IL-27 enhances peripheral B cell glycolysis of rheumatoid arthritis patients via activating mTOR signaling

Our previous study found that increased serum IL-27 could promote rheumatoid arthritis (RA) B cell dysfunction via activating mTOR signaling pathway. This study aimed to explore the effects of IL-27 on B cell metabolism and clarify the mechanisms via which IL-27 enhancing glycolysis to induce B cells hyperactivation. Peripheral CD19+ B cells were purified from healthy controls (HC) and RA patients and then cultured with or without anti-CD40/CpG and glycolysis inhibitor 2-deoxy-D-glucose (2-DG) or mTOR inhibitor rapamycin. Furthermore, the isolated CD19+ B cells were treated by HC serum or RA serum in the presence and absence of recombinant human IL-27 or anti-IL-27 neutralizing antibodies or 2-DG or rapamycin. The B cell glycolysis level, proliferation, differentiation and inflammatory actions were detected by qPCR, flow cytometry or ELISA. We found that the glycolysis in RA B cells was increased significantly compared with HC B cells. Glycolysis inhibition downregulated the proliferation, differentiation, and inflammatory actions of RA B cells. RA serum and IL-27 promoted B cell glycolysis, which could be obviously rescued by anti-IL-27 antibodies or mTOR inhibitor rapamycin. Our results suggest that the enhanced cellular glycolysis of RA B cells induced by IL-27 may contribute to B cells hyperactivation through activating the mTOR signaling pathway.


Introduction
Rheumatoid arthritis (RA) is a systemic in ammatory autoimmune disease characterized by an increase in circulating auto-antibodies, in ammatory cytokines and chemokines, and altered metabolism, which leads to chronic in ammation and irreversible joint or systemic organ damage [1]. Immunologically, the identi ed risk factors for the disease are disrupted immune cell activation and cytokine production.
Immunotherapeutic strategies have improved clinical outcomes and increased our understanding of RA pathogenesis, but there is no cure as of yet [2]. Further pathogenetic discoveries are necessary for the future of RA therapeutics.
The autoreactive B cells play an important role by secreting auto-antibodies and pro-in ammatory cytokines in the pathogenesis of RA, which largely depends on multiple cytokines and the interaction with T cells [3,4]. The ability of activated lymphocytes to engage in in ammation may be determined by metabolic reprogramming to glycolysis enhancement [5]. Increasing evidence have shown that B cell development, differentiation, and function require speci c metabolic adaptations in response to a complex signaling network in different environments [6]. According to the reports, the mammalian target of rapamycin (mTOR) is a key kinase for glucose uptake and glycolysis, which is essential for B cell proliferation, differentiation, and biological function [7][8][9]. Inhibition of mTOR with rapamycin markedly inhibits B cell proliferation and antibody responses in mice and humans [10]. PD-1 + B cells showed increased activation of mTOR and glycolytic capacity and expression of proin ammatory cytokines in RA [11]. Activated B cells differentiated into plasmablasts to produce Ig and proin ammatory cytokines, which were accompanied by enhanced mTOR activation as well as glycolysis [12][13][14]. Inhibition of glycolysis ameliorate arthritis in adjuvant arthritis rats [15]. Therefore, studying B-cell metabolism might provide new insights into the pathogenesis of RA.
Interleukin (IL)-27 is a soluble cytokine of the IL-12 family and plays pleiotropic roles in regulating in ammatory responses by activating JAK/STAT and p38 MAPK signaling pathways via binding to IL-27 receptor (IL-27R) [16]. Circulating IL-27 is elevated in RA patients and can in uence RA development by regulating synovial broblasts and various immune cell responses [16][17][18]. Our previous study has demonstrated that increased IL-27 could promote RA B cell dysfunction via activating the mTOR signaling pathway [19], which indicates that IL-27 signaling may induce RA B cell hyperactivation by controlling cellular metabolism. However, further researches still need to be done.
In the present study, we aim to analyze the glycolysis levels in peripheral B cells of RA patients and investigate the mechanisms via which IL-27 enhancing glycolysis to regulate B cell biological function. Our results will provide new insight into the understanding of aberrant B cell response contributing to RA pathogenesis.

Patients and healthy controls
The blood samples in this study were collected from RA (n=52) patients in the rheumatology department of the Second A liated Hospital of Dalian Medical University. Patients with other autoimmune diseases and metabolic diseases were excluded. At the same time, physical examination subjects of matched gender and age were selected as the healthy controls (HC, n=45) in the physical examination Center of the hospital. Serum samples of RA patients and HC were stored at -80℃. This study was approved by the Ethics Committee of the Second A liated Hospital of Dalian Medical University, and all participants signed informed consent. Clinical and laboratory characteristics of RA patients for study are listed in Table S1.

Sorting and stimulation of B cells
Peripheral blood mononuclear cells (PBMCs) were isolated with the Ficoll density-gradient centrifugation from RA patients and HC blood samples. In this study, the CD19 + B cells were puri ed from PBMCs (>95% purity) based on the manufacturer's instructions (Miltenyi, Germany). In order to study the impacts of human serum on B cells, CD19 + B cells (2.5*10 5 /well) from HC were cultured in RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS), 2% HC serum, or 2% RA serum in the presence of 0.5 mg/ml anti-CD40 antibody (R&D, USA) and 0.1 mM CpG (Miltenyi, Germany) for 24 h. To explore the process of metabolic reprogramming, CD19 + B cells (2.5*10 5 /well) from HC or RA patients were stimulated with anti-CD40 antibody/CpG in the presence or absence of 50 ng/ml recombinant human IL-27 (Peprotech, USA) for 24 or 72 h in 48-well plates. In some experiments, 50 mg/ml anti-IL-27 neutralizing antibodies (RD, USA), 1 mM 2-deoxy-d-glucose (2-DG, Solarbio, China), or 1 nM rapamycin (Solarbio, China) were added into culture system.

Flow cytometric analysis
Flow cytometric analysis was performed on CD19 + B cells. Brie y, cells were washed and suspended in 100 ml of PBS and stained with surface antigens for 30 minutes at 4℃. For intranuclear staining, cells were xed and permeabilized with an intracellular xation and permeabilization buffer set (eBioscience, USA) for 1 h followed by intranuclear staining for another 30 minutes at 4℃. The frequencies of staining-

Proliferation assays
CD19 + B cells were labeled with 2.5 mM carboxy uorescein diacetate succinimidyl ester (CFSE) (eBioscience, USA) in PBS for 10 minutes at room temperature. An excess of ice-cold RPMI 1640 medium with 10% FBS was added to the cells to quench the reaction and cells were washed extensively. CFSElabeled cells (1*10 5 /well) were cultured according to above-mentioned methods. Following 5 days of culture, cells were collected and then stained with APC-CD19 antibody. B cell proliferation was determined by ow cytometry analysis of CFSE uorescence intensity.

Glucose uptake measurement
Glucose uptake ability of PBMCs or CD19 + B cell treated as above-mentioned method were measured with uorescent D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG, Sigma, USA). Cells were incubated with 2-NBDG at a concentration of 50 mM for 30 minutes at room temperature. Then, cells were stained with APC-CD19 antibody for another 30 minutes. Cells were washed three times in PBS and acquired in a ow cytometry instrument, using the FITC channel to detect the signal from the uorescent glucose uptake tracker.

RNA extraction and quantitative PCR (qPCR)
Total RNA was extracted from CD19 + B cells using Trizol reagent (Accurate Biotechnology Co., Ltd, China), and cDNA was transcribed using a SuperScript II RT kit (Applied Biosystems, China) according to manufacturers' instructions. Expression levels of each gene were determined by qPCR using speci c primers, and mRNA levels in each sample were normalized to the relative quantity of b-actin gene expression. The primers used in the study are listed in Table S2. 2.7 ELISA CD19 + B cells were cultured accordingly, secreted IgG and IgM in the culture supernatants were quantitated by ELISA (SenBeiJia Bio, China) according to the instructions of the manufacturer.
Meanwhile, the IL-6 and IL-10 in supernatants were also detected by ELISA kits (RD, USA).

Lactate assay
CD19 + B cells were cultured accordingly and the supernatants were collected. The serum of RA patients and HC as well as the culture supernatants were diluted properly for the measurement of lactate levels with a Lactate Assay Kit II (Abbkine Scienti c, China) according to the protocol supplied by the manufacturer.

Statistical analysis
Data were summarized as means ± standard error of the mean (SEM). All statistical analyses were performed with the GraphPad Prism software (GraphPad, USA). All data within and between groups were compared using an independent samples t-test. A p-value<0.05 was considered signi cantly different.

RA peripheral CD19 + B cells display enhanced glycolysis, which contributed to B cell hyperactivities
Metabolic reprogramming is crucial for the survival and speci c functions of immune cells [20]. Our previous studies have shown altered peripheral B cell homeostasis and functions in RA patients [19].
Accordingly, in the present study, we will explore whether RA B cells have glycolysis changes, which induces aberrant RA B cells responses. CD19 + B cells from RA patients or HC were treated with or without anti-CD40/CpG and 2-DG for 24 h (Fig. 1a). The results indicated that RA CD19 + B cells had signi cantly increased expression of glycolysis-related molecules, including glucose transporter (Glut) 1, Glut3, hexokinase (HK) 2, HK3, lactate dehydrogenase (LDH), phosphate fructose kinase (PFK) 1/2 and pyruvate dehydrogenase kinase (PDK)-1 in comparison to HC B cells (Fig. 1b). RA B cells also showed higher glucose uptake capacity than HC B cells with or without anti-CD40/CpG stimulation (Fig. 1c). The levels of lactate in the B cell culture supernatant and plasma of RA patients were signi cantly higher than that of HC (Fig. 1d). Taken together, RA B cells display enhanced glycolysis compared to HC B cells.
To link glucose metabolism to RA B cell function, RA B cells were treated with glycolysis inhibitor 2-DG, a glucose analog, to block glycolysis by inhibiting HK2 activity. We found that 2-DG treatment obviously inhibited B cell proliferation and activation (Fig. 1e). More importantly, the frequencies of CD19 + CD138 + plasma cells (Fig. 1f) and supernatant levels of IgM, IgG and cytokines IL-6 and IL-10 decreased signi cantly in the 2-DG-treated B cell culture system (Fig. 1g). These ndings demonstrate that enhanced glycolysis may promote RA B cell hyperactivities, which can be restored by glycolysis inhibition.
3.2 IL-27 play a key role in RA in ammatory setting which can contribute to enhanced glycolysis of B cells Our previous studies have showed that IL-27 could induce the imbalance of B cell subsets and B cell dysfunction via the activation of mTOR signal. Base on the fact of mTOR regulating glycolysis, in this study, we wonder whether IL-27 can promote the glycolysis of RA peripheral B cells. As expected, we found that IL-27 signi cantly promoted the expression of glycolysis-related molecules and glucose uptake capacity, as well as lactate production of B cells in vitro culture. And anti-IL-27 antibody treatment inhibited IL-27-induced enhanced B cell glycolysis (Fig. 2a-c).
Given that the activities of immune cells are related to the microenvironment, whether IL-27 is a key cytokine in RA in ammatory setting which can contribute to metabolic disorders of B cells. We cultured HC B cells in a medium containing RA or HC serum. The results showed that in comparison to HC serumtreated B cells, RA serum-treated B cells showed signi cantly increased expression of glycolysis-related molecules, enhanced glucose uptake capacity, and up-regulated lactate production in the supernatants ( Fig. 3a-c). More importantly, in the presence of anti-IL-27 antibodies, the expression of glycolysis-related rate-limiting enzymes (Fig. 3d), the glucose uptake capacity (Fig. 3e) and supernatant lactate levels ( Fig.   3f) were all signi cantly decreased in RA serum-treated B cells. The above results indicate that IL-27 play a vital role in RA in ammatory setting which can contribute to enhanced glycolysis of B cells.

IL-27 enhance RA B cell glycolysis via activating mTOR signaling
To explore the relationship between mTOR signaling and glycolysis of RA B cells, we detected gene expressions of glycolysis-related molecules in RA B cells treated with or without mTOR speci c inhibitor rapamycin. Our data showed that compared to untreated RA B cells, rapamycin-treated RA B cells exhibited remarkably reduced expression of glycolysis-related molecules (Fig. 4a), decreased glucose uptake capacity and lactate production (Fig. 4b-c). As a key transcription factor, hypoxia-inducible factor (HIF)-1α plays a crucial role in regulating cellular metabolism by inducing the expression of glycolytic enzymes. We found that rapamycin signi cantly reduced the level of HIF-1α in RA B cells (Fig. 4d). Taken together, mTOR signaling plays a critical role in enhancing RA B cell glycolysis.
We further explored whether IL-27 enhanced glycolysis by activating the mTOR signaling pathway, which ultimately leads to B cell dysfunction. mTOR inhibitor rapamycin was added to IL-27-stimulated B cells culture system. The results showed that the genes expression of glycolysis-related molecules (Fig. 4e), glucose uptake capacity (Fig. 4f) and lactate secretion (Fig. 4g), and HIF-1α expression (Fig. 4h) in B cells induced by IL-27 were signi cantly inhibited in the presence of rapamycin. These results suggest that IL-27 may enhance RA B cell glycolysis via activating the mTOR signaling pathway.

Discussion
In the present study, we found that B cell glycolysis of RA patients was signi cantly increased, which results in B cell dysfunction. In addition, IL-27 contributed to B cell glycolysis enhancement, which could be rescued by the blockade of IL-27 or mTOR signaling pathway. All these results indicated that RA B cell dysfunction might be linked to the glycolysis enhancement induced by IL-27 via mTOR signaling pathway.
Studies have demonstrated that distinct immune cells can utilize different basic metabolic pathways to match their needs for speci c functions depending on the cytokine environment [21]. Our previous study found that IL-27 in RA serum induced mTOR signaling activation in B cells [19]. mTOR is a master regulator of B cell development, survival, and function by promoting the shift from primarily oxidative metabolism to glycolytic metabolism [9,22,23]. Torigoe M and collages found that the enhanced mTOR1 activation and glycolysis is crucial for the differentiation of unswitched memory B cells into plasmablasts and the production of Ig and cytokines [12]. Rapamycin or 2-DG can inhibit the proliferation, differentiation into plasma/plasmablasts and production of IgG and IgM of B cells from Sjögren's syndrome patients [22]. Consistent with these ndings, our present study found that the enhanced glycolysis contributed B cells hyperactivation. However, delicate metabolic reprogramming of different B cell subsets in the chronic in ammatory environment of RA should be investigated in the future.
Although it has been known that glycolysis is crucial for B cell expansion and antibody production, the molecular mechanisms underlying metabolic reprogramming to modulate B cell fate and function remain poorly de ned. Studies reported that the speci c immune responses may be linked to the metabolitesderived post-translational modi cation and speci c enzyme inhibition [24,25]. Lactate, the nal product of glycolysis, has been shown to be proin ammatory by promoting Th17 cell differentiation in autoimmune disorders [25,26]. A variety of metabolic cofactors, such as adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD), can be transcriptional regulators via post-translational modi cation of transcription factors [27]. Various metabolic enzymes, such as GAPDH and lactic dehydrogenase (LDH), have been shown to play non-canonical roles in regulating gene expression, in ammatory cytokine production, cell cycle progression, DNA damage repair, and cell apoptosis [28,29]. Our present results showed that the increased glycolysis induced RA B cells to differentiate into antibodyproducing cells and secrete high levels of IgM, IgG, and IL-6. Whereas, the underlying molecular mechanisms need further research.
IL-27 plays diverse regulatory roles in different immune disorders [30]. Studies indicated that IL-27R de ciency delayed the development and reduced the severity of arthritis in mice by decreasing IFN-γ secretion and the increased IL-27 can contribute to RA development by activating synovial broblast to express high levels of adhesion molecules and chemokines [18,31]. On the contrary, exogenous IL-27 treatment signi cantly attenuated collagen-induced arthritis by decreasing serum IL-6 and IgG2a levels and blocking IFN-γ and IL-17 production of CD4 + T cells [32]. In addition, studies reported that IL-27 signaling could limit Th1 cell-mediated host tissue damage by inhibiting glycolysis [33]. And IL-27R de ciency could lead to severe liver pathology by exacerbating Th1-mediated immune responses via increasing glycolytic metabolism in CD4 + T cells during infection [34,35]. While, the other study found that loss of IL-27 signaling resulted in decreased mitochondrial function, with no corresponding increase in glycolysis of T cells during vaccination [36]. In this study, we found that the serum IL-27 enhanced the glycolysis of B cells via activating mTOR signaling pathway, which led to B cell hyperactivities in RA patients. All these data indicate that it is too simple to de ne a clear-cut role for a speci c cytokine underlying distinct in ammatory situations. The exact role of IL-27 in B cell metabolism and biological effects during the evolution of RA requires more investigation.