2‐Deoxy‐D‐glucose impedes T cell–induced apoptosis of keratinocytes in oral lichen planus

Abstract Oral lichen planus (OLP) is a T cell–mediated immunoinflammatory disease. Glycolysis plays an essential role in T‐cell immune responses. Blocking glycolytic pathway in activated T cells represents a therapeutic strategy for restraint of immunologic process in autoimmune disorders. 2‐Deoxy‐D‐glucose (2‐DG) has been widely used to probe into glycolysis in immune cells. This study was aimed to explore the role of glycolysis inhibition by 2‐DG on regulating immune responses of OLP‐derived T cells. We observed that lactic dehydrogenase A (LDHA) expression was elevated in OLP lesions and local T cells. 2‐DG inhibited the expression of LDHA, p‐mTOR, Hif1α and PLD2 in T cells; meanwhile, it decreased proliferation and increased apoptosis of T cells. T cells treated by 2‐DG showed lower LDHA expression and elevated apoptosis, resulting in a reduced apoptotic population of keratinocytes that were co‐cultured with them, which was related to the decreased levels of IFN‐γ in co‐culture system. Rapamycin enhanced the effects of 2‐DG on immune responses between T cells and keratinocytes. Thus, these findings indicated that OLP‐derived T cells might be highly dependent upon high glycolysis for proliferation, and 2‐DG treatment combined with rapamycin might be an option to alleviate T‐cell responses, contributing to reducing apoptosis of keratinocytes.


| INTRODUC TI ON
Oral lichen planus (OLP) is a chronic inflammatory immune disease of unknown aetiology and has been recognized as an oral potentially malignant disorder (OPMD). 1,2 The typically histopathological characteristics of OLP are dense subepithelial infiltration of lymphocytes and liquefactive degeneration in the basal keratinocytes. 2 T cell-mediated dysfunctional immunity has been widely supposed to be the main pathogenesis of OLP. 3,4 Our previous studies demonstrated that Th1-biased responses participated in the development of OLP. 5,6 Specifically, T helper (Th) cells are activated after antigen presentation by major histocompatibility complex II (MHC-II) molecules and then produce pro-inflammatory cytokines, including interferonγ (IFNγ) and interleukin-2 (IL-2). 4 These cytokines together with MHC-I molecules on keratinocytes activate cytotoxic T cells, which further induce the apoptosis of keratinocytes. 4 The metabolic reprogramming in T-cell immune responses is characterized by a switch to aerobic glycolysis, also known as 'Warburg effect' that was first described in cancer. 7 Blocking aerobic glycolysis is pernicious to proliferation and differentiation of effector T cells while beneficial for development of regulatory T cells. 8 Our recent study showed that activated T cells required glycolytic metabolism to increase glucose utilization, during which Glut1 expression was elevated, thereby promoting T-cell proliferation and differentiation. 9 In this process, glucose-derived pyruvate was guided into glycolytic flux by lactic dehydrogenase A (LDHA), rather than into tricarboxylic acid cycle by pyruvate dehydrogenase (PDH). 9,10 Moreover, molecular pathways, including mammalian target of rapamycin (mTOR), hypoxia-inducible factor 1α (HIF1α) and phospholipase D2 (PLD2), motivate glycolytic metabolism in T cells. [11][12][13] We have found that the expression of p-mTOR, HIF1α and PLD2 was elevated in peripheral or local T cells of OLP, and mTOR/HIF1α/PLD2 axis could upregulate the phosphorylation of LDHA in T cells. 9,14,15 Besides, mTOR pathway could dynamically respond to extracellular glucose signals, then correspondingly regulate T-cell glycolysis, and finally support Tcell proliferation and differentiation. 9 These findings suggested that deregulated mTOR pathway might disturb the glycolytic metabolism in T cells, thereby leading to the dysfunctional immunity of OLP.
Lactic dehydrogenase activity has been investigated in OLP lesions in 1965 16 ; however, LDHA expression and the role of glycolysis in T cells from OLP remains unknown to date.
Recently, immunometabolism has been a burgeoning field. 8,17,18 Targeting aerobic glycolysis turns into an attractive therapeutic strategy for restraint of immunologic process in autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). 7,10 This therapy does not have broad toxicity in normal tissues that depend on oxidative metabolism. 7  In this study, the expression of LDHA in OLP lesions and local T cells was investigated by immunohistochemistry and immunofluorescence staining. Then, the effects of 2-DG on OLP-derived T cells were assessed by detecting the expression of LDHA and mTOR pathway, T-cell proliferation, and cell apoptosis. Finally, the coculture system was established with 2-DG pretreated OLP-derived T cells and LPS-stimulated keratinocytes to explore the modulation of 2-DG on immune responses between T cell and keratinocytes.

| Patients
Sixteen patients with diagnosis of OLP and ten healthy controls were enrolled prospectively in this study. They were referred to the Department of Oral Medicine, School and Hospital of Stomatology, Wuhan University. Diagnosis of OLP was made according to the clinical and histopathological findings. 2 Healthy controls came from patients without any impairment on oral mucosa or systemic diseases.
Other inclusion and exclusion standards were consistent to our previous study. 9 The clinical characteristics of samples were shown in Appendix S1 and Appendix S2. This research was carried out on the principles of the Declaration of Helsinki. It has been approved by the Ethical Committee Broad of School and Hospital of Stomatology, Wuhan University (no. 2015C35). The patients' informed consent was obtained before collection of tissue specimens and peripheral blood.

| Immunohistochemistry and immunofluorescence
The formalin-fixed, paraffin-embedded oral mucosal tissues were The fluorescence contrast parameters were described in detail in Appendix S3.
Next, T cells were sorted from PBMCs using BD IMag™ Human T Lymphocyte Enrichment Set-DM (Catalog # 557874) by IMag™ Human oral keratinocytes (cell line named OKF4, 5 × 10 4 cells/ml) were seeded in a 24-well plate with keratinocyte serum-free medium (K-SFM, Catalog # 10744019, Thermo) at 37°C with a 5% CO 2 . In the logarithmic growth phase, keratinocytes were stimulated with 10 μg/ ml LPS (Catalog # L2654, Sigma) for 36 h to simulate a local inflammatory microenvironment of OLP and resuspended with fresh medium before co-culture.

| Immunocytochemistry
Paraformaldehyde-fixed cells were smeared on the slides, permeabilized with Triton X-100 after dying at room temperature, blocked with bull serum albumin (3%), and incubated with a primary antibody against LDHA (Proteintech) and then Cy3-conjugated secondary antibody (Catalog # GB21303, Servicebio). Nuclei were stained with DAPI. The cell smears were observed under a fluorescence microscope (NIKON ECLIPSE C1, Japan) and an imaging system (NIKON DS-U3). The images were captured with CaseViewer (3DHISTECH).

| Western blotting
Protein extraction was performed with RIPA buffer (Catalog # P0013B, Beyotime) by centrifugation at 12,000 × g for 20 min.
Proteins were separated by 8%, 10% or 12% polyacrylamide gel electrophoresis and transferred on polyvinylidene fluoride membranes.
After blocking with 5% skimmed milk, membranes were incubated

| Cell proliferation assay
Primary T cells at a density of 5 × 10 4 cells/well were seeded into 96well plates for 24 h. After treatment, the cells were incubated with the Cell Counting Kit-8 (CCK-8, Catalog # CK04, Dojindo) reagent for 2.5 h at 37°C. Cell viability was quantified by optical density (OD) value at an absorbance wavelength of 450 nm.

| Cell apoptosis assay
Either T cells or keratinocytes were resuspended at a density of 10 6 cells/ml approximately for the apoptosis assay and assessed using an Annexin V-FITC/PI Apoptosis Detection Kit (Catalog # KGA108, KeyGEN) by flow cytometry (Beckman). The results were expressed as the percentage of apoptotic (Annexin V positive) cells.

| Cytokines assay
The concentrations of IFNγ, IL-4 and IL-17 in the supernatants of co-culture system were measured using human IFNγ, IL-4 and IL-17 Co., Ltd).

| Statistical analysis
Statistical analyses were performed, and graphs were generated using the Graphpad Prism 5 (Graphpad Software). The data were reported as the mean ± SEM. Significant differences between groups were evaluated by Mann-Whitney test, unpaired t test and one-way ANOVA. p < 0.05 was regarded as statistical significance level. F I G U R E 1 LDHA protein was highly expressed in OLP lesions and local T cells. Representative images of immunohistochemical staining and immunofluorescent staining for LDHA in OLP lesions (n = 12) and normal oral mucosa tissues (n = 10). (A) LDHA immunoreactivity was observed in the epithelium and infiltrated lymphocytes of OLP lesions. The statistical data were reported using mean optical density (MOD) of at least three randomly captured fields at ×400 magnification. Bar: 100 μm for ×200, 50 μm for ×400. (B) Abundant red fluorescence signals for CD3 overlapped with the green signals for LDHA in OLP lesions. b1: single channel images for DAPI (blue), CD3 (red), LDHA (green); b2: merged images. Bar: 100 μm for ×200

| OLP-derived T cells were dependent upon high glycolysis and mTOR pathway for proliferation
2-DG, a glycolytic inhibitor, was used to study the effects of glyco-

| 2-DG treatment combined with rapamycin alleviated T-cell responses, contributing to the reduced apoptosis of keratinocytes in coculture system
In OLP lesions, colloid bodies were seen in basement membrane of OLP tissues rather than in healthy controls (Appendix S5). Colloid Combined treatment of 2-DG and rapamycin resulted in a higher apoptosis rate of OLP-derived T cells and lower apoptotic events of keratinocytes after co-culture (Figure 5b & c). Specifically, 2-DG and/ or rapamycin mainly increased the early apoptosis of T cells, while T cells predominantly induced the late apoptosis of keratinocytes (Appendix S6).

| IFNγ level in co-culture system was decreased after inhibiting glycolysis and mTOR pathway in OLP-derived T cells
Cytokines play an important role in T cell-mediated keratinocyte apoptosis. 4 Considering the involvement of Th1, Th2 and Th17 cells in the immunopathogenesis of OLP, the levels of IFNγ, IL-4 and IL-17 in medium of co-culture system were detected. Results showed that IFNγ level was increased when keratinocytes were co-cultured with OLP-derived T cells (Figure 6a), which was corresponded to the results of apoptotic analysis for T cells and keratinocytes in Figure 5.

| DISCUSS ION
The abnormal metabolic state of T cells interferes in cellular biology and functions, thereby results in dysfunctional immune responses, ultimately leads to chronic inflammation in autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). 10 It has been reported that CD4 + T cells in SLE have increasing abnormal mitochondria which cause hyperactivated metabolism, and naïve T cells in RA undertake the pentose phosphate pathway and possess anti-glycolysis effects. [20][21][22] In different T cellmediated diseases, metabolic status is different. However, little is known about metabolic state of T cells in OLP. In this study, data showed that expression of LDHA was elevated in OLP lesions and local T cells compared with healthy tissues, which was consistent the results reported by Shklar that lactic dehydrogenase activity was elevated in OLP lesions. 16 LDHA is a glycolytic enzyme that converts glucose-derived pyruvate into lactic acid. 10 With lactate production, NAD + pools would backfill and then maintain glycolytic flux. 23 Therefore, it was demonstrated that glycolysis mediated by LDHA was hyperactivated in T cells of OLP.

2-DG is considered as an inhibitor of glucose metabolism.
Concretely, 2-DG can be phosphorylated by hexokinase into 2-deoxyglucose-6-phosphate, which cannot continue to be metabolized in the energy payoff phase of glycolysis. 24 Competitive nature of 2-DG over glucose utilization and inaccessibility of phosphorylated product into glycolysis can reduce substrates for the glycolytic flux, thus decreasing the production of ATP. [24][25][26] Intracellular ATP can active mTOR pathway in a variety of ways. 27 Reyes et al.
found that 2-DG induced the depletion of cellular ATP, contributing to the inhibition of mTOR as well as its downstream 4E-BP1. 25 Our previous findings revealed that the activation of mTOR pathway in T cells varied with glucose concentration, demonstrating mTOR serves as an important metabolic sensor. 9 Besides, inhibition of mTOR pathway could reduce the expression of Hif1α in T cells, 14 which is a major transcriptional regulator of LDHA. Either downregulation of mTOR or Hif1α could lead to the inhibition of LDHA. 28,29 In this study, 2-DG at concentration of 1 and 5 mM both remarkably inhib- IFNγ is an important cytokine from Th1 cells. We found that the levels of IFNγ in serum and local tissues of OLP were higher than healthy controls, 6 and the transcriptional expression of T-bet was increased in the peripheral and infiltrating T cells of OLP. 5,30 The induction of T-bet is dependent on IFNγ signalling and critical for Th1 differentiation, contributing to the immunologic dissonance in OLP. 31 Moreover, IFNγ increases CD40 expression on keratinocytes and promotes these cells releasing chemokines, such as CXCL9, CXCL10 and CXCL11, which may effectively mediate T cells recruitment. 32 CD40-expressed cells in OLP lesions were reported to be relatively prone to apoptosis. 33 34 Additionally, IFNγ-inducible CCL27 secretion from keratinocytes is involving in the recruitment of memory T cells into epithelium, which might be associated with the recurrence and chronicity of OLP. 35 In the present study, keratinocytes that were co-cultured with OLP-derived T cells had a larger number of apoptotic populations than ones cultured alone. Besides, IFNγ was highly expressed in co-culture supernatant of keratinocytes and OLP-derived F I G U R E 5 Blocking glycolysis and mTOR pathway in OLP-derived T cells resulted in a reduced apoptotic population of keratinocytes that were co-cultured with them. (A) The co-culture system was established with OLP-derived T cells and human oral keratinocytes via transwell inserts. T cells were pretreated with DMSO (at 0.1% v/v), 5 mM 2-DG or 100 nM rapamycin for 24 h. Then, T cells were placed in the upper compartment with fresh medium; LPS-stimulated keratinocytes were cultured in the lower compartment. They were co-cultured for 24 h for apoptosis analysis. The co-culture medium was consisted of 500 μl human T-cell robust expansion kit and 500 μl K-SFM. For T cells, DMSO at a concentration of 0.1% (v/v) was used as the solvent control group. For keratinocytes, cells cultured without T cells were used as a blank control. (B) Flow cytometry assay was used to detect the apoptosis of OLP-derived T cells and keratinocytes in co-culture system. B1: representative images for T-cell apoptosis; B2: representative images for keratinocyte apoptosis. (C) The histogram of apoptosis rates of T cells and keratinocytes in co-culture system. C1: for T cells, the apoptotic populations in 2-DG group and Rap group were larger than DMSO group, and the apoptotic populations in 2-DG +Rap group were larger than 2-DG group and Rap group. C2: for keratinocytes, the apoptosis rate was increased in DMSO T-cell group compared to Blank group, while decreased in 2-DG T-cell group and Rap T-cell group compared to DMSO T-cell group. The apoptosis rate in 2-DG +Rap T-cell group was lower than that in 2-DG T-cell group and Rap T-cell group. *p < 0.05; **p < 0.01; ***p < 0.001 F I G U R E 6 IFNγ level in co-culture system was decreased after blocking glycolysis and mTOR pathway in OLP-derived T cells. (A, B, C) ELISA kits were used to detect the levels of IFNγ, IL-4 and IL-17 in co-culture system. IFNγ level was increased in DMSO T-cell +keratinocyte group compared to keratinocyte group, while decreased in 2-DG T-cell +keratinocyte group and Rap T-cell +keratinocyte group compared to DMSO T-cell +keratinocyte group. IFNγ level in 2-DG +Rap T-cell +keratinocyte group was lower than that in 2-DG T-cell +keratinocyte group and Rap T-cell +keratinocyte group. *p < 0.05; **p < 0.01 F I G U R E 7 Diagram of the role of 2-DG on OLP-derived T cells. OLP T cells are highly dependent upon high glycolysis for proliferation. 2-DG blocking glycolysis leads to the inhibition of mTOR pathway, which is responsible for the expression of LDHA. T cells treated by 2-DG have lower LDHA expression and elevated apoptosis, resulting in a reduced apoptotic population of target cells, namely keratinocytes, which is related to the decreased levels of IFNγ in the microenvironment. Rapamycin enhanced the effects of 2-DG on immune responses between T cells and keratinocytes T cells. Collectively, it was postulated that IFNγ was involved in the interaction between OLP-derived T cells and keratinocytes.
Glycolysis has been shown to be indispensable for Th1 differentiation and signature cytokine IFNγ production. 36 It was shown that CD4 + T cells in the absence of LDHA produced less IFNγ. 37 LDHA promoted IFNγ expression through an epigenetic mechanism of histone acetylation, indicating a critical role for LDHA-mediated glycolysis in promoting Th1 responses. 37 Moreover, our previous study demonstrated that blocking mTOR pathway could diminish LDHA phosphorylation and reduce T-bet expression in T cells, suggesting that mTOR-dependent glycolytic pathway in T cells might serve as a metabolic target to regulate Th1 responses. 9 The current data showed that IFNγ level was increased when keratinocytes were cocultured with OLP-derived T cells. When OLP-derived T cells were pretreated with 2-DG or rapamycin, the IFNγ levels in co-culture system were decreased, and combined treatment showed the strongest effects on the IFNγ level. These findings suggested that both blocking glycolysis and mTOR pathway in OLP-derived T cells could effectively reduce IFNγ production then weaken the induction for keratinocyte apoptosis (Figure 7).
In conclusion, OLP-derived T cells might be highly dependent upon high glycolysis for proliferation. 2-DG treatment combined with rapamycin might be an option to alleviate T-cell responses, contributing to the reduced apoptosis of keratinocytes, which was related to the decreased levels of IFNγ in co-culture system.

ACK N OWLED G EM ENTS
This work was supported by grants from National Natural Science

CO N FLI C T S O F I NTE R E S T
The authors declared no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available from the corresponding author upon reasonable request.