with various concentrations of FX11 for 24 h, it was seen that the cell survival was reduced above FX11 concentrations of 2 µM (Fig. 1A); we thus decided to treat the cells with FX11 concentrations below 2 µM. We treated RAW 264.7 cells with LPS to activate the inflammatory response in the presence or absence of FX11. Inhibition of LDHA did not alter the expression of LDHA. However, a slight decrease in LDHA expression was found in samples treated with 0.5 µM FX11, regardless of LPS treatment (Fig. 1B). When the cells were treated with LPS, the lactate levels in the media increased, and FX11 treatment reduced the lactate levels to values lower than those observed in the untreated control (Fig. 1C). This suggests that FX11 pharmacologically inhibited LDHA in RAW 264.7 cells. The transcriptional induction of cytokines including TNFα, IL-1β, and IL-6 by LPS was suppressed when the cells were co-treated with FX11 (Fig. 1D-F). The most notable effect was found in IL-6. These results suggest that reduced lactate levels correlate with the suppression of the LPS-induced expression of cytokines in macrophages.

Inflammatory response induced by bacterial infection activates cytokine production and inflammatory mediators such as nitric oxide (NO) and eicosanoids (17,18). Anti-inflammatory effects were indicated by the suppression of iNOS and COX-2 expression, leading to decreased NO and PGE2 production, respectively (19). In contrast, heme oxygenase 1 (HO-1) downregulates the expression of cytokines such as TNF-α, IL-1β, and IL-6, and reduces NO production. Thus, HO-1 is suggested as an anti-inflammatory mediator (2). To determine the degree of inflammation regulated by lactate, we examined the effects of FX11 on the transcriptional expression of iNOS and COX-2. Inhibition of LDHA by FX11 downregulated the LPS-induced expression of iNOS and COX-2 (Fig. 2A and B). However, the expressions of iNOS, LDHA, and HO-1 proteins were not changed (Fig. 2D), and the production of nitrite (Fig. 2C) and PGE2 was not altered (data not shown). Only COX-2 was downregulated after treatment with 1 µM FX11.

Since FX11 transcriptionally altered the expression of iNOS, COX-2, HO-1, and inflammatory cytokines, we investigated whether the anti-inflammatory effects of FX11 inhibit the phosphorylation of MAP kinases in LPS-induced inflammatory response. We found that the phosphorylation of JNK, ERK, and p38 was reduced by FX11 treatment (Fig. 2E). These results suggest that FX11 downregulates the transcriptional expression of iNOS and COX-2, and inhibits the phosphorylation of MAP kinases.

To support the role of lactate production in anti-inflammatory response, we suppressed LDHA expression by siRNA and examined its effects on the expression of inflammatory genes and signal mediators. LDHA-specific siRNA (siLDHA) downregulated LDHA expression and siLDHA also reduced the LPS-mediated protein induction of iNOS and COX-2 (Fig. 3A). However, the protein levels of HO-1 were not altered. The transcriptional upregulation of IL-6, iNOS, and COX-2 by LPS was attenuated by siLDHA (Fig. 3B-D). Consistent with these expression results, the production of IL-6 and nitrites was reduced when siLDHA was transiently transfected into the cells (Fig. 3E and F). We then examined the effects of genetic downregulation of LDHA on MAP kinase signaling. Although we did not find any changes in the phosphorylation of JNK and ERK, the phosphorylation of p38 was slightly reduced by siLDHA (Fig. 4A). These results suggest that siLDHA-mediated genetic suppression resulted in anti-inflammatory effects by the downregulation of inflammatory gene expression and inhibition of p38 phosphorylation.

Alteration of inflammatory gene expression was found in LDHA-specific siRNA transfected RAW 264.7 cells. To determine the general effects of LDHA suppression, we performed microarray gene analysis using mRNA from both LPS-treated and untreated siLDHA-transfected cells. Gene ontology functional analyses (Fig. 4B) and pathway analysis (Fig. 4C) revealed that LDHA silencing by siRNA is implicated in diverse signaling pathways. The downregulated genes were clearly enriched in ‘cytokine activity’ (GO: 0005125), including TNF signaling pathway, chemokine signaling, cytokine-cytokine receptor interaction, and TLR signaling pathway, when we compared cells treated with LPS alone and those treated with LPS plus siLDHA. These results suggest that LDHA knockdown is associated with suppression of cytokine-related inflammatory responses.

We found that the expression of inflammatory and immune-related genes was mainly altered (Fig. 5A). To confirm the involvement of LDHA in inflammation, we measured the expression of CXCL10, which was downregulated as shown in the microarray analyses. We found that pharmacologic or genetic inhibition of LDHA by FX11 treatment or siLDHA transfection, respectively, downregulated CXCL10 in LPS-treated cells (Fig. 5B and C). These results were consistent with those of the microarray analyses. These results suggest that lactate metabolism is positively correlated with inflammatory and immune responses.

To determine the clinical relevance of LDHA inhibition, we treated PBMCs isolated from healthy controls and patients with Behçet's disease with FX-11 in the presence or absence of LPS pre-treatment for 6 h. Behçet's disease is an autoimmune disease characterized by ulceration of the mouth and genitals, inflammation in the eye, and arthritis. IL-1β and TNFα were significantly upregulated by LPS treatment (Fig. 6). When cells were treated with FX11 in the presence of LPS, the expression of IL-1β and TNFα was suppressed in both PBMCs from healthy controls (Fig. 6A) and from patients with Behçet's disease (Fig. 6B). These results suggest that LDHA suppression downregulates the inflammatory response in PBMCs isolated from clinical subjects.