Metformin abrogates pathological TNF-α-producing B cells through mTOR-dependent metabolic reprogramming in polycystic ovary syndrome

B cells contribute to the pathogenesis of polycystic ovary syndrome (PCOS). Clinically, metformin is used to treat PCOS, but it is unclear whether metformin exerts its therapeutic effect by regulating B cells. Here, we showed that the expression level of tumor necrosis factor-alpha (TNF-α) in peripheral blood B cells from PCOS patients was increased. Metformin used in vitro and in vivo was able to reduce the production of TNF-α in B cells from PCOS patients. Administration of metformin improved mouse PCOS phenotypes induced by dehydroepiandrosterone (DHEA) and also inhibited TNF-α expression in splenic B cells. Furthermore, metformin induced metabolic reprogramming of B cells in PCOS patients, including the alteration in mitochondrial morphology, the decrease in mitochondrial membrane potential, Reactive Oxygen Species (ROS) production and glucose uptake. In DHEA-induced mouse PCOS model, metformin altered metabolic intermediates in splenic B cells. Moreover, the inhibition of TNF-α expression and metabolic reprogramming in B cells of PCOS patients and mouse model by metformin were associated with decreased mTOR phosphorylation. Together, TNF-α-producing B cells are involved in the pathogenesis of PCOS, and metformin inhibits mTOR phosphorylation and affects metabolic reprogramming, thereby inhibiting TNF-α expression in B cells, which may be a new mechanism of metformin in the treatment of PCOS.

Metformin used in vitro and in vivo was able to reduce the production of TNF-α in B 29 cells from PCOS patient. Administration of metformin improved mouse PCOS 30 phenotypes induced by dehydroepiandrosterone (DHEA) and also inhibited TNF-α 31 expression in splenic B cells. Further, metformin induced metabolic reprogramming 32 of B cells in PCOS patients, including the alteration in mitochondrial morphology, the 33 decrease in mitochondrial membrane potential, ROS production and glucose uptake. polycystic ovaries and chronic oligo-/anovulation, which can lead to infertility 47 (Rotterdam, 2004). Accumulating evidence indicates that PCOS occurs under chronic 48 inflammation, leading to ovarian dysfunction and metabolic disorders (Hu et al., 49 2020). Such inflammation is characterized by elevated levels of inflammatory 50 cytokines (eg, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interferon-γ 51 (IFN-γ)) in the serum, follicular fluid and ovary (Qin et al., 2016;Xiong et al., 52 2011). Published results have reported that CD3 + CD4 + T cells of follicular fluid 53 produce high levels of IFN-γ and IL-2 in women with PCOS and splenic macrophages 54 release more TNF-α in rats with PCOS (Figueroa et al., 2012;Qin et al., 2016). In 55 chronic inflammatory and autoimmune diseases, such as diabetes mellitus, multiple 56 sclerosis, and systemic lupus erythematosus (SLE), pathologic cytokine-producing B 57 cells have been verified to play important roles in these diseases (Arkatkar et al., 2017;58 Jagannathan et al., 2010;Li et al., 2015). In a mouse models of SLE, IL-6 production 59 by B cells drives autoimmune germinal center formation and accelerates disease 60 progression (Arkatkar et al., 2017). B cells expressing IFN-γ inhibit Treg cell 61 differentiation and exacerbate autoimmune experimental arthritis (Olalekan et al., 62 2015). Our previous studies demonstrated that the peripheral proportion and activity 63 of CD19 + B cells were increased in women with PCOS and dehydroepiandrosterone 64 (DHEA)-induced morphological changes to mouse ovaries were prevented by CD19 + 65 B cell depletion (Xiao et al., 2019), demonstrating that CD19 + B cells contribute to 66 the pathogenesis of PCOS. However, the pathogenic role of B cells in PCOS remains 67 unclear and little is currently known about the inflammatory cytokines expression by 68

B cells from PCOS. 69
Metformin is a widely used oral medication to treat type 2 diabetes (T2D) and also 70 involved in other pharmacological actions, including antitumor effect, antiaging effect 71 and neuroprotective effect (Bai et al., 2021;Bharath et al., 2020). Published results 72 have suggested that metformin not only reduces chronic inflammation through 73 reducing hyperglycemia and increasing insulin sensitivity, but it also has direct 74 anti-inflammatory effects. For example, it has been shown that metformin reduces 75 Th17 cytokine production in older subjects (Bharath et al., 2020), decreases the 76 proinflammatory late/exhausted memory B cell subset and B cell TNF-α mRNA 77 expression in obesity and T2D patients (Diaz et al., 2017). The effects of metformin 78 are thought to be mediated primarily through regulation of the activity of adenosine 79 monophosphate-activated protein kinase (AMPK), the mechanistic target of 80 rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K), which are major 81 regulators of metabolic stress responses. Metformin suppresses systemic 82 autoimmunity in Roquin san/san mice through inhibiting B cell differentiation via 83 regulation of AMPK/mTOR signaling (Lee et al., 2017). Metformin inhibits the 84 proliferation of rheumatoid arthritis fibroblast-like synoviocytes via inhibiting mTOR 85 phosphorylation through PI3K/AKT signaling (K. Chen et al., 2019). Meanwhile, 86 metformin exerts inflammation-inhibitory effects by altering intracellular metabolic 87 processes. Recent evidence has suggested that metformin alleviates the 88 aging-associated Th17 inflammation by improving mitochondrial bioenergetics and 89 reducing oxidative stress via lowering ROS production (Bharath et al., 2020). At 90 present, metformin is also widely used among women with PCOS and reduce insulin 91 resistance, increase ovulation and improve clinical pregnancy outcomes (Morley et al., 92 2017). We hypothesize that pathological B cells may exist in PCOS patients, and 93 metformin may alleviate PCOS symptoms by regulating the activity of pathological B 94

cells. 95
Here, we showed for the first time that the increase of pathological B cells 96 producing TNF-α was associated with PCOS. Peripheral blood (PB) B cells from 97 women with PCOS produced higher levels of TNF-α, and the percentage of TNF-α + 98 cells in B cells was positively associated with serum AMH levels. However, the 99 expression of TNF-α in B cells was reduced after oral administration of metformin in

TNF-α production by pathological B cells in women with PCOS 115
Inflammation plays an essential role in the pathogenesis of PCOS. To define 116 PCOS-associated cytokine profiles, we first quantified the levels of serum 117 inflammatory cytokines in 25 women with PCOS and 13 age-matched control subjects. 118 Results showed that the level of TNF-α was higher in women with PCOS ( Figure 1A). 119 Our previous report demonstrated the proportion and activity of CD19 + B cells were 120 significantly increased in women with PCOS (Xiao et al., 2019), thus we questioned 121 whether the abnormal B cells are responsible for the increase of serum TNF-α in 122 women with PCOS. The results showed that B cells isolated from PB of women with 123 PCOS expressed higher amounts of TNF-α protein than those from the controls 124 ( Figure 1B). We also quantified TNF-α expression in B cells after stimulation with B 125 cell-targeted activator (CpG, CD40L plus anti-IgM/IgA/ IgG) by flow cytometry and 126 found that the percentage of TNF-α + cells in CD19 + B cells was notably higher in 127 women with PCOS than that in control subjects ( Figure 1C). More TNF-α was 128 secreted into the culture supernatants ( Figure 1D). AMH is solely secreted by 129 granulosa cells (GCs) of the pre-antral and small antral ovarian follicles and could be 130 an effective indicator for the diagnosis of PCOS (Teede et al., 2019). We analyzed the 131 relationship between the percentage of TNF-α + cell in B cells and the level of serum 132 AMH by Pearson's correlation analysis, and the results showed that the percentage of 133 TNF-α + cells in CD19 + B cells was positively associated with serum AMH levels 134 ( Figure 1E). To further investigate the possible role of TNF-α in GCs, luteinized GCs 135 from healthy individuals were isolated and cultured in RPMI-1640 containing 10% 136 FBS with or without TNF-α. We found that TNF-α could significantly increase the 137 expression of IL-6, IL-8, CCL2 and CCL20 mRNAs in GCs ( Figure 1F). Together, 138 these data indicate that the increase of pathological B cells producing TNF-α is 139 associated with PCOS. 140 were determined by two-tailed Mann-Whitney U-test and data are presented as 189 medians with interquartile ranges. *P < 0.05; **P < 0.01. 190 The online version of this article includes the following figure supplement(s) for 191 figure 2: Source data 2. Data points for graphs in Figure 2 and its supplements.

Metformin inhibits mTOR phosphorylation in pathological B cells 195
Metformin is known to affect AMPK/PI3K/mTOR mediated signaling(K. Chen et al.,196 2019; Lee et al., 2017). In order to explore the molecular mechanism of metformin 197   Figure 4E). However, metformin had no effect on mitochondrial mass ( Figure  248 4F). In addition, ROS production is considered to be a biomarker of mitochondrial 249 respiration. Excessive ROS can induce oxidative stress, ultimately causing 250 mitochondrial dysfunction and Th17 cytokine production (Bharath et al., 2020). crosslinking (Doughty et al., 2006). We would like to know whether metformin 283 affects glucose uptake of pathological B cell in women with PCOS. 2-NBDG is a 284 fluorescent glucose analog, which is commonly used to assess glucose uptake by cells. 285 As shown in Figure 5A The online version of this article includes the following figure supplement(s) for 358 figure 6: Source data 6. Data points for graphs in Figure 6.  The online version of this article includes the following figure supplement(s) for 397 figure 7: Source data 7. Data points for graphs in Figure 7. 398 399

Effect of metformin on B cells from DHEA-induced PCOS mice 400
We further evaluated the effects of metformin on the production of TNF-α, molecular 401 mechanism and metabolism in B cells isolated from spleen of DHEA-induced PCOS 402

mice. The percentage of TNF-α + cells in CD19 + B cells was notably higher in mice 403
with PCOS than that in control groups and metformin inhibited the expression of 404 TNF-α in pathological B cells ( Figure 8A). Simultaneously, metformin significantly 405 increased the phosphorylation level of AMPK (S487) and inhibited the 406 phosphorylation levels of PI3K p85 (Tyr458) and mTOR (S2481) in splenic B cells 407 from mice with PCOS ( Figure 8B). These results were consistent with the results in 408 women with PCOS. We next examined cellular metabolites by mass spectrometry. 409 Metformin increased the accumulation of glycolytic lactate and TCA cycle 410 intermediates (succinate, fumarate, oxaloacetate) in splenic B cells ( Figure 8C-D).  (Xiao et al., 2019). B cells plays a role in chronic inflammatory and 451 autoimmune diseases by secreting proinflammatory cytokines (Shen et al., 2015). 452 Interestingly, we found that peripheral blood B cells from women with PCOS and 453 splenic B cells from DHEA-induced PCOS mice both produced higher levels of 454 TNF-α, and the percentage of TNF-α + cells in human peripheral CD19 + B cells was 455 positively associated with the serum AMH levels. It is reported that TNF-α-deficient 456 B cells were less efficient than wild type B cells to amplify IFN-γ expression by T 457 cells during the course of the Th1 inflammatory response to Toxoplasma gondii 458 infection (Menard et al., 2007). Atherosclerosis is reduced in mice with 459 TNF-α-deficient B cells; B cells-derived TNF-α is a key cytokine that promotes 460 atherosclerosis development through augmenting macrophage TNF-α production to 461 induce cell death and inflammation that promote plaque vulnerability (Tay et al., 462 2016). These findings suggest that pathological TNF-α-producing B cells may 463 contribute to the develop of PCOS by promoting inflammatory response. 464 In this study, we propose a novel mechanism that metformin alleviates PCOS by In summary, we found that pathological TNF-α-producing B cells are involved in 526 the pathological process of PCOS. Metformin inhibits mTOR phosphorylation and 527 affects metabolic reprogramming, and then inhibiting TNF-α expression in 528 pathological B cells (Figure 9). These data presented here may provide new insights 529 into the mechanism of metformin in the treatment of PCOS and specific targeting 530 TNF-α-producing B cells may be a potential therapy. 531  Cell proliferation was measured by loading of cells with 1 M CFSE (eBioscience). 631 All measurements described above including intracellular cytokine levels, cell 632 proliferation, apoptosis, mitochondrial mass and membrane potential, and ROS 633 production as well as 2-NBDG uptake were conducted using a fluorescence-activated 634 cell sorting (FACS) Aria I cytometer (Becton Dickinson) and analyzed using FACS 635  (60 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead 689 citrate using standard methods. Stained grids were examined and photographed using 690 a HT7800 transmission electron microscope (HITACHI). The length and width of 691 mitochondria were calculated using ImageJ software (NIH). 692