3.1 The differential expressions of TLR2, TLR4 and TLR9 in gastric cancer cell lines
As RNA and protein expression of TLR2, TLR4 and TLR9 were previously shown highly expressed in gastric tissue human GC tissue13–14 and tumor tissue from gp130F/F mouse stomach, a mouse model of gastric cancer that spontaneously develops gastric intestinal type hyperplasia at early 6 weeks of age15 compared with matched adjacent nontumoral or wild-type stomach tissues, we firstly examined mRNA and protein expression level of TLR2, TLR4 and TLR9 in a panel of human gastric cancer cell lines. Indeed, TLR2, TLR4 and TLR9 were differentially expressed in the panel of GC cells. MKN1 and MKN7 had the most TLR2 mRNA expression while TLR4 expression was abundantly presented in AGS, MKN1 and N87 (Fig. 1A, B). Most GC lines exhibited TLR9 expression at transcriptional level whereas the expression in MKN28 and SNU601 was higher than that of other lines (Fig. 1C). In addition, the levels of all TLRs protein expression were confirmed by Western blot using specific antibodies as shown in Fig. 1D-F. We then selected MKN1 and AGS as the representatives of lines with high TLR2 and TLR4 expression respectively. NUGC4 was chosen to stand for the line with high TLR9 expression in the following experiments.
3.2 TLRs activation in GC cell lines mediates OXPHOS and glycolysis
To evaluate whether TLRs regulates metabolic alterations in human GC epithelial cells, representative lines mentioned above were stimulated with TLR ligands (P3C (Pam3CSK4), LPS or CpG) indicated for 24 h and subjected to bioenergetic functional assays. The high TLR2 expressing line, MKN1 had an increased basal oxygen consumption rate (OCR) (Fig. 2B, column 1) and the OCR induced by the proton ionophore (uncoupler) FCCP (measuring maximal respiration capacity) in response to P3C (Fig. 2A). Spare respiration capacity (SRC), the parameter calculated by the difference between basal and maximal OCR was also elevated, reflecting the enhanced ability of cells dealing with increased energy needs (Fig. 2B, column 3). Furthermore, the extracellular acidification rate (ECAR), representing glycolytic activity (Fig. 2B, column 2) and lactate production, which measures acid production in cell media (Fig. 2G, column 1) were consistently increased in response to P3C. Unlike TLR2 activation, the ECAR in LPS treated AGS with high expression of TLR4 was downregulated with no change in L-lactate production. LPS induced TLR4 activation had no effect on OXPHOS and lactate despite of considerable changes of ECAR and FCCP induced SRC (Fig. 2C, D, G). In contrast, both OXPHOS and glycolysis were strongly inhibited in NUGC4, the line with high expression of TLR9. As shown in Fig. 2E-G, basal and maximal OCR, basal ECAR and SRC were all downregulated in NUGC4 cell after 24 h stimuli of CpG. We then examined the ROS production following TLR activation using fluorescence signals. Surprisingly, the reduction of ROS production was observed in P3C treated MKN1 regardless of the augmented mitochondrial activity whereas ROS in NUGC4 was decreased in the presence of CpG (Fig. 2H). We confirmed that these observations were not due to any difference in cell viability (Supp Fig. 1A). Therefore, these data suggest diverse metabolic phenotypes induced following the activation of different TLRs signaling pathways.
3.3 TLR2 promotes OXPHOS and glycolysis in GC cell lines rather than other TLRs
Besides the effect of these TLR ligands in respective TLR high expressing cells, whether these cell lines were influenced by other two TLR agonists was considered as well. All these ligands were used in the same concentration indicated before. In MKN1, LPS and CpG reduced the ability of OXPHOS and glycolysis based on OCR, ECAR and SRC (Fig. 3A-B). In AGS, P3C and CpG only had impact on SRC and showed an opposite alteration (Fig. 3C-D). P3C greatly stimulated the ability of OXPHOS and glycolysis in NUGC4 cell, another line with detectable expression of TLR2 (Fig. 1A, D) while LPS had no effect (Fig. 3E-F). Lactate and ROS production were shown in Fig. 3G-H which indicated that TLR2 agonist P3C had greatest change in these cell lines.
3.4 TLR2 agonists from different origins induced comparable metabolic phenotypes in GC cells
As Pam3CSK4 induced greater metabolic changes in GC cells, we further tested whether different TLR2 ligands derived from whole pathogen lysates or synthetic lipoproteins give rise to similar effects on MKN1 cell. FSL-1, a synthetic diacylated lipopeptide recognized by a TLR2/6 heterodimer, or P3C, mainly recognized through a TLR1/2 heterodimer were added at the indicated concentration (FSL1: 1 µg/ml; P3C: 10 ug/ml ) for 24 h. Consistently, FSL-1 or P3C induced TLR2 activation promoted both mitochondrial activity and glycolysis as OCRs, SRC, ECAR as well as lactate production were all increased as shown in Fig. 4A-B, whereas the significant reduction of ROS was detected by fluorescent signal in MKN1 supernatant following FSL-1 or P3C stimulation (Fig. 4F). In addition, we had comparable observations of the changes of metabolic phenotypes for NUGC4 in response to P3C and FSL-1 treatment (Supp Fig. 2A-B).
We next evaluated the influences of whole pathogen lysate induced TLR2 activation on cellular levels of oxidative stress and glycolytic activity. Heat Killed Listeria monocytogenes (HKLM), an intracellular Gram-positive bacterium prepared in a specific approach which was mainly indicated to target TLR2 according to the literature16. Two doses (107 cells/ml and 108 cells/ml) are used most commonly in the literature and recommended by the manufacturer. Consistent with those two synthetic TLR2 agonists, OCR, ECAR, SRC and lactate production were all significantly increased in MKN1 cell in a dose-dependent manner (Fig. 4C-D). Furthermore, cellular ROS levels were markedly reduced in both HKLM-treated MKN1 cells (Fig. 4H). In addition, similar observations were detected in NUGC4 in response to higher dose of HKLM (Supp Fig. 2C-D). Lactate production (Supp Fig. 2E, G) and cellular ROS levels (Supp Fig. 2F, H) of HKLM-treated NUGC4 cells showed a similar trend to MKN1. However, cell viability was unchanged in any agonists stimulated cell lines compared to non-stimulated control group (Supp Fig. 1A-D). Collectively, these data suggest that activation of conserved TLR2 signaling by either synthetic ligands or bacteria derived agonist in GC cell lines with detectable TLR2 expression causes comparable metabolic alterations, including enhanced OXPHOS and glycolysis, increased acid production, and reduced ROS levels.
3.5 TLR2 activation promotes the expression of key genes in control of bioenergetic processes and redox systems
To explore the potential mechanism of TLRs activation in metabolic reprogramming11, we examined the expression of key genes involving in mitochondrial function, glycolysis and oxidative phosphorylation etc. As shown in Fig. 5A, qPCR analyses of synthetic ligand-treated MKN1 cells revealed that genes involving in glycolysis, such as pyruvate kinase isozyme M2 (PKM2) and 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFR3) were significantly elevated. P3C stimulation appeared to have greater effects on OXPHOS genes such as cyclic AMP-responsive element-binding protein 1 (CREB1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) (Fig. 5B). Furthermore, genes located on mitochondrial chromosome such as mitochondrially encoded ATP synthase 6 (MT-ATP6), mitochondrial cytochrome b (MT-CYTB) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) were elevated as well (Fig. 5C).
Similarly, low or high dose of HKLM stimulations in MKN1 cells led to significant elevated expression for genes encoding hypoxia-inducible factor 1 alpha (HIF1α), pyruvate kinase isozyme M2 (PKM2), hexokinase 2 (HK2) (Fig. 5D). Likewise, PPARGC1A, the gene promoting OXPHOS, were uniquely increased in two doses of HKLM treated cells (Fig. 5E). And the expression of all mitochondrially encoded genes we studies was dramatically upregulated in the presence of HKLM (Fig. 5F). However, the only expression of HIF1A and PFKFB3 were increased in AGS, the high TLR4 expressing cell line or NUGC4, the high TLR9 expressing cell line treated with LPS and CpG respectively (Supp Fig. 3A, B) regardless of any alteration in bioenergetic assays observed (Fig. 2C-F).
To delineate the intricate antioxidant system contributing to the reduction of ROS following TLR2 activation, we also analyzed the key genes implicated in other redox systems including manganese-dependent SOD2 gene, glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS). qPCR showed that 24-hour stimulation from all agonists had strong transcriptional SOD2 induction in high TLR2 expressing lines, MKN1 cells (Fig. 5G). Consistently, SOD2 protein exhibited a significant increase in MKN1 and NUGC4 upon various ligands-induced TLR2 activation but not in response to any other TLR agonists (Fig. 5G-H). By contrast, SOD2 expression displayed no change in AGS regardless of any TLR activations (Fig. 5H). Taken together, key genes from multiple redox systems involves in mediating TLR2 dependent bioenergetic alterations.