Kir2.1 is highly expressed and regulates the membrane potential in primary macrophages
Using RNA-sequencing (RNA-seq) analysis, we found that KCa3.1 (encoded by Kcnn4), THIK1 (encoded by Kcnk13), TWIK2 (encoded by Kcnk6), and Kir2.1 (encoded by Kcnj2) were most strongly expressed in bone-marrow-derived macrophages (BMDMs) (Fig. 1A). KCa3.1 plays a role in macrophage activation and migration (Feske et al., 2015), while TWIK2 and THIK1 have recently been shown to be required for NLRP3 inflammasome activation by promoting K+ efflux (Di et al., 2018; Madry et al., 2018). We thus focused on the inwardly-rectifying K+ channel Kir2.1. Under physiological conditions, Kir channels generate a large K+ conductance at potentials negative to K+ equilibrium potential (EK) but also permit a relatively small current flow at potentials slightly more positive to EK which is essential to stabilize the resting membrane potential (Vm) (Hibino et al., 2010; Miyazaki et al., 1974; Sakmann and Trube, 1984). Among different tissues and cell types, the expression of Kir2.1 was relatively high in macrophages and other specialized macrophages including osteoclasts and microglia (Figure S1A). To examine whether primary macrophages exhibit functional Kir2.1 channels, we performed whole-cell patch-clamp recordings from peritoneal macrophages subjected to voltage-ramps from − 120 to + 60 mV. Ba2+ (BaCl2) is usually used to block the Kir channels and we found a robust Ba2+-sensitive Kir current in the current-voltage relationship (Figure S1B). ML133 is a selective Kir2 blocker, with an IC50 of 1.9 µM for Kir2.1 (Wang et al., 2011; Wu et al., 2010). In HEK293 cells transfected with Kir2.1, ML133 potently inhibited the Kir2.1 current of both a large inward current component and a slight outward current component (Edwards and Hirst, 1988; Sakmann and Trube, 1984) (Figure S1C). Consistently, ML133 inhibited the endogenous Kir2.1 currents in both resting and LPS-stimulated peritoneal macrophages (Figs. 1B, 1C, 1F, and S1D).
Because knockout of Kir2.1 is lethal in the neonate (Zaritsky et al., 2000), its role in immune cells has not been reported using Kir2.1-deficient immune cells. We deleted Kir2.1 selectively in myeloid cells by generating Lyz2-cre-Kcnj2f/f mice and found the ability of bone marrow cells to differentiate ex vivo into macrophages in the absence of Kir2.1 was normal in light of the expression of the macrophage surface markers CD11b and F4/80 (Figure S1F). The proliferation of cultured Kir2.1-deficient BMDMs was also normal (Figure S1G). However, Kir2.1 deficiency greatly impaired the Kir2.1 currents in both resting and LPS-stimulated peritoneal macrophages (Figs. 1D, 1E, 1F, and S1E), and ML133 showed no additional effect (Figs. 1D, 1E, and 1F).
Kir2.1 was reported to stabilize the resting Vm of many cell types including cardiac myocytes (Sakmann and Trube, 1984), vascular smooth muscle cells (Karkanis et al., 2003; Park et al., 2008; Quayle et al., 1993), endothelial progenitor cells (Quayle et al., 1993; Zhang et al., 2019) and microglial cells (Gattlen et al., 2020). We first adopted a real-time dynamic detection of peritoneal macrophage Vm for 3 min by patch clamp experiments because of a relatively slow inhibitory effect of ML133 on Kir2.1 (Figs. 1B and 1C). The Vm of peritoneal macrophages (-38.4 ± 2.8 mV ) was not changed when Kir2.1 was blocked by ML133 (-39.4 ± 3.4 mV) in the resting state. However, the Vm of LPS-stimulated macrophages was dramatically changed from − 42.8 ± 2.9 mV to a much more depolarized Vm of + 12.3 ± 1.9 mV by ML133, and to a lesser extent, to -29.7 ± 2.3 mV due to Kir2.1 deficiency (Figs. 1G and S1H). Moreover, the effect of ML133 was abolished in Kir2.1-deficient macrophages (Figs. 1G and S1H), further indicating the specificity of ML133 on Kir2.1. We also performed patch clamp recording for a short period of time of 30 seconds and found the Vm of LPS-stimulated macrophages (-31.9 ± 1.6 mV) was depolarized to -17.3 ± 1.1 mV due to Kir2.1 deficiency (Fig. 1H). Of note, the Vm of resting macrophages was also depolarized by Kir2.1 deficiency under this condition (Fig. 1H), suggesting a discrepancy in the depolarization of resting macrophages between ML133 (acute blockade) and Kir2.1 deficiency (long-term absence). Together, we conclude that Kir2.1 plays a critical role in regulating the membrane potential of inflammatory macrophages.
Kir2.1 loss-of-function suppresses LPS- and infection-induced inflammatory cytokines and pathological inflammation
To explore the role of Kir2.1 in LPS-induced inflammation, we first treated LPS-stimulated peritoneal macrophages with ML133 and found little cytotoxicity of ML133 (Figure S2A). Strikingly, ML133 dose-dependently inhibited the LPS-induced IL-1β, but not the TNF-α (Figs. 2A and 2B). Blockade of Kir2.1 by Ba2+ gave similar results (Figure S2B). To more broadly explore the effect of ML133, we performed RNA-seq analysis in BMDMs and found that, while Il1b was the gene most decreased by ML133, a cluster of LPS-induced inflammatory genes including Il1a, Il18, Il12a, and Il6 were also decreased, but Tnf was still not affected (Figs. 2C and S2C). Gene set enrichment analysis (GSEA) of the RNA-seq data showed a striking enrichment of ‘inflammatory response’ after ML133 treatment (Fig. 2D). Next, we used several genetic strategies to further investigate the role of Kir2.1 in LPS-induced inflammation. Silencing of Kcnj2 by small-interfering RNAs (siRNAs) in BMDMs or stably expressing shRNAs in immortalized BMDMs (iBMDMs) suppressed the LPS-induced IL-1β, but not the TNF-α (Figures S2D-S2G). Peritoneal macrophages from Lyz2-cre-Kcnj2f/f mice similarly showed a reduction in LPS-induced IL-1β compared to wild-type mice (Fig. 2E). We also treated Kir2.1-depleted peritoneal macrophages with ML133 and found the inhibitory effect of ML133 on LPS-induced IL-1α and IL-1β was greatly impaired, further suggesting the specificity of ML133 through Kir2.1 (Figures S2H). To address the function of Kir2.1 in vivo, we used an LPS-induced sepsis model which largely reflects the inflammatory functions of monocytes/macrophages. ML133 or Kir2.1 deficiency significantly decreased the serum levels of IL-1β and increased the survival of mice (Figs. 2F-2I). The decreased TNF-α levels in Lyz2-cre-Kcnj2f/f mice are probably due to the contribution of IL-1β to TNF-α production in vivo (Fig. 2H) (Tannahill et al., 2013). When infected with Gram-negative bacteria, such as Escherichia coli and S. typhimurium (strain SL1344), ML133 or Kir2.1 deficiency similarly led to decreased IL-1β and IL-1α production both in vitro and in vivo, with less effect on the TNF-α (Figs. 2J, 2K, S2I, and S2J). Last, to strengthen the evidence for an anti-inflammatory effect of Kir2.1 blockade in samples from patients, we used synovial fluid cells from gouty patients. Gout is an inflammatory form of arthritis and IL-1 inhibitors are effective in treating patients with acute and chronic gout (Jiang et al., 2017). We found that ML133 prevented IL-1β production in freshly-isolated synovial fluid cells from gouty patients (Fig. 2L). These data together indicate that Kir2.1 stimulates LPS-induced inflammation and reveal a potential anti-inflammatory strategy by targeting Kir2.1.
Kir2.1 promotes LPS-induced glucose uptake and consumption in inflammatory macrophages
Given the inconsistent inhibition of LPS-induced IL-1β and TNF-α, we predicted that Kir2.1 blockade would not affect the general signaling pathways mediated by TLR4, including the NF-κB and MAPK activation essential for both cytokines upon LPS stimulation. As predicted, ML133 or Kir2.1 deficiency had little effect on LPS-induced NF-κB and MAPK activation (Figures S3A-S3C). Accumulating evidence indicates that the Warburg effect of aerobic glycolysis plays critical roles in driving inflammatory macrophages, in particular the LPS-induced IL-1β production (Adamik et al., 2013; O'Neill et al., 2016). We thus further investigated whether Kir2.1 drives inflammatory macrophages by modulating LPS-induced metabolic reprogramming. We first measured real-time changes in the extracellular acidification rate (ECAR) and found ML133 or Kir2.1 deficiency led to a decrease in the LPS-induced long-term commitment to glycolysis (Fig. 3A). Moreover, both unbiased metabolomics profiling and a targeted metabolomics approach revealed that ML133 decreased the LPS-induced accumulation of metabolites representing aerobic glycolysis (Figs. 3B and 3C), suggesting a role of Kir2.1 in supporting a constant glycolysis during inflammation. Akt-mTORC1 signaling and HIF-1α activation were reported to modulate LPS-induced aerobic glycolysis and IL-1β production (Everts et al., 2014; Mills et al., 2016; Mills et al., 2018; Palsson-McDermott et al., 2015b; Tannahill et al., 2013). We found ML133 or Kir2.1 depletion had little effect on the early LPS induction of Akt-mTORC1 signaling in light of the phosphorylation of Akt and S6 (Figures S3A, S3B, and S3D). Moreover, GSEA of RNA-seq data showed that ‘mTORC1 signaling’ and ‘HIF-1α targets’ were not particularly enriched by ML133 (Figures S3E and S3F). Given that macrophages require a constant supply of extracellular glucose to support their intracellular metabolic reprogramming (Freemerman et al., 2014; Gamelli et al., 1996), we next considered the possibility that Kir2.1 promotes glucose import during LPS stimulation. Strikingly, LPS-induced glucose uptake in peritoneal macrophages was dose-dependently inhibited by ML133 in vitro (Fig. 3D). Similar results were obtained in peritoneal macrophages from Lyz2-cre-Kcnj2f/f mice compared to wild-type macrophages (Fig. 3E). Furthermore, we assessed this effect of Kir2.1 in vivo by measuring glucose uptake of macrophages in peritoneal exudate cells (PECs) and inflammatory monocytes in peripheral mononuclear cells (PBMCs) after intraperitoneal LPS challenge. Consistently, LPS-induced glucose uptake by these cells was significantly lower in Lyz2-cre-Kcnj2f/f mice than that in wild-type mice (Figs. 3F and 3G). Together, these data suggest that Kir2.1 promotes glucose uptake and consumption in inflammatory macrophages.
Kir2.1 supports glycolysis offshoots and SGOC metabolism in inflammatory macrophages and its loss-of-function leads to an amino acid starvation phenotype
A key mechanism for LPS-induced glycolysis is the induction of dimeric PKM2, which enters into a complex with HIF-1α to drive IL-1β expression (Palsson-McDermott et al., 2015a). As HIF-1α activation was minimally affected by ML133 (Figure S3F), we considered other mechanisms underlying the impaired glycolysis by Kir2.1 blockade. While often represented as a linear metabolic flux, glycolysis-derived carbons also feed into several offshoots and integrate into different biosynthetic pathways (Chaneton et al., 2012; Keller et al., 2012). The pentose phosphate pathway (PPP) generates pentose sugars for nucleotide synthesis, and another three-step offshoot, the serine synthesis pathway (SSP) diverts glucose-derived carbons into serine, which is further integrated into the serine, glycine, one-carbon (SGOC) metabolic network that includes the folate and methionine cycles (Newman and Maddocks, 2017; Yang and Vousden, 2016) (Fig. 4A). Strikingly, unbiased metabolomics profiling revealed that the key metabolites 3-phosphoserine (3PS, representing the SSP) and ribose 5-phosphate (R5P, representing the PPP), increased in response to LPS but were decreased by ML133 (Fig. 4B). In addition, the enzymes mediating the three-step SSP – Phgdh, Psat1, and Psph – (Fig. 4A), and genes previously described as master regulators of the SSP (Yang and Vousden, 2016), such as Atf4 and Mdm2, were all upregulated by ML133 (Fig. 4C), showing a phenotype similar to a compensatory increase in the SSP upon serine starvation (Maddocks et al., 2013; Ye et al., 2012). Many enzymes involved in SGOC metabolism were also upregulated in ML133-treated inflammatory macrophages (Figure S4A). Moreover, GSEA of RNA-seq data showed that ML133-treated inflammatory macrophages were enriched in ‘amino acid transport’ and ‘SGOC metabolism’ (Fig. 4D). Using strategies of pharmacological inhibitor (acute blockade) and genetic depletion (long-term absence) may lead to certain discrepancies in downstream cellular and molecular mechanisms. To reveal the common mechanisms of both ML133 and Kir2.1 deficiency, we analyzed together the RNA-seq data from both ML133-treated and Kir2.1-depleted macrophages upon LPS stimulation. We found 236 overlapped downregulated and 163 overlapped upregulated genes (Fig. 4E). Pathway analysis revealed that the downregulated pathways affected by both ML133 and Kir2.1-depletion were mostly related to ‘inflammatory response’ and ‘response to LPS or IL-1’ (Fig. 4F). Strikingly, the upregulated pathways were related to ‘response to amino acid starvation’ and ‘regulation of translation’ (Fig. 4F). Among these upregulated genes, we found several master sensors and regulators in response to amino acid starvation, including GCN2, PERK, IMPACT, and SLC38A2 (Figure S4B) (Broer and Broer, 2017).
We next used U-[13C]-glucose tracers to confirm the changes in the SSP and SGOC metabolism when Kir2.1 was blocked during LPS stimulation. ML133 significantly decreased intracellular m + 6 glucose, m + 3 serine, and m + 2 glycine, suggesting that Kir2.1 supports glucose uptake and the channeling of glucose-derived carbons into the SSP upon LPS stimulation (Figs. 4G and 4H). To assess the contribution of this suppressed SSP to the anti-inflammatory phenotype of ML133, we supplemented cell penetrable 3-PG (Finder and Hardin, 1999; Yang et al., 2014), which is more channeled into the SSP from glycolysis under serine-depleted conditions (Chaneton et al., 2012; Maddocks et al., 2013; Zhang et al., 2012), and found the suppressed IL-1β production by ML133 was partially restored (Figure S4C). Of note, most of the accumulation of intracellular serine and methionine was unlabeled (m + 0) (Figures S4D and S4E), indicating their accumulation is largely fueled by exogenous import during inflammation. Strikingly, the LPS-induced accumulation of unlabeled serine, glycine, and methionine (key amino acids fueling SGOC metabolism (Locasale, 2013)) was also greatly blocked by ML133 (Fig. 4I). Similarly, we used U-[13C]-serine tracers and found a decrease of m + 3 serine, m + 2 glycine, and m + 0 methionine due to Kir2.1 deficiency, recapitulating the impeded serine and methionine uptake (Figure S4F). These results together highlight a role of Kir2.1 in the nutrient supply fueling glycolysis offshoots and SGOC metabolism during LPS-induced inflammation.
Kir2.1 supports S-adenosylmethionine generation and configures histone methylation in inflammatory macrophages
Through coupling with the methionine cycle, the SGOC metabolic network acts as an integrator of nutrient status to generate diverse outputs, including the primary methyl donor S-adenosylmethionine (SAM) (Locasale, 2013; Yang and Vousden, 2016; Yu et al., 2019). We and others have previously showed that glucose can provide carbons to feed SAM generation through de novo ATP synthesis (Maddocks et al., 2016; Newman and Maddocks, 2017; Yu et al., 2019) (Fig. 5A). Using U-[13C]-glucose, we found ML133 led to a decrease in LPS-induced m + 5 to 9 SAM (via both the PPP and SSP) and its demethylation product S-adenosylhomocysteine (SAH) (Figs. 5A and 5B), as well as m + 5 to 9 ATP (Figure S5A). The total amount of SAM was also reduced by ML133 (Figure S5B). Using U-[13C]-serine tracers, we obtained similar results that ML133 or Kir2.1 deficiency decreased the LPS-induced incorporation of U-[13C]-serine-derived carbons into m + 1 to 4 SAM, as well as the total amount of SAM (Figs. 5C, 5D, and S5C). Given that Kir2.1 blockade led to impaired nutrient uptake, exogenous glucose or amino acids (serine, glycine, or methionine) that donate carbons into SGOC metabolism only had mild rescue effects on the suppressed IL-1β production by ML133 (Figures S5D-S5F). However, addition of SAM dose-dependently restored this suppressed IL-1β production (Figs. 5E and 5F), suggesting a role of Kir2.1 in supporting LPS-induced SAM generation by providing the supply of extracellular nutrients.
SAM is the universal methyl donor for all methylation reactions in cells, which plays critical roles in the chromatin state and gene transcription (Goll and Bestor, 2005). SAM availability can directly modulate several epigenetic methylation marks required for the maintenance of downstream gene transcription (Mentch et al., 2015; Mews et al., 2014; Shiraki et al., 2014; Shyh-Chang et al., 2013). RNA-seq analysis of the expression of 183 annotated SAM-dependent methyltransferase enzymes (Kottakis et al., 2016) revealed that Kir2.1 deficiency upregulated all methyltransferases for histone H3 methylation at lysine 36 (H3K36me) (Wagner and Carpenter, 2012) compared to those for other histone methylation marks (Figs. 5G, 5H, and S5G). Among the most upregulated methyltransferase genes, ASH1L and NSD1 have specific mono- and dimethylase activity for H3K36, and SETD2 is the only reported trimethylase catalyzing the trimethylation of H3K36 (Fig. 5G) (Chen et al., 2017; Wagner and Carpenter, 2012). H3K36me3 is one of the most dynamic histone methylation marks and its main distribution appears in a wide range of gene body regions as a SAM ‘sink’ (Ye et al., 2017), making it more sensitive to SAM availability for downstream gene expression (Wagner and Carpenter, 2012). To test whether Kir2.1-induced SAM generation fuels histone methylation such as H3K36me3 upon LPS stimulation, we coupled chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) and found that LPS-induced occupancy of H3K36me3 in Il1b gene-body regions farther from the 5’ end (Adamik et al., 2013) was significantly decreased by ML133 or Kir2.1 deficiency (Figs. 5I and S5H). In addition, LPS-induced H3K36me3 enrichment in the gene-body regions of other inflammatory factors including Il1a, Il18, and Cxcl10 were also decreased (Figs. 5J and S5H). In contrast, H3K36me3 enrichment in the gene-body regions of Tnf was unaffected in these experiments (Figure S5I). We thus conclude that deregulation of histone methylation marks, at least H3K36me3, contributes to the anti-inflammatory outcome of Kir2.1 loss-of-function.
Kir2.1-mediated regulation of membrane potential orchestrates metabolic-epigenetic reprogramming in inflammatory macrophages
Given that monovalent cations such as K+ regulate the membrane potential, which indirectly controls the flux of Ca2+ and intracellular signaling pathways (Feske et al., 2015; Franchini et al., 2004; Lam and Schlichter, 2015), we initially investigated whether Kir2.1 stimulates inflammatory macrophages by modulating Ca2+ homeostasis. In LPS-stimulated BMDMs loaded with Ca2+-sensitive fluorescent dye Fluo-4-AM, we found an elevation of cytosolic Ca2+ level in response to ML133 (Figure S6A). However, when we used BAPTA or EGTA to chelate the extracellular Ca2+ or BAPTA-AM to chelate the intracellular Ca2+, the inhibitory effect of ML133 on LPS-induced IL-1β production was not affected (Figures S6B-S6F). Notably, BAPTA-AM showed an additional inhibitory effect on the production of IL-1β (Figures S6E and 6F), suggesting a separate contribution of intracellular Ca2+ in parallel to ML133. Consistent with idea, when we depleted ER Ca2+ store by thapsigargin (TG), although TG alone was able to reduce the production of IL-1β, ML133 still had a similar inhibitory efficiency on IL-1β production (Figure S6G). Moreover, the effect of ML133 was independent of the concentrations of extracellular Ca2+ (0, 1.2, 2.4, and 3.6 mM) or the other divalent cations Mg2+ and Mn2+ (Figures S6H and S6I). We thus conclude that the changes in Ca2+ homeostasis is not required for the anti-inflammatory outcome of Kir2.1 blockade.
Given the critical role of Kir2.1 in maintaining Vm of inflammatory macrophages (Figs. 1E and 1F), we considered the possibility that Kir2.1 promotes LPS-induced inflammation by its regulation of Vm. We first examine whether Vm depolarization by other means results in an analogous suppression of inflammatory macrophages. Increased extracellular K+ ([K+]e) is widely used to depolarize Vm. We found the Vm of both resting and LPS-stimulated macrophages was depolarized by elevated [K+]e, as determined by either patch clamp experiments or a membrane sensitive fluorochrome DiBAC4(3) (Eil et al., 2016) (Figures S6J and S6K). Strikingly, elevation of [K+]e similarly decreased the LPS-induced IL-1β without affecting the TNF-α (Fig. 6A), as well as glucose uptake detected by intracellular m + 6 glucose using U-[13C]-glucose tracers (Fig. 6B). The effect of elevated [K+]e was not due to an osmotic effect, as choline chloride or mannitol did not induce a similar suppression of IL-1β production (Figure S6L). Gramicidin is another widely used strategy to depolarize Vm by forming pores permeable to both K+ and Na+ in the plasma membrane (Munoz-Planillo et al., 2013). As expected, gramicidin depolarized the Vm of inflammatory macrophages (Figure S6K), and it recapitulated the suppressed LPS-induced IL-1β production and glucose uptake, but not the TNF-α (Figs. 6C and 6D). Furthermore, using U-[13C]-glucose, we found that elevated [K+]e and gramicidin led to a decrease in LPS-induced m + 3 serine, m + 5 to 9 ATP, and m + 5 to 9 SAM, as well as unlabeled m + 0 serine and methionine (Figs. 6E and 6F). Elevated [K+]e also reduced the enrichment of H3K36me3 in the gene-body regions of Il1b, Il1a, Il18, and Cxcl10 loci (Figs. 6G and 6H). Together, these data suggest that the maintenance of membrane potential may be a common mechanism that orchestrates metabolic-epigenetic reprogramming in inflammatory macrophages.
Kir2.1 supports nutrient supply by promoting the surface expression of nutrient transporters during LPS-induced inflammation
Nutrient import is the proximal step at which intracellular metabolism can be tightly regulated. Cells require adaptations in nutrient transport mechanisms to meet different metabolic demands (McCracken and Edinger, 2013). Consistent with this idea, glucose transporter expression levels are elevated in proliferating cells and in a wide variety of tumor types (Adekola et al., 2012). To investigate the mechanisms underlying the regulation of nutrient import by Kir2.1 in inflammatory macrophages, we first analyzed the RNA-seq data with the KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome pathway databases. Strikingly, apart from pathways related to inflammation, Kir2.1 blockade led to a strong enrichment for the pathways related to endocytosis, vesicle transport, and membrane trafficking (Figs. 7A and 7B). Given the critical roles of surface transporters in the regulation of nutrient import, we next evaluated the potential contribution of Kir2.1 to the surface expression of nutrient transporters fueling glucose and amino acid metabolism. Glucose transporter 1 (GLUT1) is the primary transporter that rewires glucose metabolism in LPS-stimulated macrophages (Freemerman et al., 2014). While the mRNA expression was not affected (Figure S7A), ML133 dose-dependently decreased the membrane GLUT1 expression in LPS-stimulated macrophages examined by flow cytometry (Fig. 7C). This effect was also confirmed by immunoblotting for biotinylated cell-surface proteins in ML133-treated or Kir2.1-depleted inflammatory macrophages (Figs. 7C and S7B). Consistently, this decreased membrane GLUT1 expression was recapitulated by elevated [K+]e (Fig. 7D). Among the membrane amino acid transporters, L-type amino acid transporter (LAT1) together with 4F2 cell-surface antigen heavy chain (4F2hc, also known as CD98) transport large neutral amino acids including methionine (McCracken and Edinger, 2013; Yanagida et al., 2001). Both ML133 and elevated [K+]e decreased the surface expression of CD98 in LPS-stimulated macrophages (Fig. 7E), and depolarization by gramicidin gave similar results including GLUT1 and CD98 (Figs. 7F and 7G). These results further indicated the contribution of membrane potential to the nutrient import in inflammatory macrophages.
The surface expression and activity of glucose transporters were regulated by lipid rafts-mediated trafficking and internalization (Kumar et al., 2004; Michel and Bakovic, 2007; Yan et al., 2018). Nearly 80% of GLUT4 is internalized by a lipid rafts-dependent mechanism which can be blocked by the cholesterol-chelating drug nystatin (Blot and McGraw, 2006; Ros-Baro et al., 2001). Without affecting TLR4-mediated downstream signaling upon LPS stimulation (Figure S7C), nystatin partially restored the decreased level of surface GLUT1, the suppressed glucose uptake, and IL-1β production but not TNF-α when Kir2.1 was blocked by ML133 or depleted (Figs. 7H-7K and S7D). Nystatin also rescued the enrichment of H3K36me3 in the gene-body regions of Il1a, Il1b, Il18, and Cxcl10 loci in Kir2.1-depleted inflammatory macrophages (Figure S7E). Last, nutrient transporters including GLUT1, CD98, and LAT1 are ARF6/GRP1 cargo proteins that can be recycled back to the plasma membrane via the tubular recycling endosome (Eyster et al., 2009; Finicle et al., 2018; Maldonado-Baez et al., 2013; McCracken and Edinger, 2013). Strikingly, a constitutively active mutant of GRP1 (S155D/T280D, GRP1 DD mutant), which forces the recycling of these transporters back to the plasma membrane and prevents their loss (Finicle et al., 2018; Li et al., 2012), largely restored the ML133-induced suppression of IL-1β (Fig. 7K), further strengthening the contribution of surface nutrient transporters loss to the anti-inflammatory outcome of Kir2.1 blockade.