Enhanced Fatty Acid Synthesis Leads to Subset Imbalance and IFN-γ Overproduction in T Helper 1 Cells

Recent reports have shown the importance of IFN-γ and T-bet+ B cells in the pathology of SLE, suggesting the involvement of IFN-γ-producing T-bet+ CD4+ cells, i.e., Th1 cells. This study determined the changes in Th1 subsets with metabolic shift and their potential as therapeutic targets in SLE. Compared with healthy donors, patients with SLE had higher numbers of T-bethiCXCR3lo effector cells and T-bet+Foxp3lo non-suppressive cells, which excessively produce IFN-γ, and lower number of non-IFN-γ-producing T-bet+Foxp3hi activated-Treg cells. These changes were considered to be involved in treatment resistance. The differentiation mechanism of Th1 subsets was investigated in vitro using memory CD4+ cells obtained from healthy donors and patients with SLE. In memory CD4+ cells of healthy donors, both rapamycin and 2-deoxy-D-glucose (2DG) suppressed T-bet+Foxp3- cells, and induced T-bet+Foxp3+(lo/hi) cells. Rapamycin induced IFN-γ-producing T-bet+Foxp3lo cells accompanied with enhanced lipid metabolism, whereas 2DG induced IFN-γ-non-producing T-bet+Foxp3hi cells. In memory CD4+ cells of SLE patients, inhibition of fatty acid synthesis, but not β-oxidation, suppressed IFN-γ production, and up-regulated of Foxp3 expression in T-bet+Foxp3+ cells. Metabolic regulators such as fatty acid synthesis inhibitors may improve the pathological status by correcting Th1 subset imbalance and overproduction of IFN-γ in SLE.


INTRODUCTION
Systemic lupus erythematosus (SLE) is a common autoimmune disease. That nonspecific treatment of steroids or immunosuppressants is still used as the main therapeutic modality stresses the need to explore the pathological mechanisms and design new therapeutic strategies for SLE.
IFN signature plays an important role in SLE (1). In recent years, modular transcriptional repertoire analysis has demonstrated a different type I/II IFN signature and the importance of not only type I IFN but also type II IFN (IFN-g) (2). While helper T (Th) cells are known to play an important role in the pathogenesis of SLE, their differentiation and functional abnormalities remain unclear (3).
In the 1990-2010, SLE pathology was discussed in the context of a balance between Th1 and Th2, together with the involvement of Th1 (4,5). However, Th1 is only defined as an IFN-g-producing cell, and other important markers for Th1 such as CXCR3 and T-bet, have not been simultaneously examined in these papers. Recently, the diversity of Th cells has been reported, and CXCR3, T-bet and IFN-g have been reported to be expressed not only in Th1 but also in other diverse Th subsets. In particular, it was reported that T peripheral helper (TPH) cells and Th10 cells, which attract much attention and are important for B cell help, produced IFN-g and expressed CXCR3 and T-bet (6,7). Recent studies have emphasized the roles of T-bet + B cells in SLE pathology, such as the production of autoantibodies and renal involvement (8)(9)(10)(11). TPH cells and Th10 cells were known to be correlated with T-bet + B cells in SLE (6,12).
T-bet is known as a master transcription factor that regulates Th1 differentiation, and it was reported previously that T-bet plays a positive role in the regulation of IFN-g production by effector Th1 cells (13,14). Interestingly, various new functions of T-bet as a transcription factor have been recognized in recent years, including the presence of T-bet + Foxp3 + T reg , which specifically inhibits Th1 in mice (15) and inhibition of aberrant autocrine type I IFN and its downstream signaling in Th1 (16). However, the role of T-bet-expressing CD4 + cells, i.e., Th1 subsets, in SLE pathology remains unknown.
During the process of tissue infection, lymphocytes, including Th cells, rapidly change into the effector phase upon antigen stimulation. These activities require massive energy and rapid synthesis of biological components using amino acids, lipids, and nucleic acids (17). Recent studies in rodents have demonstrated enhanced glycolysis under aerobic conditions associated with such anabolic processes in the activation of various immunocytes (18)(19)(20)(21). The differentiation regulation mechanism through immunometabolism involved in Th cell differentiation is being clarified in many studies mainly in mice; however, it is largely unknown in human, particularly in the pathology of autoimmune diseases, such as SLE.
The aim of this study was to shed light on the pathogenic process of SLE. Specifically, we determined the changes in Th1 subsets in SLE patients and the roles of these cells in the differentiation regulation mechanism via immunometabolism.

Patients
PBMCs were obtained from patients with SLE and healthy subjects. The clinical characteristics of the patients are summarized in Table 1.

Lactate Assay
CD45RAmemory CD4 + T cells were cultured alone or under stimulation/treatment for 3 days in 96-well plates. The culture medium was later collected and diluted properly for measurement of lactate concentration using Lactate Assay Kit II (BioVision, Milpitas, CA), and the protocol supplied by the manufacturer.

Cytokine Production
IFN-g, IL-2, IL-4, IL-6, and IL-17 levels in the culture media were determined by the BD Cytometric Bead Array human Flex set, according to the instructions provided by the manufacturer (BD PharMingen).

Intracellular ROS Levels
After 3-day culture, CD45RAmemory CD4 + T cells were gently washed twice with 37°C PBS, then incubated with 100 ml of 1X DCFH-DA medium solution at 37°C for 60 min. They were repeatedly washed thereafter with PBS at 37°C, then cultured for 5 min in wells each containing 100 ml of culture medium with 100 ml of 2X Cell Lysis Buffer. Finally, 150 ml of the culture mixture was transferred to a 96-well plate black culture fluorometric plates. Fluorescence was read with a fluorometric plate reader at 480/ 530 nm.

Extracellular Flux Analysis
The XF96 Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA) was used to quantify the oxygen consumption rate (OCR). CD45RAmemory CD4 + T cells stimulated for 3 days were resuspended in XF media and then plated on XF96 cell culture microplates (2 × 10 5 cells per well) coated with Cell-Tak (BD Biosciences). The OCR was measured in XF media (Agilent Technologies, Santa Clara, CA) supplemented with 1 mM sodium pyruvate, 10 mM glucose, and 2 mM L-glutamine under both basal conditions and in following the addition of 2 µM oligomycin, 2 µM carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP), and rotenone/antimycin A (Rot/AA).

Electron Microscopy
Cells were collected and immersed in 2% glutaraldehyde solution at 4°C for 4 h. The samples were washed three times with 1 mol/L phosphate buffer, followed by 1% osmium tetroxide for 2 h, dehydrated in graded concentrations of alcohol (50, 70, 80, 90, and 100%), and embedded in epoxy resin. The embedded samples were then cut into ultra-thin serial sections (80 nm, Ultramicrotome, Leica UC-7, Leica, Germany) and stained with lead citrate and uranyl acetate. The samples were subsequently visualized using an electron microscope (JEOL, JEM-1200EX, Japan) at 80 kV at the Department of Electron Microscopy Center of University of Occupational & Environmental Health.

Gas Chromatography Mass Spectrometry
2×10 5 CD45RA -CD4 + cells metabolome under different conditions were extracted with 50% acetonitrile-water. Dried powder was dissolved in 20 mg/ml pyridine in methoxylamine-HCl, followed by N-Methyl-N-(trimethylsilyl)trifluoro-acetamide (MSTFA) reagent. The metabolome was identified using GC/MS (JMS-Q1500, 7890GC, JEOL, Japan) equipped with a direct capillary column DB-5MS sized 30 m×0.25 mm×0.25 mm film thickness. The column temperature was set at 80°C for 2 min and gradually increased at 15°C/min until it reached 320°C then held for 12 min. The temperature of the injector was adjusted to 230°C and the MS transfer line was adjusted to 250°C. Helium gas was used as the carrier gas, which was injected at a constant flow rate of 1.5 ml/min. In the next step, 1 ml of the sample was injected with a solvent delay at 2 min. An autosampler (7650A) was coupled with the GC and set in split mode for automatic injection of the samples and solvents. The effluent of GC column was transferred directly into the source of the MS through the transfer line. Electron ionization mass spectrometry fragments were initiated at 70 eV within the range of 30-500 m/z at a full scan mode. The temperature of the ion source was adjusted to 200°C. NIST mass spectral libraries were used to identify the obtained mass spectra of the active molecules in the extract.

Statistical Analysis
All data were expressed as mean ± SD, unless otherwise indicated. Differences between groups were examined for statistical significance by the paired-or unpaired-t test. Pearson correlation coefficient was used to test the relation between two variables of interest. A p-value of <0.05 denoted the presence of statistical significance. Statistical analyses were conducted using the Prism software (Prism Software, Irvine, CA).

Accumulation of IFN-g Producing-CXCR3 lo T-bet hi Effector Memory CD4 + Cells in SLE
First, we used T-bet, CXCR3, and IFN-g, which are originally known as typical markers of Th1 cells, to examine possible abnormalities of Th1 cells in the peripheral blood of SLE patients. Table 1 summarizes the clinical background of the Japanese 60 participating patients. They included 5 males and 55 females, with a mean disease duration of 142 months, SLEDAI of 11.5 ± 9.0, BILAG 12.9 ± 11.0, clinical relevant organ involvement (lupus nephritis 33.3%, central nervous system 23.3%). We also enrolled 31 age-and gender-matched healthy donors (the control group) ( Table 1).
CD4 + T cells were separated into three populations of CXCR3 -T-bet -, CXCR3 hi T-bet lo , and CXCR3 lo T-bet hi cells. The percentage of CXCR3 lo T-bet hi cells among CD4 + T cells was significantly higher in SLE than the control (HD: 1.9 ± 2.8%, SLE: 5.6 ± 8.5%) ( Figure 1A). CXCR3 -T-betcells mainly consisted of CCR7 + CD45RA + naïve cells, CXCR3 hi T-bet lo cells consisted of CCR7 + CD45RAcentral memory cells and CCR7 -CD45RAeffector memory cells, and CXCR3 lo T-bet hi cells consisted mainly of CCR7 -CD45RAeffector memory cells ( Figure 1B). CXCR3 lo Tbet hi cells which did not express CD28, were CXCR5 -CCR6cells ( Figures 1C, D). The percentage of HLA-DR + CD38 + cells among CD4 + T cells was significantly higher in SLE than the control. In addition, the percentage of HLA-DR + CD38 + cells among CD4 + T cells was significantly higher in CXCR3 lo T-bet hi cells than CXCR3 -T-betand CXCR3 hi T-bet lo cells in SLE ( Figures 1E, F). CXCR3 lo Tbet hi cells were Foxp3cells ( Figure 1G). CXCR3 lo T-bet hi cells showed high potential for IFN-g production selectively in patients with SLE but not in the control ( Figure 1H).
Next, we examined the relation of CXCR3 lo T-bet hi cells to the clinical background of patients with SLE. The results showed that disease duration and treatment resistance were the parameter that significantly related to the percentage of CXCR3 lo T-bet hi cells (Figure 2).
High IFN-g Producing T-bet + Foxp3 lo Non-Suppressive Cells and Low IFN-g Non-Producing T-bet + Foxp3 hi -Activated-T reg Cells Percentages in SLE Next, we analyzed the changes in T-bet + Foxp3 + cells in SLE patients. The clinical characteristics of the patients are summarized in Supplementary Table 1. For this purpose, peripheral blood FoxP3 + CD4 + cells were divided to three subsets of CD45RA + Foxp3 lo naïve-T reg , CD45RA -Foxp3 lo non-suppressive cells, and CD45RA -Foxp3 hi activated-T reg (22). There were no differences in the percentages of CD45RA + FoxP3 lo naïve-T reg , CD45RA -FoxP3 hi activated-T reg between SLE and the control. However, the percentage of CD45RA -FoxP3 lo non-suppressive cells was significantly higher in SLE than the control ( Figure 3A). Considering in detail of these populations, the percentage of IFN-g producing-T-bet + cells among CD45RA -Foxp3 lo non-suppressive cells was higher while the percentage of IFN-g non-producing-Tbet + cells among CD45RA -FoxP3 hi activated-T reg cells was lower in patients with SLE, compared to the control (Figures 3B, C).
Activation of mTOR, which is known to induce various anabolic processes, such as aerobic glycolysis, is important in the maintenance of a balance between T eff and T reg differentiation (23). Phosphorylation of mTOR in peripheral CD4 + T cells of patients with SLE was significantly higher than the control. Phosphorylation of mTOR was significantly correlated with T-bet expression in CXCR3 lo T-bet hi CD4 + cells, but not in total CD4 + cells ( Figure 3D). These results suggest that activation of mTOR can affect the imbalance between Th1 subsets.

Rapamycin Induced IFN-g-Producing-T-bet+Foxp3lo Cells, Whereas 2DG Induced IFN-g-Non-Producing-T-bet+Foxp3hi Cells by Different Effects on Lipid Metabolism
In the next step, we examined in vitro the effects of rapamycin, a mTORC1 inhibitor, and 2DG, a glycolysis inhibitor, on cell metabolism, differentiation, and function in stimulated memory CD4 + cells obtained from HDs, as only a few CD4 + CD45RAmemory T cells were obtained from the patients with SLE. Stimulation with anti-CD3 Abs and anti-CD28 Abs induced mTOR phosphorylation and T-bet expression selectively in CD45RA -CD4 + memory cells ( Figure 4A). The addition of rapamycin or 2DG reduced the percentage of CD4 + CD25 + Foxp3 -T eff cells and increased CD4 + CD25 + Foxp3 hi T reg cells among CD45RA -CD4 + cells, with a resultant significant increase in the For T-bet and IFN-g staining, PBMCs were incubated with PMA (50 ng/ml, ionomycin (1 mg/ml) and breferdin (2.5 mg/ml) for 1 h at 37°C. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Iwata et al. Enhanced Lipid Metabolism in Th1
Frontiers in Immunology | www.frontiersin.org November 2020 | Volume 11 | Article 593103 T reg /T eff ratio (Supplemental Figure 1A). BACH2, IRF4, PRDM1, and EZH2 are transcriptional factors important for the differentiation and function of effector T reg cells (24)(25)(26)(27). Both rapamycin and 2DG induced upregulation of BACH2, IRF4, PRDM1, and EZH2 in CD45RA -CD4 + cells (Supplemental Figure 1B). Evaluation of the changes in Th1, Th2, and Th17 cytokines demonstrated that rapamycin and 2DG had different effects on these cytokines, especially their effects on Th1 cytokines. For example, anti-CD3 Abs and anti-CD28 Abs-stimulated IFN-g was more strongly inhibited by 2DG than by rapamycin. In contrast, interleukin (IL) 2 was not induced by anti-CD3 Abs and anti-CD28 Abs stimulation but strongly induced only by 2DG (Supplemental Figure 1C). Next, we assessed the effects of rapamycin and 2DG on T-bet + Foxp3 + cells differentiation. Rapamycin induced IFN-gproducing T-bet + Foxp3 lo cells. On the other hand, 2DG induced IFN-g-non-producing T-bet + Foxp3 hi cells (Figures 4B, C). Both rapamycin-induced T-bet + FoxP3 lo cells and 2DG-induced Tbet + FoxP3 hi cells produced IL-10, and the production tended to be slightly larger in the latter ( Figure 4D). Since rapamycin and 2DG have different effects on T-bet + Foxp3 + cell differentiation, we next examined the mechanism from the perspective of cell metabolism using CD4 + CD45RAmemory T cells obtained from HDs. Although rapamycin and 2DG had similar effects on the dynamics of cellular metabolism, such as aerobic glycolysis, mitochondrial function and glutaminolysis (Supplemental Figures 2, 3), their effect on lipid metabolism was different. The expression levels of fatty acid oxidation-related enzymes such as CPT1a and CPT2 were increased by both rapamycin and 2DG, while the expression levels of fatty acid synthesis-related enzymes such as FASN and SREBF1 were more highly increased by rapamycin compared with 2DG. This effect of rapamycin was coupled with larger increases in palmitic and stearic acid levels compared with 2DG, suggestive of enhancement of lipid metabolism, selectively in fatty acid synthesis (Figures 4E-G).
These results indicate that rapamycin, but not 2DG, enhanced lipid metabolism, resulting in distinct effect in T-bet + Foxp3 + cell differentiation and its IFN-g production.

Inhibition of Fatty Acid Synthesis in Memory CD4+ Cells Obtained From SLE Patients Resulted in Suppression of IFN-g Production and Up-Regulated Foxp3 Expression in T-bet+Foxp3+ Cells
We also investigated the role of dyslipidemia in the observed changes in T-bet + Foxp3 + cell differentiation and IFN-g production by memory CD4 + T cells of HDs and SLE patients. The results showed that stimulation of the memory CD4 + T cells with anti-CD3 Abs and anti-CD28 Abs induced IFN-g-producing  T-bet + Foxp3 + cells in SLE samples ( Figure 5A). Furthermore, the addition of rapamycin increased IFN-g-producing T-bet + Foxp3 + cells in HDs cells. However, unlike the results in HDs cells, IFN-gproducing T-bet + Foxp3 + cells remained unchanged by the addition of rapamycin in SLE patients ( Figures 5B, C). Moreover, IFN-gproducing T-bet + Foxp3 + cells did not change following the addition to rapamycin of etomoxir, a fatty acid b oxidation inhibitor, but was significantly inhibited by C75, a fatty acid synthesis inhibitor in both HDs and SLE patients ( Figure 5B, Supplementary Figure 4A). The addition of C75 to rapamycin increased Foxp3 expression in Tbet + Foxp3 + cells to a level similar to that seen with 2DG ( Figure 5C, Supplementary Figure 4B). Finally, we evaluated the effects of various metabolic regulators on the production of IFN-g and IL-2, which are two Th1 cytokines produced by memory CD4+ T cells in HDs and SLE. IFN-g production induced by anti-CD3 Abs and anti-CD28 Abs stimulation was inhibited by rapamycin and 2DG and further inhibited by the combination of C75 plus rapamycin in both HDs and SLE patients ( Figure 5D, Supplementary Figure 4C). Interestingly, 2DG, but not anti-CD3 Abs and anti-CD28 Abs stimulation, induced IL-2 production in both HDs and SLE patients ( Figure 5D).

DISCUSSION
We investigated the changes in Th1 subsets in SLE and their involvement in SLE pathology. Our results showed abundance of T-bet hi CXCR3 lo effector cells and T-bet + Foxp3 lo non-suppressive cells (which produce large amounts of IFN-g) in SLE, compared with deficiency of T-bet + Foxp3 hi activated-T reg cells (which do not produce IFN-g). These changes were considered to be involved in treatment resistance. In the in vitro arm of the study, we showed that treatment of stimulated memory CD4 + cells with rapamycin and 2DG resulted in suppression of T-bet + Foxp3cells and induction of T-bet + Foxp3 +(lo/hi) cells. Interestingly, rapamycin alone enhanced lipid metabolism and induced IFN-g-producing T-bet + Foxp3 lo cells, whereas 2DG induced IFN-g-non-producing T-bet + Foxp3 hi cells. In memory CD4 + cells of SLE patients, inhibition of fatty acid synthesis suppressed IFN-g production and enhanced Foxp3 expression in T-bet + Foxp3 + cells. Thus, our results demonstrated that SLE is associated with IFN-g overproduction by Th1 cells and Th1 subset imbalance due to metabolic abnormalities including enhanced fatty acid synthesis.
In the 1990-2010, SLE pathology was discussed in the context of a balance between Th1 and Th2, together with the involvement of Th1 (4,5). However, Th1 is only defined as an IFN-g-producing cell, and other important markers for Th1 such as CXCR3 and T-bet, have not been simultaneously examined in these papers. Recently, the diversity of Th cells has been reported, and CXCR3, T-bet, and IFN-g have been reported to be expressed not only in Th1 but also in other diverse Th subsets. In particular, it was reported that TPH and Th10, which attract much attention and are important for B cell help, produced IFN-g and expressed CXCR3 and T-bet (6,7). In our study, the simultaneous confirmation of CXCR3, T-bet, and IFN-g expression in CD4 + cells revealed that CXCR3 lo T-bet hi cells, rather than CXCR3 hi T-bet lo cells, overproduced IFN-g and was closely involved in the pathogenesis of SLE, including treatment resistance (Figures 1, 2). In addition, our results suggest that not only the aerobic glycolysis but also fatty acid synthesis may be involved in the subset imbalance and overproduction of IFN-g in Th1 cells ( Figure 5D).
In 2002, Gergely and colleagues (28) were the first to describe the metabolic changes in CD4 + T cells in SLE, including increased mitochondrial membrane potential (hyperpolarization) and increased ROS production. Activation of mTORC1 in CD4 + T cells has also been reported in a lupus mouse model as well as in SLE patients (29,30), suggesting that differentiation to Th1 and Th17 is enhanced by mTORC1 in wild-type and autoimmune mice. Previous studies also discussed the importance of lipid metabolism in Th17 differentiation (31). Furthermore, various metabolic abnormalities have been described in CD4 + T cells of SLE patients, but many aspects of SLE pathology remain unknown (32,33). Our study highlighted the pathogenic roles of IFN-g overproduction and imbalance of T-bet + Foxp3and T-bet + Foxp3 + cells, and that abnormal fatty acid synthesis is an important metabolic abnormality mediating the above changes.
The functional properties of mTORC1 include enhancement of mRNA translation, ribosome biogenesis, and glycolysis in the downstream, as well as inhibition of catabolic metabolism, such as autophagy, fatty acid oxidation, and oxidative phosphorylation in the mitochondria (34,35). Rapamycin is known as an mTORC1 inhibitor whereas 2-deoxy-D-glucose (2DG) is a glycolysis inhibitor. Studies in experimental animals have demonstrated that mTORC1 activity and inhibition of aerobic glycolysis by rapamycin and 2DG are associated with suppression of T eff cell and induction of T reg cells (36)(37)(38). However, the effect of rapamycin and 2DG to human CD4 + T cells, especially memory CD4 + T cells remained unclear. In addition, there is no consensus on the role of mTORC1 in the induction of T reg differentiation (39,40). In related studies, it was demonstrated that T reg cells induced by selective mTORC1 deficiency by deletion of TSC and phosphatase and tensin homolog (PTEN) and persistent activation, lacked immunosuppressive capacity (40,41). Furthermore, mTORC1 deficient mice exhibited inhibition of T reg induction, setting a series of inflammatory processes (42). Several studies reported that T-bet + T reg cells inhibit Th1 differentiation and Th1-related autoimmunopathologies (15,(43)(44)(45)(46), while others indicated that these cells do not possess such suppressive activity (47). Thus, fractionation of (T-bet + ) Foxp3 + CD4 + cells includes populations of plastic and non-functional or rather proinflammatory cells. It is suggested that rapamycin may promote the differentiation of these cells.
It has been reported that CD45RA -Foxp3 lo non-suppressive T cells are increased in SLE patients and that these cells produce IFN-g and IL-2 and exhibit little suppressive activity (48). On the other hand, CD45RA -Foxp3 hi cells (activated-Treg) have not been found to be consistent (48,49). In the present study, CD45RA -Foxp3 lo non-suppressive T cells were increased in patients with SLE as previously reported ( Figure 3A). By focusing on Th1 markers such as T-bet, IFN-g, we found that T-bet + IFN-g + CD45RA -Foxp3 lo nonsuppressive T cells were increased, while T-bet + IFN-g - Frontiers in Immunology | www.frontiersin.org November 2020 | Volume 11 | Article 593103 Foxp3 hi CD45RAactivated-Treg cells were decreased ( Figures 3B,  C), and the imbalance of these subsets was caused by enhanced fatty acid synthesis (Figures 5B, C).
Although the mTOR signaling inhibitors, including rapamycin, have been reported to be important metabolic control drugs in SLE patients, it has limited effectiveness (50). Our study demonstrated that, unlike rapamycin, 2DG induced IFN-g non-producing T-bet + Foxp3 hi activated-T reg cells differentiation and IL-2 production. In this regard, previous studies reported that a decrease in serum IL-2 level in SLE patients is associated with inhibition of T reg function with consequent enhancement of inflammatory pathology (51,52). In fact, previous studies showed that administration of a small amount of soluble IL-2 was associated with an increase in T reg cells with a resultant control of SLE disease activity (53). Evidence suggests that the primary cells that produce IL-2 are activated Th cells (54,55). Our study demonstrated that 2DG induced the production of IL-2 from memory CD4 + cells in patients with SLE ( Figure 5D), suggesting that 2DG does not only correct the abnormality of Th1 subsets through the control of abnormal cell metabolism but also improve the pathological status of SLE through the induction of IL-2 production.
Limitations of this study were that a limited number of memory CD4 + T cells can be isolated from the peripheral blood in patients with SLE, and therefore, detailed analysis of the mechanisms occurring in the memory CD4 + T cells derived from the patients was unfeasible. Furthermore, the annual incidence of SLE without treatments for the disease is too limited to allow us to perform these studies.
Taken together, SLE patients exhibited IFN-g overproduction by Th1 cells and subset imbalance of these cells, suggesting the involvement of Th1 cells in SLE pathology such as treatment resistance. Metabolic regulators, particularly fatty acid synthesis inhibitors, may be therapeutically beneficial in SLE by correcting these abnormalities.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article (and its supplementary information files).

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Human Ethics Review Committee of the University of Occupational and Environmental Health, Japan, and each subject provided a signed consent form (H29-045). The patients/participants provided their written informed consent to participate in this study.