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GABA transporter sustains IL-1β production in macrophages

GAT2 links metabolism to DNA methylation to orchestrate macrophage-dependent inflammation.


INTRODUCTION
There is an increasing interest about the link between nervous sys tem and immune system based on the discovery that neurons, neuro transmitters, and/or neuropeptides shape the function of immune cells (1,2). The aminobutyric acid (GABA)ergic system [e.g., GABA, GABA receptors, glutamic acid decarboxylase (GAD), and GABA transporters (GATs)] is a crucial family of the central nervous sys tem. It has been well documented that various peripheral immune cells express GABAergic components, e.g., macrophages express GAD and GABA receptors, and T cells express GAD, GATs, and GABA receptors (3,4). These findings indicate that GABAergic sys tem may be closely associated with the activation and function of immune cells. Notably, more interest has been highlighted on the effect of GATs [i.e., GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and GAT4/BGT1 (SLC6A12)] on T cells. For example, GAT1 deficiency enhances T cell-mediated responses (5,6), and GAT2 deficiency promotes T helper 17 (T H 17) cell differentiation during infection (4). Nonetheless, whether innate immune cells (macrophages in particular) express GATs and the roles of GATs in innate immune cell-mediated responses are still unknown.
Macrophages are of the principal innate immune cells and show high diversity and plasticity in response to microenvironmental variations, thereby exerting diverse functions (7,8). For example, upon stimulation with proinflammatory signals, like lipopolysac charide (LPS) [or plus interferon (IFN)], macrophages produce large amounts of proinflammatory cytokines [e.g., interleukin1 (IL1)] and perform bactericidal and antitumor activities. However, defects in macrophage phenotypic switch, such as the prolonged activation of a proinflammatory property in macrophages, are asso ciated with various macrophageassociated diseases, including sepsis, infectioninduced inflammatory diseases, and highfat diet (HFD)induced obesity (9)(10)(11). Thus, there is an emerging field that attracts increasing attention to study the contributors to shape the polariza tion of macrophages, such as environmental signals, cellular signaling pathways, and cellular metabolism (12,13). However, we are still lack ing a complete understanding of the functions of GABAergic system, especially about GATs, in macrophage polarization and function.

GAT2 deficiency dampens IL-1 secretion from proinflammatory macrophages
We then explored the influence of GAT2 deficiency on the function of activated macrophages. There were lower mRNA expressions of inducible nitric oxide synthase (iNOS), IL1, and IL18 in LPS/ IFN-stimulated KO macrophages compared to the WT control (Fig. 1C). Besides, IL1 (both LPSinduced and LPS/IFN-induced) secretion (Fig. 1D) and the abundance of cleaved IL1 and IL18 were reduced in proinflammatory macrophages with GAT2 abla tion (Fig. 1, E and F). Moreover, small interfering RNA (siRNA) was used to suppress endogenous GAT2, and we found that GAT2 knockdown considerably inhibited IL1 secretion in LPS/IFNstimulated PEMs (fig. S2, A and B). To avoid cellular heterogeneity of conventional PEMs, we next chose ANA1 cells to examine the function of GAT2. GAT2 silencing in ANA1 cells also lowered the LPS/IFN-induced IL1 production ( fig. S2C). GAT2 deficiency significantly lowered IL6 production while had little effect on the production of TNF in LPS/IFN-stimulated macrophages (fig .  S2D). The inconsistent results of IL1 and TNF indicate that GAT2 deficiency affects macrophage function in cytokinespecific manner.
Mammalian target of rapamycin (mTOR) signaling is involved in macrophage polarization and required for IL1 production (17). We found that GAT2 deficiency had little effect on the phosphor ylation of mTOR ( fig. S2E) and 4EBP1 (fig. S2F) while highly en hanced the phosphorylation of S6K (as readout of mTORC1) and Nmyc downstream regulated gene 1 (NDRG1) [as readout of se rum and glucocorticoidinduced kinase 1 (SGK1) activity that is a downstream node of mTORC2 signaling] ( fig. S2F), indicating that the inhibition of LPS (or plus IFN)-induced IL1 by GAT2 de ficiency may through mTORindependent pathways.
We then explored the NFB signaling and NLRP3 inflam masome, both are responsible for the production of IL18 and IL1 in proinflammatory macrophages (18). Notably, GAT2 deficiency inhibited the activation of NFB signaling (based on the lower ratio of pp65/p65) in LPS/IFN-stimulated macrophages (Fig. 1, E and F) and decreased the protein abundance of inflammasome components (e.g., NLRP3 and caspase1) (Fig. 1, G and H). Further more, GAT2 deficiency selectively inhibited the mRNA expression of ASC ( fig. S2G), suggesting that GAT2 deficiency dampens the activation of NLRP3 inflammasome at transcriptional level. Likewise, GAT2 silencing reduced the mRNA expression of ASC ( fig. S2H). Immunoprecipitation and immunofluorescence analysis also showed that GAT2 deficiency in macrophages blocked NLRP3-ASC-caspase1 complex formation, mainly lowering the abundance of ASC (Fig. 1, I and J). These results suggest that GAT2 deficiency negatively mod ulates macrophage NLRP3 inflammasome activation in the context of proinflammatory stimulation.
Considering that GAT2 expression was substantially increased in IL4-stimulated WT macrophages ( fig. S2I), we sought to deter mine whether GAT2 deficiency affects the activation of macrophage stimulated with IL4. GAT2 deficiency reduced the mRNA expres sions of Arg-1 and IL-10 ( fig. S2J) and lowered the protein abun dance of Arg1 and CD206 (fig. S2K); however, it had little effect on IL10 secretion ( fig. S2L). Moreover, GAT2 deficiency had no effect on signal transducers and activators of transcription-6 activation, which is responsible for M2 macrophage polarization ( fig. S2M) (19). Thus, whether GAT2 dictates IL4-stimulated macrophage activa tion remains to uncover. Together, GAT2 deficiency dampens the function of proinflammatory macrophages, especially lowers the secretion of IL1 through blocking NLRP3-ASC-caspase1 com plex formation.

GAT2 deficiency promotes OXPHOS in proinflammatory macrophages
To explore the nature of GAT2 ablation-induced the inhibition of IL1 secretion, we performed RNA sequencing (RNAseq) analysis on LPS/IFN-stimulated macrophages with or without GAT2 de ficiency. There was a significant difference about the transcriptomic profile between KO macrophages and WT macrophages ( Fig. 2A). Further analysis demonstrated that GAT2 deficiency affected totally 959 genes (fold change > 2; P < 0.05; 355 upregulated; 604 down regulated) in LPS/IFN-stimulated macrophages (Fig. 2B), enrich ing in immune system, signaling transduction, and metabolism (list hits > 5; P < 0.05) ( Fig. 2C and fig. S3, A to C). For example, the NFB signaling pathway and NODlike receptor signaling pathway were highly enriched ( fig. S3, A to C), which were consistent with our previous results ( Fig. 1, E to J).
We next assessed cellular bioenergetics with seahorse analysis and found that unstimulated KO macrophages became more "oxi dative" as compared with unstimulated WT macrophages, on the basis of higher OCR (oxygen consumption rates; a measure of OXPHOS) in basal respiration, ATP production, maximal respiration, The basal respiration, ATP production, maximal respiration, and spare capacity of WT and KO resting/proinflammatory macrophages (n = 10). Results represent three independent experiments. Data were analyzed by unpaired t test and represented as means ± SD unless indicated. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. and spare capacity (Fig. 2, G and H). Notably, GAT2 deficiency en hanced OXPHOS in LPS/IFN-stimulated macrophages (Fig. 2I) and seven in IL4-stimulated macrophages ( fig. S3D). However, the results of ECAR (extracellular acidification rate; a measure of glycol ysis) were comparable between KO and WT macrophages ( fig. S3, E to G). Collectively, GAT2 deficiency alters the transcriptomic pro files of LPS/IFN-stimulated macrophages and especially switches proinflammatory macrophage cellular metabolic status toward to the OXPHOS.  S4, A, D, and E). However, NNC052090 (an inhibitor for GAT4) treatment in LPS/IFN-stimulated KO macro phages did not affect the IL1 secretion ( fig. S4H), ruling out the possibility that lower IL1 secretion came from the increased GAT4 expression. Moreover, the involvement of decreased gluta mate in IL1 secretion from LPS/IFN-stimulated KO macro phages was also ruled out on the basis of the results that glutamate supplementation had little effect on IL1 secretion from both WT and KO proinflammatory macrophages ( fig. S4I).
The cellular metabolism is highly associated with the functional output of macrophages (21). To explore the influence of GAT2 de ficiency in cellular metabolic profiles in macrophages, we performed gas chromatography (GC)-3Q-mass spectrometry (MS) method (4) to identify the metabolites between WT macrophages with or without LPS/IFN treatment, between WT and KO resting macro phages, as well as between LPS/IFN-stimulated WT and KO macrophages. There was a significant difference about the cellular metabolites between unstimulated WT macrophages and LPS/ IFN-stimulated WT macrophages ( Fig. 3A and fig. S5A), in which 12 metabolites were upregulated, while 44 metabolites were downregulated after LPS/IFN stimulation (e.g., GABA and suc cinate). These metabolites were enriched in arginine and proline metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, riboflavin metabolism, and caffeine metabolism ( Fig. 3A and fig. S5B). Likewise, there were also signifi cant differences about the cellular metabolite profile between un stimulated WT and KO macrophages ( Fig. 3B and fig. S5, C and D) and LPS/IFN-stimulated WT/KO macrophages ( Fig. 3C and  fig. S5, E and F). Notably, tryosine, hypoxanthine, betaine, deox yguanosine, citraconic acid, glyceric acid, and kynurenic acid were increased in LPS/IFN-stimulated KO macrophages compared to those in WT control (Fig. 3C).
We next studied the effect of these increased metabolites in IL1 secretion from LPS/IFN-stimulated macrophages by adding each of them to the medium during macrophage polarization. Hypoxan thine and deoxyguanosine were decreased in WT proinflammatory macrophages compared to WT resting macrophages (Fig. 3D), sug gesting that hypoxanthine and deoxyguanosine might inhibit the function of proinflammatory macrophages. Deoxyguanosine and especially hypoxanthine decreased the IL1 secretion from both LPS/IFN-stimulated WT and KO macrophages (Fig. 3, E and F). Like GAT2 deficiency, hypoxanthine had little effect on TNF pro duction ( fig. S5, G and H). Moreover, the IL1 secretion was not significantly influenced by other increased metabolites (e.g., kynurenic acid, citraconic acid, and betaine) ( Fig. 3E and fig. S5, I and J). The levels of glycerophosphocholine were reduced in LPS/IFNstimulated KO macrophages (Fig. 3C), but glycerophosphocholine supplementation could not promote IL1 secretion from both WT macrophages and KO macrophages (fig. S5, K and L). Notably, IRG1/ACOD1 (immuneregulated gene 1 or aconitate decarboxy lase 1) mediates itaconate production and is important for regulat ing the proinflammatory state of M1 macrophages (22). Here, we found that GAT2 deficiency mildly inhibited IRG1 expression and itaconate production in LPS/IFN-stimulated macrophages (fig. S5, M and N). Therefore, we envisioned that GAT2 deficiency inhibits IL1 production in LPS/IFN-stimulated macrophages through hypoxanthine.
We next asked whether hypoxanthine could also alter cellular bioenergetics in proinflammatory macrophages. In LPS/IFNstimulated WT macrophages, hypoxanthine (8 or 800 M) treatment promoted OXPHOS (Fig. 3, L and M, and fig. S5, P and Q) but had little effect on glycolysis (fig. S5, R and S), which is similar to GAT2 deficiency ( Fig. 2I and fig. S3G). Hypoxanthine supplementation also substantially increased the mRNA expressions of OXPHOS related genes with dosagedependent manner, including Cox6b1, Sdhc, Ndufa7, and Ndufb9 ( Fig. 3N and fig. S5T). Collectively, GAT2 deficiency decreases IL1 production in proinflammatory macrophages through hypoxanthine.

GAT2 deficiency decreases IL-1 secretion via betaine-hypoxanthine axis
To explore the underlying mechanism by which GAT2 deficiency promotes hypoxanthine accumulation in macrophages, we further analyzed the hypoxanthine metabolism in macrophages since there is a core purine metabolism in proinflammatory macrophages (24). There was a significant alteration about the hypoxanthine metabolism both in unstimulated macrophages and LPS/IFN-stimulated macrophages after GAT2 deficiency, resulting in accumulation of  S6B). GAT2 deficiency also affected the enzyme activity and mRNA expressions of xanthine oxidase (XOD) and xanthine dehydrogenase (XDH) in LPS/IFN-stimulated macrophages, which mediate the metabolism of hypoxan thine into xanthine and the production of reactive oxygen species (ROS) (Fig. 4A and fig. S6C) (25).
Intriguingly, we observed that the intracellular betaine levels were increased in both unstimulated and LPS/IFN-stimulated KO macrophages (Figs. 3, B and C, and 4A). This is not unexpected because LPS/IFN-stimulated KO macrophages have higher ex pression of GAT4 ( fig. S4G), which transports both GABA and . Results represent two (H and J to L), three (B to D), or four (G) independent experiments. Data were analyzed by unpaired t test (A, B, C, D, F, and G) or two-way ANOVA with Bonferroni correction (G to L) and represented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
betaine. GAT2 deficiency also increased GAT4 expression in IL4stimulated macrophages ( fig. S6D), leading to intracellular accumu lation of hypoxanthine ( fig. S6E). Considering that GAT4 blockade and betaine supplementation during M1 macrophage polarization could not alter IL1 secretion (figs. S4H and S5J), thus, we envi sioned that GAT2 deficiency induces intracellular accumulation of betaine in resting macrophages by the compensated expression of GAT4, and betaine is converted into hypoxanthine by PNP to lower IL1 production in LPS/IFN-stimulated macrophages.
To test this, first, betaine was transfected into macrophages be fore LPS/IFN stimulation. Betaine delivery increased intracellular levels of betaine and hypoxanthine and the activity of PNP (Fig. 4, B and C, and fig. S6, F to H). Notably, intracellular transfection with betaine extremely reduced the secretion of IL1 from LPS/IFNstimulated WT macrophages (Fig. 4D) and the intracellular abun dance of inflammasome components, pp65 and IL1 (Fig. 4, E and F). Then, we treated LPS/IFN-stimulated WT macrophages with BCX1777 after betaine transfection; BCX1777 decreased the activity of PNP and intracellular level of hypoxanthine ( Fig. 4G and  fig. S6, H and I) but rescued the production of IL1 (Fig. 4H). Sim ilarly, PNP overexpression in WT macrophages lowered the IL1 secretion, while PNP silencing in KO macrophages rescued IL1 production ( Fig. 4I and fig. S6J).
Subsequently, to support the importance of GAT4 in mediating intracellular accumulation of betaine in macrophages with GAT2 deficiency, we knocked down the GAT4 in KO bone marrowderived macrophages (BMDMs) by means of siRNA delivery ( fig. S6K). The GAT4 silencing in KO BMDMs remarkably rescued IL1 production ( fig. S6L). Subsequently, we knocked down the GAT2, GAT4, and GAT2 combined with GAT4 in RAW264.7 cells, respectively. It should be noted that RAW264.7 cells lack ASC expression (26); thus, the concentration of IL1 in the culture me dium measured by enzymelinked immunosorbent assay (ELISA) is low ( fig. S6M) and may reflect that of pro-IL1 rather than the active form of IL1. Even so, we found that the change of extracel lular IL1 was consistent with those of the intracellular IL1 by Western blot analysis (fig. S6, M and O). Therefore, we used ELISA for measuring the extracellular IL1 in further experiment with RAW264.7 cells. To simulate the physiological situation in vivo, we supplemented ~50 M betaine [20 to 70 M in normal human plasma (27,28); 50 to 100 M in mouse plasma (29)] into medium during the whole transfection and/or polarization progress. The results also demonstrated the critical roles of GAT4 in the intracellular accu mulation of betaine (Fig. 4, J and K) and reduced IL1 production in GAT2deficient macrophages ( Fig. 4L and fig. S6P). Notably, GAT2 silencing without betaine supplementation had lower extra cellular and/or intracellular level of betaine while higher IL1 pro duction in comparison to GAT2 silencing in the presence of betaine, highlighting the irreplaceable role of betaine in determining IL1 production in LPS/IFN-stimulated macrophages (Fig. 4, J to L). Together, our results highlight that GAT2 deficiency boosts betaine hypoxanthine metabolic pathway to lower IL1 production in proinflammatory macrophages.

Hypoxanthine decreases IL-1 secretion via KID3
Then, we want to know the underlying mechanism by which hypo xanthine regulates inflammasome activation and OXPHS in macro phages, especially hypoxanthinemediated expressions of OXPHOSrelated genes. According to our previous observations that the mRNA encoding for Cox6b1, Ndufa7, and Ndufb9 are in creased both in LPS/IFN-stimulated KO and WT macrophages supplemented with hypoxanthine (Figs. 2F and 3N), thus, these genes are the upmost interest in the current study. In silico analysis shown that these genes are regulated by the TF KID3, CPBP, and PRRX2 (Fig. 5A). On the basis of the standard that resting macro phages, LPS/IFN-stimulated KO macrophages and WT macro phages supplemented with hypoxanthine should have similar alteration in the expressions of TFs, compared to LPS/IFNstimulated WT macrophages, we took KID3 as the key TF (Fig. 5,  B and C). Similarly, we also found that GAT2 silencing inhibited KID3 expression (fig. S7A). Of note, BCX1777 treatment signifi cantly increased KID3 expression while decreased Cox6b1, Ndufa7, and Ndufb9 expressions in LPS/IFN-stimulated KO macrophages (Fig. 5D), suggesting that hypoxanthine inhibits the expression of KID3, which negatively regulates the expressions of these OXPHOS related genes. In addition, PNP silencing increased KID3 expression while inhibited Ndufb9 expression; nevertheless, PNP overexpres sion showed contrary results ( fig. S7, B and C). We also demonstrated that hypoxanthine treatment decreased the mRNA expressions of IL1 and KID3 while increased the expression of Ndufb9 in LPS/ IFN-stimulated RAW264.7 cells (fig. S7D).
To further validate the roles of KID3 in regulating mRNA ex pressions of these OXPHOSrelated genes, we transfected WT macro phages and KO macrophages with siKid3 and Kid3containing plasmid to inhibit and enhance the expression of KID3, respectively ( fig. S7E). Like GAT2 deficiency or hypoxanthine supplementation, KID3 inhibition reduced production and expression of IL1 (Fig. 5E  and fig. S7F) and promoted the expressions of both Ndufa7 and Ndufb9 in LPS/IFN-stimulated WT macrophages (Fig. 5F). Similar to PNP inhibition in KO macrophages, KID3 overexpression increased production and expression of IL1 in KO macrophages (Fig. 5E  and fig. S7F) and decreased the expressions of both Ndufa7 and Ndufb9 in LPS/IFN-stimulated KO macrophages (Fig. 5F). These results indicate that the expressions of Ndufa7 and Ndufb9 are neg atively regulated by KID3. Unexpectedly, KID3 silencing reduced the mRNA expressions of NLRP3 and ASC, while KID3 overexpres sion promoted the mRNA expressions of NLRP3 and ASC ( fig. S7G), suggesting that these two genes might be positively controlled by KID3. To identify the role of KID3 in determination of gene expres sion, we conducted dual luciferase report system assay and found that Ndufb9 was negatively modulated by KID3 in a direct way (Fig. 5G), suggesting that KID3 affects OXPHOS by directly target ing the expression of OXPHOSrelated genes. Collectively, hypo xanthine inhibits the expression of KID3, which regulates the expressions of genes associated with OXPHOS and inflammasome.

Hypoxanthine inhibits KID3 through methylation
Next, we sought to reveal the underlying mechanism by which KID3 is inhibited in LPS/IFN-stimulated KO macrophages or hypoxanthine supplementation. We focused on the methylation of KID3 because betaine metabolism is accompanied by the produc tion of SAM (functions as the main methyl donor), which could be metabolized into hypoxanthine (24,30). Notably, GAT2 silencing and GAT2 deficiency improved the intracellular level of SAM in LPS/IFN-stimulated macrophages (Figs. 4A and 5H and fig. S7H). Hypoxanthine increased the intracellular level of SAM in LPS/ IFN-stimulated WT macrophages, while BCX1777 blocked the accumulation of SAM in LPS/IFN-stimulated KO macrophages  t test (B) or two-way ANOVA with Bonferroni correction (C, D to I, L, and N to P). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (Fig. 5H). These results indicated that hypoxanthine positively reg ulates the accumulation of SAM in proinflammatory macrophages. Of note, we found that the relative abundance of MAT2A, the main enzyme that favors in the SAM de novo synthesis from methionine in mammalian cells (31), was much higher in LPS/IFN-stimulated KO macrophages or WT macrophages with hypoxanthine supple mentation but lower in LPS/IFN-stimulated KO macrophages with BCX1777 treatment (Fig. 5I). Likewise, PNP overexpression in WT macrophages and PNP silencing in KO macrophages affected the MAT2A expression ( fig. S7I).
To better understand how hypoxanthine specifically improves the enzymatic activity of MAT2A, we performed in silico docking analysis between MAT2A as receptor and hypoxanthine as the ligand. Intriguingly, we found that hypoxanthine could bind to MAT2A through van der Waals with two amino acid residues (Ser 206 and Arg 249 ), hydrogen bonds with Ser 247 , and orthophosphate (Pi) cation/sigma with Phe 250 of MAT2A (Fig. 5J). It has been well demonstrated that adenosine occupies the active site of MAT2A through several hydrogen bonds with Ser 247 and Arg 249 , as well as  stacking between the purine base of adenine and Phe 250 of MAT2A (32). Adenosine and hypoxanthine have identical chemical properties ( fig. S7J), implying that hypoxanthine could also occupy the active site and hydrogen bond to MAT2A, thereby enhance its enzymatic activity. To firmly validate our hypothesis, we conducted sur face plasmon resonance (SPR) assay about hypoxanthine and human MAT2A protein [since no available commercial recombinant murine MAT2A protein, and the MAT2A is highly conserved between mouse and human ( fig. S7K)]. The results showed that hypo xanthine directly interacted with MAT2A in a dosedependent manner (Fig. 5K).
These data suggested that the DNA hypermethylation may be involved in the reduced expression of KID3 after GAT2 deficiency or hypoxanthine supplementation. This hypothesis had been demon strated by the following evidence: (i) The relative abundance of DNA methyltransferases (i.e., DNMT1, DNMT2, DNMT3a, and DNMT3b) were much higher both in LPS/IFN-stimulated KO macrophages and WT macrophages with hypoxanthine supple mentation, but lower in LPS/IFN-stimulated KO macrophages with BCX1777 treatment (Fig. 5L). (ii) The accumulation of hypo xanthine could enhance DNA total methylation level of KID3 (Fig. 5M). (iii) The specific DNA methyltransferase inhibitors azacitidine and decitabine (33) both highly increased KID3 and ASC expression and IL1 production (Fig. 5, N and O) while in hibited Ndufb9 expression in LPS/IFN-stimulated KO macro phages (Fig. 5O). (iv) The blockage of MAT2A by PF9366 (31) reversed the inhibitory effect of hypoxanthine on IL1 production in WT proinflammatory macrophages (Fig. 5P). In summary, these data illustrate that GAT2 deficiency promotes hypoxanthine accumu lation, which inhibits KID3 expression via the enhanced methylation.

GAT2 deficiency attenuates LPS-induced sepsis, Pasteurella multocida-induced pneumonia, and HFD-induced obesity in mice
We then asked whether GAT2 deficiency suppresses the inflamma tory responses in vivo. We first investigated the effect of GAT2 de ficiency on LPSinduced sepsis (a model that largely reflects the inflammatory functions of monocytes/macrophages). The survival rate of KO mice after intraperitoneal injection of LPS was signifi cantly increased ( fig. S8A). GAT2 deficiency inhibited mRNA ex pressions and abundance of inflammatory cytokines (especially IL1 and TNF) in the jejunum and liver of mice ( fig. S8, B and C). GAT2 deficiency significantly lowered numbers of macrophages (marked by F4/80) in the lung and liver of mice under LPS chal lenge ( fig. S8D). After deletion of macrophages using clodronate loaded liposome, the survival rates of WT and KO mice were comparable under LPS challenge ( fig. S8E).
We next addressed the suppressive role of GAT2 deficiency in inflammation in vivo using a model of P. multocida (PmCQ2)infected pneumonia. Likewise, the KO mice were less sensitive to PmCQ2 infection in overall survival assays (Fig. 6A). GAT2 defi ciency substantially reduced mRNA expressions of IL1 and TNF in the lung of mice (Fig. 6B) and attenuated infectioninduced pneumonia (Fig. 6C) and decreased levels of IL6, IL1, and TNF in the lung and serum (Fig. 6, D and E). Our previous study reported that PmCQ2 infection triggers inflammatory responses in mouse lung depending on macrophages and neutrophils (34). Here, GAT2 deficiency significantly lowered the numbers of macrophages (marked by CD68), rather than neutrophils (marked by Ly6G) in the lung of mice (Fig. 6, F and G). To verify that GAT2 deficiency inhibits host inflammatory responses by regulating the functions of alveolar macrophages, the alveolar macrophages in vivo were deleted using clodronateloaded liposome. In this model, GAT2 deficiency had no effect on the numbers of alveolar macrophages (Fig. 6H) and mRNA expressions and secretion of IL1 in the lung (Fig. 6I) and serum (Fig. 6J) of mice.
The above two models indicate that GAT2 deficiency could mit igate macrophagemediated acute inflammation in vivo; however, whether GAT2 deficiency affects macrophagemediated chronic inflammation (e.g., obesity) is still unknown. To uncover this, we fed mice with a HFD for 12 weeks. As expected, HFDtreated KO mice had a lower body weight, liver weight, and adipose tissue weight than those in HFDtreated WT mice (Fig. 7, A and B). GAT2 deficiency prevented insulin resistance, although no significantly effect on glucose intolerance was observed in mice after HFD treat ment ( fig. S8, F and G). GAT2 depletion reduced the production of inflammatory cytokines in adipose tissues and serum ( Fig. 7C and  fig. S8H), suggesting a suppressive action of GAT2 deficiency on metabolic syndrome-related inflammation in vivo. In addition, GAT2 deficiency remarkably reduced the numbers of macrophages in adipose tissues (Fig. 7D), especially the ratio of iNOS + (M1like): CD206 + (M2like) macrophages (Fig. 7E). Of note, in our experi mental models, GAT2 deficiency decreased TNF level, which is not consistent with the finding in vitro, which is probably due to the contribution of IL1 to TNF production in vivo (35). Collectively, these results indicate that GAT2 deficiency ameliorates macrophage mediated inflammatory responses in vivo.

DISCUSSION
Mounting evidence shows the crosstalk between the nervous sys tem with various immune cells to regulate host homeostasis and immunity. Intriguingly, both central and enteric nervous system can physiologically contribute to the inflammatory responses through different mediators (e.g., neurotransmitters) at homeostasis and during pathogen infection (36,37). However, whether GABAergic system has implications for controlling macrophagemediated in flammatory responses are not well characterized. This study reported that GAT2 affects IL1 production in macrophages. During pathogen infection, sympathetic neurons could polarize muscularis macro phages toward a tissueprotective phenotype (38); nevertheless, the mecha nism whereby sympathetic neurons alter macrophage polarization is not clear. We provided evidence that GAT2 deficiency inhibited M1 macrophage polarization by suppressing inflammasome activa tion and enhancing OXPHOS capacity. In addition, previous study showed that the activation of GABAergic system in macrophages enhances autophagy activation, phagosomal maturation, and anti microbial responses against mycobacterial infection (3).
It is noteworthy that macrophages arise from monocytes and that monocyte migration could be markedly suppressed by GABA B receptor agonist (39); thus, it is interesting to investigate whether GABA signaling is decisive for the development and maturity of Data were analyzed by unpaired t test and represented as means ± SD unless indicated. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. macrophages. Moreover, other GABAergic components, including GABA A R, GAD65, and GABA, were also observed in mouse macro phages (3,15). The impacts of these GABAergic components in macrophage function need further study. As recently reviewed (40), the activation of macrophages increases the levels of the GABAergic components and GABA could induce inward currents (although smaller in amplitude and slower to rise and decay) in macrophages. These findings suggest that GABAergic signaling links to the multifarious functions of macrophages despite the underlying mechanisms remain elusive.
fascinating study has demonstrated that mast cells could express GAT1 (45). These findings support the importance of GABAergic signaling in modulating the biology of innate immune cells. Con sidering that GATs are the pivotal components of GABAergic sys tem and most of the studies have centered on the GATs in T cells thus far, future additional works should be focused on GATs in the modulation of innate immune cell functions (e.g., mast cells). Nota bly, similar to GABA, glutamate (excitatory amino acid), aspartate (excitatory amino acid), and glycine (inhibitory amino acid) can also function as the amino acid neurotransmitters; hence, it is meaningful to further investigate the crosstalk between the nervous system (e.g., glutamatergic and glycinergic signaling) and the im mune system.
A purine nucleotide cycle is active in M1 macrophages, which could supply tricarboxylic acid (TCA) cycle intermediates (e.g., fumarate) to synchronize mitochondrial activity with glycolysis by balancing electron transfer into mitochondria (24). However, we did not find any alterations of TCA cycle intermediates from LPS/ IFN-stimulated KO macrophages in comparison to the WT con trol, indicating that GAT2 deficiency switches cellular bioenergetics of proinflammatory macrophages independent of TCA cycle inter mediates. Our data demonstrated that GAT2 deficiency boosts the levels of intracellular purine metabolites (especially hypoxanthine) to function as the pivotal metabolite to enhance OXPHOS by pro moting Ndufb9 expression in LPS/IFN-stimulated macrophages. Mechanistically, we confirmed that GAT2 deficiency increases PNPmediated intracellular hypoxanthine level by improving beta ine level involving the compensatory increase of GAT4 expression, which serves as the betaine transporter in the cells (15). These data highlight the importance of GABA transporters in directing im mune cell function. Moreover, hypoxanthine can funnel into the salvage pathway for purine nucleotides (46), whether hypoxanthine can support the proliferation of macrophages is still unknown. Likewise, inosine (the metabolic precursor of hypoxanthine) could serve as an alternative carbon source for CD8 + T cell function under glucose restriction (47), whether hypoxanthine could exploit the similar effects in macrophages warrants further investigation.
Masses of C2H2 zinc finger family (the largest class of TFs in mammals) members are key transcriptional regulators involved in immune cell development and function (48)(49)(50). Unexpectedly, we noticed that KID3 (also termed as Zfp354c, one of the C2H2 zinc finger protein) displays discrepant roles in targeting Ndufb9 and ASC to regulate their expression. The possible reasons may be as follows: (i) Other factors that share large amino acid sequence sim ilarity and/or DNA sequence identity with KID3 could also bind to GCrich regulatory regions of Ndufb9 and ASC. (ii) KID3 co operates with epigenetic control mechanisms (e.g., acetylation and methylation) to exert different transcriptional activities. Virtually, emerging evidence propose that epigenetic regulation drives immune responses and that SAM supports proinflammatory macrophages (24,30). We suggested that the occurrence of the enhanced methyl ation evidenced by GAT2 deficiency or hypoxanthine supplemen tation facilitates betaine to generate maximum SAM. Now, we concluded that GAT2 deficiency increases DNA total methylation in the promoter region of KID3 to silence its expression to augment OXPHOSrelated gene (chiefly Ndufb9) expression, thereby lower ing IL1 production in LPS/IFN-stimulated macrophages. How ever, other underlying mechanisms (e.g., cofactors and signaling) connecting to KID3 in orchestrating proinflammatory macrophage function (especially in guiding ASC activation) are not fully elucidated.
It is mentionable that GAT2 deficiency had no effects on IKK and IB while significantly inhibiting p65 phosphorylation, which is probably due to the fact that GAT2 deficiency reduces the activity of preexisting NFB nuclear complexes, inducing transcriptional inactivation (51). ASC is an adapter protein that is required for the formation of the inflammasome, contributing to macrophage acti vation (52)(53)(54). Upon inflammasome activation, ASC forms the ASC speck, a supramolecular aggregate of ASC dimers, which serves as another platform for the activation of Caspase1 (55,56). More over, phosphorylation of ASC is critical for speck formation and caspase1 activation (26). Now, hypoxanthine highly reduces the mRNA expression of ASC, dampening the formation of NLRP3-ASC-caspase1 complex. However, it is interesting to know whether hypoxanthine affects the NLRP3 inflammasome activation through protein posttranslation modification.
We have delineated the influences of systemic knockout of GAT2 in macrophage functions; additional work will be highly help ful by exploring the macrophage functions with Slc6a13 fl/fl LysMcre mice, in which GAT2 was deleted specifically in macrophages (although we have conducted macrophagedeleting experiments). Moreover, although we have validated that GAT2 deficiency enhances betaine/SAM/hypoxanthine metabolic pathway in proinflammatory macrophages, it is critical to perform a stable isotopebased meta bolomic approach to identify the metabolic route of betaine. This study lacks a stable isotopebased metabolomic approach to identify the metabolic route of betaine (e.g., usage of [ 13 C] betaine or [ 15 N] betaine) to hypoxanthine for following reasons: (i) Only the methyl group (CH3) from betaine attaches to the methionine sulfur atom in SAM, which then physically latches on to certain areas along the DNA strand to inactive genes (called "methylation") and cannot participate in the downstream metabolism. (ii) All carbon atoms and nitrogen atoms in hypoxanthine entirely originate from adenosine.
Collectively, we have elucidated that GAT2 affects macrophage IL1 production. Mechanistically, GAT2 deficiency boosts intra cellular accumulation of hypoxanthine, which not only blocks NLRP3-ASC-Caspase1 complex formation but also enhances the expression of OXPHOSrelated genes via reducing expression of TF KID3 through the methylation (Fig. 8). We postulate that metabolic licensing of DNA methylation provides another layer of control that serves to finetune the immune cell activation. Our study expands the role of the nervous system in host innate immune responses by showing that GAT2 tailors IL1 production in proinflammatory macrophages. Thus, manipulation of GABAergic system could be a strategy to tackle macrophageassociated diseases.

Bacterial strains
Bovine P. multocida serotype A strain CQ2 (PmCQ2) was generally grown in Martin's broth agar containing 5% horse serum at 37°C for 24 hours.
Cell lines RAW264.7 cells and ANA1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and kept in a 5% car bon dioxide (CO 2 ) incubator at 37°C. Cells were polarized for 12 hours toward proinflammatory macrophages with LPS (1 g/ml) plus IFN (20 ng/ml) or left unstimulated and used as resting macrophages.

Mice
All animal care and experimental procedures used in this study were approved by the Laboratory Animal Ethical Commission of the South China Agricultural University. GAT2 −/− mice (KO) have previously been described (4). All experiments were performed using littermate control mice (WT). Six to 8weekold mice were used for all experiments and were age and gendermatched for individ ual experiments.

Murine primary PEMs
Murine primary PEMs were prepared as described in previous studies with some minor modifications. Briefly, 2 to 4 days after the injection of 4% thioglycolate, macrophages were flushed from peritoneal cavity with phosphatebuffered saline (PBS) and cultured in complete DMEM. After adherent purification, PEMs were polarized for 12 hours to ward proinflammatory macrophages with LPS (1 g/ml) or plus IFN (20 ng/ml) or toward antiinflammatory macrophages with IL4 (20 ng/ml) or left unstimulated and used as resting macrophages.

Bone marrow-derived macrophages
Murine BMDMs were prepared as described in previous study with some minor modifications (57). Briefly, murine bone marrow cells were flushed from tibias and femurs with icedcold RPMI 1640 and cultured in complete RPMI 1640 medium supplemented with mac rophage colony-stimulating factor (10 ng/ml) to generate BMDMs. BMDMs were polarized for 24 hours toward proinflammatory macro phages with LPS (100 ng/ml) plus IFN (20 ng/ml) or left unstim ulated and used as resting macrophages.

Deletion of macrophages in mice
Macrophage deletion was performed as previously described with some minor adjustments (34,58). Briefly, the mice were completely anesthetized and intraperitoneally or intratracheally administered 200 l of empty liposomes or liposome clorophosphate.

In vivo LPS challenge
Mice with or without clodronateloaded liposome treatment were injected intraperitoneally with LPS (100 mg/kg body weight) for the sepsis model. Survival rates were measured in all groups after injec tion. Mice were sacrificed, and the serum, jejunum, and liver were collected for further experiments.

In vivo PmCQ2 infection
Mice with or without clodronateloaded liposome treatment were infected by an intraperitoneal injection with PmCQ2 at a dose of 2.2 × 10 6 colonyforming units/ml according to the previous study (34). Survival rates were investigated in all groups after infection. Mice were killed to collect the serum and lung, and the samples were stored at −80°C until processing.

HFD-induced obesity model
Sixweekold male mice were maintained on HFD for 12 weeks. For glucose tolerance test, mice were injected intraperitoneally with glucose (2 g/kg body weight) after 16hour fasting. Blood glucose was determined by AccuChek glucose meters (Roche) at different time points. For insulin tolerance test, mice were injected with re combinant human insulin (0.75 U/kg body weight) after 4hour fasting, and blood glucose was also determined at different time points. Moreover, serum and adipose tissues were sampled for fur ther analysis.

Reverse transcription polymerase chain reaction
The total RNA of the samples was extracted using TRIzol (Invitrogen, USA) based on the manufacturer's instructions. RNA was reverse transcribed to complementary DNA by using the Primer Script TM RT reagent Kit (Takara, Qingdao, China) under the manufacturer's recommendations. RTPCR was conducted via SYBR Green on the Quant Studio 6 RealTime PCR System (Thermo Fisher Scientific, America). Fold change was assessed using the 2 −∆∆Ct method using actin as a reference gene.

Western blotting
Immunoblotting analysis was performed following the standard procedures. The tissues and cells were washed with icedcold PBS and lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Beyotime). Protein con centration was quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime), and an equal amount of protein was subjected to SDS-polyacrylamide gel electrophoresis (SDSPAGE). Proteins were transferred onto polyvinylidene difluoride membranes, fol lowed by incubations with primary and secondary antibodies, and visualized using chemiluminescent reagent. The optical density of the signals on the film was quantified by ImageJ software.

Detection of cytokines and mediators
Culture supernatants from cell culture and serum and supernatants from homogenized tissue or cell samples were collected, and levels of cytokines (e.g., IL1, IL6, and TNF) and mediators (e.g., ROS and xanthine) were measured by commercial kits following the protocols.

Coimmunoprecipitation
PEMs were treated as indicated and collected, then lysed with lysate on ice for 1 hour, and followed by centrifugation at 12,000g at 4°C for 30 min. Subsequently, the supernatants were harvested and in cubated with the appropriate antibody at 4°C overnight and precip itated with protein A/G agarose beads (Santa Cruz Biotechnology) for another 4 hours at 4°C. The beads were washed with the lysate for three times and centrifuged at 1000g at 4°C. The immunopre cipitated proteins were separated by SDSPAGE, and Western blot ting was conducted with the indicated antibodies.

Immunofluorescence
Immunofluorescence analysis was conducted according to the stan dard protocols. The fixed, permeabilized, and sliced samples were blocked by 5% bovine serum albumin (BSA), followed by incuba tion with primary and secondary antibodies. Stained cells and tis sues were viewed using a confocal fluorescence microscope (Zeiss).
In silico docking analysis Molecular docking between MAT2A and hypoxanthine was con ducted using the AutoDock Vina tool without the incorporation of water molecules. Interactions between hypoxanthine and the residues of the binding sites in MAT2A are shown using a twodimensional diagram by Discovery Studio 4.5.

SPR (OpenSPR) assay
Binding experiments was performed via an SPR (OpenSPR) local ized SPR (LSPR) biosensor (Nicoya Lifesciences). Briefly, the COOH sensor chip (Nicoya, #SENAU10010COOH) was activated by injecting 200 l of 1ethyl3(3dimethylaminopropyl) carbodiimide (EDC)/Nhydroxysuccinimide (NHS) (1:1). Subsequently, MAT2A was diluted in immobilization buffer [10 mM acetate (pH 3.5)] and immobilized on the chip at a flow rate of 20 l/min for 4 min. The free carboxyl groups were deactivated using 200 l of blocking buffer (Nicoya) followed by achieving a stable baseline. Then, the buffer matched hypoxanthine (0, 100, 200, 400, and 800 M) was succes sively injected into the flow cell at a rate of 20 l/min. Following a 4min interaction time, the dissociation was recorded for an ad ditional 3 min. The data were analyzed using TraceDrawer 1.8 (Ridgeview Instruments AB, Sweden).

RNA sequencing
PEMs were polarized into proinflammatory macrophages and col lected. RNA extraction, quality verification, library preparation, and sequencing were performed by NovoGene (Beijing). Briefly, clean data were obtained by filtering lowquality reads from raw data and mapped to the reference genome. Differential expression analysis was performed with DESeq2.GO, and KEGG pathway en richment analysis of the DEGs (FC > 2; P < 0.05) were performed.

Seahorse assay
Realtime measurements of OCR and ECAR were performed using an XFe24 Extracellular Flux Analyzer as per the manufacturer's in structions (Seahorse Agilent). PEMs (2 × 10 5 cells per well) were plated in XFe24 plates and polarized as described above toward pro inflammatory macrophages with or without hypoxanthine treatment or toward antiinflammatory macrophages or left unstimulated and used as resting macrophages. Subsequently, macrophages were washed and incubated for 1 hour in XF assay medium [nonbuffered DMEM (pH 7.4) + 10 mM glucose + 2 mM lglutamine] in a non CO 2 incubator at 37°C. OCR was measured under basal conditions and after the addition of the following drugs:100 M oligomycin, 100 M carbonyl cyanide ptrifluoromethoxyphenylhydrazone, and 50 M rotenone plus antimycin A. ECAR was assayed under basal conditions and after the addition of the following drugs:1 mM glucose, 1 mM oligomycin, and 1 mM 2deoxyglucose.

Amino acid analysis
The levels of amino acids in supernatants from cell culture and lysed cells were analyzed with isotope dilution liquid chromatography-MS (LCMS) methods.

Metabolite profiling analysis
PEMs (1 × 10 7 cells per well) were plated in sixwell plates and po larized as described above toward proinflammatory macrophages or left unstimulated and used as resting macrophages. Macrophages were then used to perform metabolomics assay at Shanghai Biotree Biotech Co. Ltd. Briefly, for each sample, 1000 l of extract solution (acetonitrile:methanol:water = 2:2:1) was added. The samples were homogenized at 35 Hz for 4 min and sonicated for 5 min with three cycles in iced water bath followed by incubating at −40°C for 1 hour and centrifuging at 12,000g for 15 min at 4°C. The supernatant (800 l) was transferred to a fresh tube and dried in a vacuum concentrator at 37°C. Subsequently, the dried samples were reconstituted in 100 l of 50% acetonitrile by sonication on ice for 10 min. The constitu tion was then centrifuged at 12,000g for 15 min at 4°C, and 75 l of supernatant was transferred to a fresh glass vial for LCMS analysis.
Measuring the enzyme activity of PNP For measuring the enzyme activity of PNP, the cells were washed with icedcold PBS and collected; then, the cells were repeatedly frozen and thawed for several times [note that the freezethaw pro cess was initiated with 30 min of freezing process at a −80°C refrig erator until completely frozen, followed by a thawing process under room temperature (25°C)]. Last, the enzyme activity of PNP was measured according to the manufacturer's recommendation (Andy gene, AD3407Mo).

Gene silencing or overexpression
Gene silencing or overexpression in macrophages was achieved via lipofection using the Lipofectamine 3000 transfection reagent ac cording to the manufacturer's protocol.

Intracellular transfection of betaine
PEMs were prepared as described above. For betaine delivery, first, macrophages were cultured in 1× basic DMEM containing a FuGENE HD Transfection reagent (5 l/ml) with or without 1 mM betaine and kept in a 5% CO 2 incubator at 37°C for 45 min. Then, the above medium was replaced with the normal complete DMEM, and macrophages were incubated at 37°C in 5% CO 2 for 24 hours. Last, macrophages were stimulated as described above.

Luciferase reporter assay
The promoter of mouse Ndufb9 was produced by PCRbased am plification and subcloned into the pPRORBReport1 to form lucif erase reporter plasmids. PEMs (2 × 10 4 cells per well) were plated in 96well plates and cotransfected with 50 ng of the luciferase reporter plasmid and 50 ng of pEXRBMammKid3 plasmid or the control vector. After 36 to 48 hours, the luciferase activity was determined by the DualGlo Luciferase Assay System in accordance with the manufacturer's instructions.

MassARRAY methylation assay
PEMs (2 × 10 6 cells per well) were plated in sixwell plates and po larized as described above toward proinflammatory macrophages and treated as indicated in the legend. Then, the DNA samples of macrophages were isolated and used to perform methylation assay at Shanghai OE Biotech. Co. Ltd. Briefly, the potential CpG islands in the KID3 promoter region were predicated by the website (www. ebi.ac.uk). The primer sets for the methylation analysis of the KID3 promoter region were designed by EpiDesigner software (http:// epidesigner.com; primer: 5′aggaagagagTAGGTATAATGAAGG GTTAAGGGGT3′; 5′agtaatacgactcactatagggagaaggct AAT CAAAAAAAACCTCAAACATACC3′). Subsequently, the quantitative gene specific methylation analysis was conducted on the Sequenom MassARRAY platform, including bisulfite treatment of DNA, amplification of DNA, Shrimp Alkaline Phosphatase (SAP) treatment, RNA transcription, basespecific cleavage by ribonucle ase A, and matrixassisted laser desorption/ionization-timeof flight analysis. Last, the methylation data of individual units were generated and analyzed by EpiTYPER software (SEQUENOM).

Histopathological examination
Mouse lung samples were fixed in 4% paraformaldehyde (PFA) at 4°C overnight and dehydrated and embedded into paraffin. Prepared sections (5 m) were stained with hematoxylin and eosin using standardized procedures. Histopathological scoring was performed by a pathologist blinded to all groups according to the previous study (59).

Immunohistochemistry
Briefly, mouse lung, liver, and adipose tissues were collected and washed with icedcold PBS and fixed in 4% PFA at 4°C overnight. The fixed samples were dehydrated in graded ethanol, embedded in paraffin, sliced and blocked by 2% BSA, followed by incubation with appropriate primary and secondary antibodies. The positive areas of the sample were measured using ImageJ software.

Statistical analysis
All statistical analyses were performed using Prism 6.0 software (GraphPad), and results are represented as means ± SEM or SD. Survival rates of mice were evaluated using KaplanMeier anal ysis (Prism 6.0). Data between two groups were analyzed by un paired t test (Prism 6.0) if the data were in Gaussian distribution and had equal variance, by unpaired t test with Welch's correction (Prism 6.0) if the data were in Gaussian distribution but with un equal variance, or by nonparametric test (MannWhitney U test, Prism 6.0) if the data were not normally distributed. Data among more than two groups were analyzed by the oneway analysis of variance (ANOVA) followed by Dunnett multiple comparisons (Prism 6.0) if the data were in Gaussian distribution and had equal variance or analyzed by KruskalWallis followed by Dunn's multi ple comparisons (Prism 6.0) if the data were not normally distributed. The Gaussian distribution of data was analyzed by D'Agostino Pearson omnibus normality test (Prism 6.0) and KolmogorovSmirnov test (Prism 6.0). The variance of data was analyzed by homogeneity of variance test (SPSS 22.0) or BrownForsythe test (Prism 6.0). Differ ences with P < 0.05 were considered significant.