The cGAS-cGAMP-STING Pathway: A Molecular LinkBetween Immunity and MetabolismJuli Bai and Feng Liu

Diabetes 2019;68:1099–1108 | https://doi.org/10.2337/dbi18-0052

It has been appreciated for many years that there isa strong association betweenmetabolism and immunityin advanced metazoan organisms. Distinct immune sig-natures and signaling pathways have been found notonly in immune but also in metabolic cells. The newlydiscovered DNA-sensing cGAS-cGAMP-STING path-way mediates type I interferon inflammatory responsesin immune cells to defend against viral and bacterialinfections. Recent studies show that this pathway isalso activated by host DNA aberrantly localized in thecytosol, contributing to increased sterile inflammation,insulin resistance, and the development of nonalcoholicfatty liver disease (NAFLD). Potential interactions of thecGAS-cGAMP-STING pathway with mTORC1 signaling,autophagy, and apoptosis have been reported, sug-gesting an important role of the cGAS-cGAMP-STINGpathway in the networking and coordination of theseimportant biological processes. However, the regula-tion, mechanism of action, and tissue-specific role ofthe cGAS-cGAMP-STING signaling pathway in meta-bolic disorders remain largely elusive. It is also unclearwhether targeting this signaling pathway is effective forthe prevention and treatment of obesity-induced met-abolic diseases. Answers to these questions wouldprovide new insights for developing effective therapeuticinterventions for metabolic diseases such as insulin re-sistance, NAFLD, and type 2 diabetes.

Formaintenance of normal physiological function, a livingorganism needs to obtain nutrients from the environ-ment and convert it into energy through its metabolicsystem. Meanwhile, the organism has to protect itselffrom attacks of potential pathogenic invaders. Evolution-arily, it is not surprising that the functions of the immuneand metabolic systems are closely linked and coordinated.It is well known that an effective immune response is

highly energy dependent in order to activate innateimmunity and promote adaptive immunity in responseto various environmental insults. Under conditions ofenergy insufficiency such as famine, prolonged and in-tensive physical activities, and overloaded neuronal andcardiovascular functions, the immune responses in anorganism may be sacrificed, leading to increased infec-tions and other immune-related defects (1). On the otherhand, overnutrition may lead to overactivated immunity,resulting in inflammation and related metabolic diseasessuch as insulin resistance and type 2 diabetes (2). Tomaintain homeostasis, an organism thus codevelops theimmune and metabolic systems to adapt to environmen-tal changes. Although metabolic cells and tissue-residentimmune cells exert distinct functions, numerous studieshave already demonstrated that these cells undergo in-tensive and dynamic cross talks to coordinate their actionin preserving homeostasis. Emerging evidence accumu-lated over the past several years also reveals the presenceof distinct immune signatures and signaling pathways inkey metabolic cells and vice versa, further signifying anintegral link between immune activity and metabolicfunction.

Activation of the cGAS-cGAMP-STING Pathway inImmunityOver the past several years, a key DNA immune responsepathway, the cGAMP synthase (cGAS [also known asMb21d1])–cGAMP–stimulator of interferon genes (STING[also called TMEM173, MITA, MPYS, and ERIS]) pathway,has been discovered in immune cells. The cGAS-cGAMP-STING pathway was originally identified as a signalingcascade that is activated by double-stranded DNA (dsDNA)during pathogen infections. cGAS senses viral and bacterialdsDNA aberrantly localized in the cytosol independent ofits sequence context (3–5), and binding dsDNA promotes

Department of Pharmacology, UT Health San Antonio, San Antonio, TX

Corresponding author: Feng Liu, liuf@uthscsa.edu

Received 20 January 2019 and accepted 4 March 2019

© 2019 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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cGAS oligomerization and activation (3,6,7). In addition toplaying a critical role in antiviral immune response, cGAShas also been shown to be involved in some other impor-tant biological processes such as macular degeneration (8),cellular senescence (9–11), myocardial infarction–relatedinflammation (12), and macrophage transformation (13).Activated cGAS catalyzes the formation of 2939-cGAMP,a cyclic dinucleotide (CDN) composed of adenosine andguanosine linked via two phosphodiester linkages. Apartfrom cGAMP, STING is also activated by CDNs such ascyclic di-AMP or cyclic di-GMP from bacteria (14).CDNsand 2939-cGAMP bind to the endoplasmic reticulum(ER)-localized STING, which promotes STING dimeriza-tion and translocation from the ER to perinuclear punc-tuate structures (14,15). During the trafficking process,STING recruits and activates TANK binding kinase1 (TBK1), stimulating phosphorylation and nuclear trans-location of the transcription factor interferon regulatoryfactor 3 (IRF3), and to a lesser extent nuclear factor-kB(NF-kB), which can also be activated by IkB kinase (IKK)(16,17), leading to the production of type 1 interferons(IFNs) and many other inflammatory cytokines (18)(Fig. 1). By binding to the IFN-a/b receptor on thecell membrane of target cells, IFNs promote the expres-sion of proteins involved in inhibiting viral replicationand thus enhances the protective defenses of the im-mune system (7,19). In addition to initiation of IFNsignaling in cells in which it is produced by cGAS, there issome evidence showing that 2939-cGAMP is able topromote downstream signaling in neighboring cellsvia distinct mechanisms such as gap junction–, membranefusion–, or viral particle–mediated transfer (20–22),thus mediating the cross talk between immune cells andtheir targeting cells. Intriguingly, activation of the cGAS-cGAMP-STING pathway could also be detected in non-immune cells such as mouse embryonic fibroblasts andadipocytes (23,24), suggesting that activation of this path-way may have broader roles in addition to immune defensefunctions.

Activation of the cGAS-cGAMP-STING Pathway bySelf-DNAIn a healthy cell, host DNA normally resides in the nucleusor mitochondria. However, under certain pathophysiologicconditions such as DNA instability and/or mitochondrialstress, genomic DNA and/or mtDNA may be released intothe cytoplasm, where it serves as a danger-associatedmolecular pattern to trigger immune responses. Westet al. (23) found that, via heterozygosity of the mitochon-drial transcription factor A (TFAM), disruption of mtDNAstability promoted mtDNA release into the cytosol, whereit activated the cGAS-cGAMP-STING pathway and in-creased IFN gene expression. mtDNA-mediated activationof the cGAS-cGAMP-STING pathway is also observed byBax/Bak-induced permeabilization of mitochondrial outermembrane (25,26). Besides mtDNA, recent studies showthat the cGAS-cGAMP-STING pathway could also be

activated by genomic DNA such as ruptured micronucleiand double-strand broken DNA of the primary nucleuscaused by genomic instability and/or DNA damage(19,27–29). In addition to genomic DNA and mtDNA,phagocytic DNA inadequately digested in the lysosomeshas been shown to activate the cGAS-cGAMP-STINGpathway (7,19,30,31) (Fig. 1). These results demon-strate that self-DNA is an important source of sterileinflammatory response induction that has been widelystudied in many of the autoimmune disease and cancers.These findings raise an interesting question as towhether self-DNA–induced sterile inflammation is as-sociated with metabolic disorders.

Activation of the cGAS-cGAMP-STING Pathway inObesity-Induced Inflammation and Metabolic DiseasesObesity is associated with various metabolic diseases suchas type 2 diabetes, nonalcoholic fatty liver disease(NAFLD), cardiovascular disease, and many types of can-cer. Numerous studies have shown that chronic sterileinflammation in adipose tissue plays a key role in medi-ating obesity-induced insulin resistance and its associatedmetabolic diseases (32,33). However, the precise mecha-nisms by which obesity causes inflammation remain to befully elucidated.

During the past several years, evidence has accumulatedto suggest an important role of the cGAS-cGAMP-STINGpathway in regulating inflammation and energy homeo-stasis. The expression levels and/or activities of compo-nents in this signaling cascade, including cGAS, STING,and TBK1, are significantly upregulated under obesityconditions in mice (24,34,35). Activation of the cGAS-cGAMP-STING pathway in mouse adipose tissue could betriggered by high-fat diet (HFD)–induced mtDNA release,leading to an increase in chronic sterile inflammatoryresponse (24). HFD-induced obesity and activation ofthe cGAS-cGAMP-STING pathway are prevented by adi-pose tissue–specific overexpression of disulfide bond Aoxidoreductase-like protein (DsbA-L), a chaperone-like andmitochondrial localized protein whose expression in adi-pose tissue is greatly suppressed by obesity (36). Alterna-tively, knockout of DsbA-L in adipose tissue impairedmitochondrial function, increased mtDNA release, andactivated the cGAS-cGAMP-STING pathway, leading toincreased inflammation and exacerbated obesity-inducedinsulin resistance (24) (Fig. 2). These findings reveal thatactivation of the cGAS-cGAMP-STING pathway may me-diate obesity-induced inflammation and metabolic dys-function, beyond its well-characterized roles in innateimmune surveillance. Consistent with this view, globalknockout of the cGAS-cGAMP-STING downstream targetTBK1 (37) or IRF3 (38), or pharmacological inhibition ofIkB kinase e (IKKe) and TBK1 by amlexanox, reduced bodyweight, enhanced insulin sensitivity, and improved glucosetolerance in obese mice and in a subset of patients withtype 2 diabetes (34,39). However, it should be noted thatwhile fat-specific knockout of TBK1 increased energy

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expenditure and attenuated HFD-induced obesity, it alsoexaggerated adipose tissue inflammation and insulin re-sistance, suggesting that TBK1 may have a feedback role inregulating obesity-induced inflammation (35) (Fig. 2). In-deed, activation of TBK1 has been found to reduce NF-kBactivity and inflammation by promoting phosphorylation-dependent degradation of NF-kB–inducing kinase (NIK),an upstream kinase of IKKs (35). TBK1 has also been shownto attenuate cGAS-cGAMP-STING–mediated response bypromoting STING ubiquitination and degradation (40)(Fig. 2). These findings explain the bidirectional rolesof TBK1 in regulating inflammation (35). Nevertheless,it remains to be determined what role cGAS-cGAMP-STING signaling, which activates TBK1, may play ininflammation, insulin resistance, and energy expenditurein metabolic cells.

The Potential Role of the cGAS-cGAMP-STINGPathway in NAFLDIn addition to mediating obesity-induced insulin resistancein adipose tissue, activation of the cGAS-cGAMP-STINGpathway has also been implicated in other metabolicdiseases including NAFLD. NAFLD is characterized byhepatic steatosis, which contributes to the developmentof nonalcoholic steatohepatitis (NASH), a potentially pro-gressive liver disease that may lead to cirrhosis and he-patocellular carcinoma. There is some evidence suggestingthat the innate immune response contributes to NAFLDand NASH (41–44). However, the underlying mechanismsby which the innate immune response promotes NALFD

remain elusive. Luo et al. (42) and Yu et al. (45) recentlyindependently found that activation of the liver cGAS-cGAMP-STING signaling pathway may mediate overnutri-tion-induced NAFLD and/or NASH. Indeed, STING levelswere higher in liver tissues from NAFLD human patientscompared with those without NAFLD. In addition, themRNA levels of cGAS and STING are elevated in NASHmouse livers (42,46). Furthermore, the phosphorylationstates of TBK1 and IRF3, two downstream targets of thecGAS-cGAMP-STING pathway, were significantly higher inlivers of mice fed an HFD, which is coupled with NAFLD(42). Consistent with these findings, cGAS-cGAMP-STING–dependent activation of TBK1 in hepatocytes pro-motes the formation of insoluble p62/sequestosome1 (SQSTM1) aggregates, a critical marker of NASH (47).On the other hand, STING deficiency attenuates steatosis,fibrosis, and inflammation in livers of mice fed with eithermethionine- and choline-deficient diet or HFD (42,45).Interestingly, both systemic or myeloid cell–specific knock-out of STING increased resistance to HFD-induced ormethionine- and choline-deficient diet–induced hepaticsteatosis, inflammation, and/or fibrosis in mice (42,45).In addition, transplantation of bone marrow cells fromcontrol mice to STING knockout mice restored HFD-induced severity of steatosis and inflammation (42), sug-gesting that the improved metabolic phenotypes in theSTING knockout mice were due to STING deficiency inliver-resident macrophages rather than in hepatocytes.These results are consistent with the finding that STINGis not present in human and murine hepatocytes but is

Figure 1—Activation and regulation of the cGAS-cGAMP-STINGpathway in cells. The cGAS is activated by viral and bacterial DNA aswell asmtDNA and phagocytosed DNA aberrantly localized in the cytosol. Activated cGAS uses ATP and GTP as substrates to catalyze theformation of the second messenger, cGAMP, which binds to STING localized on the ER membrane. The binding of cGAMP to STINGpromotes STING translocation to the Golgi apparatus. During the translocation, STING recruits and activates TBK1, which in turn catalyzesthe phosphorylation and nuclear translocation of IRF3, and to a lesser extent NF-kB, which can also be activated by IKK, leading to increasedsynthesis of IFN and other inflammatory genes.

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expressed at high abundance in hepatic nonparenchymalcells (48). Indeed, there is some evidence showing thathepatocytes do not express STING (45,49) and that byfacilitating hypoxia-induced autophagy in hepatocytes,cGAS protects the liver from ischemia-reperfusion injuryvia a STING-independent mechanism. The lack of STINGin human hepatocytes also explains why hepatitis virus hasadapted to specifically replicate in hepatocytes (48). It iswell known that selective pressures in evolution promotethe development of an effective immune surveillancesystem to ensure survival in the face of pathogen invasion.While at early stages infection initiates various biochemicalprocesses such as glucose release from stored glycogen,glycogenolysis, and gluconeogenesis, the glucose synthesisability of the infected body may be greatly impaired at laterstages of overwhelming infection, leading to hypoglycemia(50). Because hypoglycemia is detrimental to an organism,in the frame of evolution there is no host survival advan-tage for chronic pathogen infection. Therefore, a successfulimmune response is often short-lived, resulting in thetermination of the pathogen-induced response quicklyto ensure an organism’s survival. However, the lack ofSTING in hepatocytes results in type 1 IFN deficiency inresponse to hepatitis virus infection, which facilitateshepatitis viruses to escape from immune detection andcauses not only acute but also chronic inflammation in theliver, leading to consequent hepatitis, cirrhosis, and he-patocellular carcinoma (48), which is often accompaniedwith metabolic dysfunction such as insulin resistance(51,52). Activation of the cGAS-STING pathway by over-expression of STING specifically in hepatocytes signifi-cantly suppressed the replication of hepatitis virus in vivo(48,53). Nevertheless, there are some reports showingthat STING is present in hepatocytes and that knockingdown either STING or IRF3 in hepatocytes alleviated lipidaccumulation, hepatic inflammation, and apoptosis (54–56).These findings raise a possibility that some of the NAFLDphenotypes observed in the whole-body STING knockoutmice may result from STING deficiency in the hepatocytesof the mice. Further studies are needed to clarify thisdiscrepancy.

The Cross Talk Between the cGAS-cGAMP-STING andthe mTORC1 Signaling PathwaysThe mechanistic target of rapamycin complex 1 (mTORC1)is a nutrient sensor that integrates energy, hormonal,metabolic, and nutritional inputs to regulate cellular me-tabolism, growth, and survival. Activation of the mTORC1signaling pathway promotes anabolic processes such asprotein, nucleotide, fatty acid, and lipid biosynthesis whileinhibiting catabolic processes such as lipolysis and autoph-agy (57). Hasan et al. (58) recently found that chronicactivation of the cGAS-cGAMP-STING signaling pathwayis associated with reduced mTORC1 signaling in metabol-ically relevant tissues such as liver, fat, and skeletal muscleof the three-prime repair exonuclease 1 knockout (Trex12/2)mice, concurrently with increased inflammation and altered

metabolic phenotypes such as reduced adiposity and in-creased energy expenditure. Interestingly, STING defi-ciency in the Trex12/2 mice rescued both inflammatoryand metabolic phenotypes, but IRF3 deficiency onlyrescued inflammation, suggesting that a component down-stream of STING but upstream of IRF3 in the cGAS-cGAMP-STING pathway may play a role in regulatingmetabolism. Consistent with this view, they found thatTBK1, the downstream target of STING, directly inhibitedmTORC1 signaling by interacting with the mTORC1 com-plex (58) (Fig. 3). This result is in agreement with thefindings of others that TBK1 inhibits mTORC1 activity inprostate cancer cells (59,60) and in an experimental au-toimmune encephalomyelitis model (60). Nevertheless,one study reported that TBK1 may activate, rather thaninhibit, mTORC1 through site-specific phosphorylation ofmTOR at Ser2159 in response to epidermal growth factorbut not insulin treatment (61). On the other hand, there issome evidence showing that mTOR may inhibit the cGAS-cGAMP-STING antiviral pathway. Meade et al. (62)recently identified a cytoplasmically replicating poxviruses–encoded protein, F17, that binds and sequesters Raptorand Rictor. The binding of F17 to Raptor promotesmTORC1-mediated suppression of STING activity andIRF3 translocation to the nucleus. The binding of F17 toRictor, on the other hand, facilitates mTORC2-mediatedcGAS degradation. By disrupting the mTORC1-mTORC2cross talk, F17 inhibits cGAS-cGAMP-STING signaling

Figure 2—Activation of the cGAS-cGAMP-STING pathway medi-ates obesity-induced inflammation andmetabolic disorders. Obesityreduces the expression levels of disulfide bond A oxidoreductase-like protein (DsbA-L) in adipose tissue, leading to mitochondrialstress and subsequent mtDNA release into the cytosol. Aberrantlocalization of mtDNA in the cytosol activates the cGAS-cGAMP-STING pathway, leading to enhanced inflammatory gene expressionand insulin resistance. Phosphorylated and activated TBK1 exertsa feedback inhibitory role by promoting STING ubiquitination anddegradation or stimulating phosphorylation-dependent degradationof NF-kB–inducing kinase (NIK), thus attenuating cGAS-cGAMP-STING–mediated inflammatory response.

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and thus retains the benefits of mTOR-mediated stimulationof viral protein synthesis. Likewise, the mTOR down-stream effector ribosomal protein S6 kinase 1 (S6K1)has been found to interact with STING in a cGAS-cGAMP–dependent manner, and the interaction promotesTBK1-mediated phosphorylation of STING and recruit-ment of IRF3 for antiviral immune responses (63). Ofnote, the interaction of S6K1 with STING is mediated bythe kinase domain but not the kinase function of S6K1,suggesting a mTORC1-independent regulation of S6K1on STING signaling (Fig. 3). Taken together, all thesefindings indicate a complex cross talk between the cGAS-cGAMP-STING and themTORC1 signaling pathways. Furtherinvestigations will be needed to clarify these controversiesand to elucidate the molecular details as well as the metabolicconsequences of the cross talk between the cGAS-cGAMP-STING and the mTORC1 signaling pathways.

The cGAS-cGAMP-STING Pathway andAutophagy/MitophagyIn addition to cross talking to mTORC1, the cGAS-cGAMP-STING pathway has also been found to interactwith the autophagy machinery in innate immuneresponses (Fig. 4). Autophagy exerts its quality controlfunction by sequestering damaged organelles and proteinaggregates and invading intracellular pathogens in the

cytoplasm for lysosomal-mediated degradation (64). Thisprogrammed survival pathway also acts as a recyclingsystem to maintain essential protein biosynthesis duringstress conditions, such as nutrient insufficiency, growthfactor depletion, and pathogen invasion (65). Thus,autophagy is important for the maintenance of the met-abolic homeostasis of a cell, and its dysregulation mightcontribute to the development of metabolic disorders(66–68).

Autophagy is initiated with the activation of the ULK1complex, which is inhibited by mTORC1 and promotedby nutrient deprivation and AMPK activation (67–69) (Fig.4). Autophagy is also regulated by a multiprotein com-plex comprising beclin-1, a mammalian ortholog of theyeast autophagy-related gene 6 (Atg6), vacuolar proteinsorting 34 (VPS34), and autophagy/beclin-1 regulator 1(AMBRA1), which favors the nucleation of autophagosomeprecursors (67,70). Activation of the cGAS-cGAMP-STINGpathway has been found to prompt ubiquitin-mediatedautophagy that delivers bacteria to autophagosomes fordegradation (71). Similarly, cGAS protects hepatocytesfrom ischemia-reperfusion injury–induced apoptosisin vivo and in vitro through an induction of autophagy inmouse hepatocytes (49). How cGAS stimulates autophagyin hepatocytes is currently unknown but appears to bemediated by a STING-independent mechanism (49). In-terestingly, cGAS has been found to competitively bindbeclin-1 to dissociate the negative autophagy factor rubi-con from the beclin-1–phosphatidylinositol 3-kinase classIII (PI3KC3) autophagy complex, leading to PI3KC3 acti-vation and subsequent autophagy induction (72,73). Thisfinding provides a possible explanation for the finding thatcGAS promotes autophagy via a STING-independentmechanism. It is interesting to note that in addition tostimulating autophagy, the interaction between cGAS andbeclin-1 negatively regulates cGAS enzyme activity inimmune cells such as RAW264.7 and L929 cells, thuspromoting cytosolic DNA degradation and preventingoveractivation of the cGAS-cGAMP-STING pathway–mediated IFN responses and persistent immune stimula-tion (72). Activation of the cGAS-cGAMP-STING pathwayalso promotes an autophagy-dependent negative feedbackregulation of STING, which is mediated by cGAMP-induceddephosphorylation of AMPK and activation of ULK1 thatphosphorylates and promotes autophagosome-dependentdegradation of STING (74) or by TBK1-p62/SQSTM1–dependent ubiquitination and degradation of STING(40), providing a mechanism to prevent the persistenttranscription of innate immune genes (Fig. 4). However,a very recent study showed that upon binding cGAMP,STING translocated from the endoplasmic reticulum tothe endoplasmic reticulum-Golgi intermediate compart-ment, which served as a membrane source for LC3lipidation, leading to autophagosome formation andautophagy (75). This study reveals that the STING-inducedactivation of autophagy is mediated by a mechanism thatis dependent on the Trp-Asp (W-D) repeat domain

Figure 3—The interplay between the cGAS-cGAMP-STING path-way with mTORC1 signaling. Knockout of three-prime repair exo-nuclease 1 (Trex1), which degrades DNA in the cytosol, leads to theactivation of the cGAS-cGAMP-STING pathway and suppression ofthe mTORC1 activity in mice. The cGAS-cGAMP-STING pathwaymay inhibit mTORC1 activity through a TBK1-dependent mecha-nism. Conversely, the cGAS-cGAMP-STING pathway may be inhib-ited by mTORC1-dependent suppression of STING activity andIRF3 translocation or by mTORC2-mediated cGAS degradation.Of note, the kinase domain but not the kinase function of riboso-mal protein S6 kinase 1 (S6K1) is essential for S6K to interactwith STING, which facilitates TBK1-mediated phosphorylation ofSTING and recruitment of IRF3 for antiviral immune responses.

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phosphoinositide-interacting protein (WIPI2) and autophagy-related gene 5 (ATG5), but independent of TBK1, ULK, orVPS34-beclin kinase complexes. Interestingly, p62/SQSTM1has been shown to interact with mTOR and Raptor, whichis critical for mTOR recruitment to lysosomes and for aminoacid signaling–induced activation of S6K1 and 4EBP1(76). However, it remains unknown whether the cGAS-cGAMP-STING signaling–induced and TBK1-mediatedphosphorylation of p62/SQSTM1 plays a role in regulatingmTORC1 signaling and function.

Mitophagy is a selective form of autophagy that mit-igates inflammation by removing damaged mitochondriafrom cells (77). Mitochondrial damage induces mitophagyby promoting the accumulation of the ubiquitin kinasePINK1 on the outer membrane of the damaged mitochon-dria, which in turn phosphorylates Parkin at Ser65, leadingto the activation of this E3 ubiquitin ligase and subsequentdegradation of ubiquitinated substrates (78). A recentstudy showed that Parkin and PINK deficiency promotedmtDNA release and activation of the cGAS-cGAMP-STINGpathway in mouse heart tissue, leading to a strong in-flammatory phenotype (77). These results support a role ofPINK/Parkin-mediated mitophagy in restraining cGAS-cGAMP-STING signaling and innate immunity. By quan-titative proteomics analysis, Richter et al. (79) recentlyfound that the cGAS-cGAMP-STING downstream targetTBK1 phosphorylated several mitophagy receptors such asoptineurin (OPTN) and p62 (SQSTM) at their autophagy-relevant sites, which creates a signal loop amplifyingmitophagy (Fig. 4). However, while these findings reveal

a link between the cGAS-cGAMP-STING pathway andautophagy/mitophagy, the detailed biochemical mecha-nism underlying the link remains largely elusive, espe-cially in metabolic tissues. Seeking answers to thesequestions would be an attractive subject for furtherinvestigation.

The cGAS-cGAMP-STING Signaling and ApoptosisApoptosis is a programmed cell death process that pro-vides a mechanism to maintain organismal homeostasis(80). Apoptosis is regulated by prodeath proteins such asBak and Bax and prosurvival proteins such as BCL-2 andBCL-XL. Bak/Bax activation promotes mitochondrial out-er-membrane permeabilization and the release of apopto-tic proteins such as cytochrome c to the cytosol, leading tofurther activation of the downstream pathway of intrinsicapoptosis through initiator and executioner caspases cas-cade (81) (Fig. 5). Activation of caspases, which is a hall-mark of apoptosis, has been found to not only promote celldeath but also prevent dying cells from triggering a hostimmune response (82). However, while apoptosis has longbeen known as an immunologically silent form of celldeath, the molecular basis underlying the suppression ofimmune responses remains unknown. Several recent stud-ies suggest that caspase-mediated inhibition of the cGAS-cGAMP-STING signaling pathway may contribute to thesilencing of immune process in apoptotic cells. In agree-ment with this, TBK1 phosphorylation and IFNa-stimulated gene expression are increased in caspase-9knockout or caspase-3/-7 double knockout mice and cells

Figure 4—The cross talk between the cGAS-cGAMP-STINGpathway and autophagy/mitophagy. Autophagy is initiatedwith the activation ofULK1 complex. ULK1-induced activation of beclin-1 complex favors the nucleation of autophagosome precursors and promotes autophagy.Mitophagy is a selective form of autophagy. Activation of the cGAS-cGAMP-STING pathway may stimulate autophagy via a cGAS/beclin-1interaction–dependent mechanism. The cGAS-cGAMP-STING pathway also promotes autophagy/mitophagy by TBK1-dependent phos-phorylation and activation of receptors OPTN and p62 (SQSTM). Alternatively, activation of the cGAS-cGAMP-STING pathway promotesan autophagy-dependent negative feedback regulation by 1) the interaction of cGAS with beclin-1, which in turn inhibits cGAS activity;2) cGAMP-induced activation of ULK1, which promotes STING degradation by phosphorylation at Ser366; and 3) TBK1-p62/SQSTM1–dependent ubiquitination and degradation of STING. PINK/Parkin-induced mitophagy also restrains cGAS-cGAMP-STING signaling andinnate immunity by mitophagy-mediated mtDNA clearance.

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(25). Constitutive activation of the type I IFN responsewas alsoobserved in caspase-9–deficient mouse embryonic fibroblasts(26). In addition, inhibition of caspases led to increasedphosphorylation of TBK1 and IRF3 in Bax/Bak-sufficient cellsbut not in Bax/Bak knockout cells. Furthermore, the caspasedeficiency–induced IFNb response was prevented by knockingout cGAS or STING (25). These findings suggest that apoptosismay suppress immune responses by inhibiting the cGAS-cGAMP-STING pathway. However, the precise biochemicalmechanism(s) by which caspases negatively regulate thecGAS-cGAMP-STING pathway remains unclear but couldresult from multiple redundant processes such as atten-uated gene expression, cleavage and inactivation of a com-ponent or components of the type I IFN productionpathway, and caspase-mediated degradation of mtDNA,thereby disrupting its interaction with cGAS, thus prevent-ing the activation of the cGAS-cGAMP-STING signalingpathway and its downstream IFN action (26). Consis-tent with this, Wang et al. (83) found that cGAS couldbe cleaved by several inflammatory caspases includingcaspase-1, which can be activated by mtDNA release–induced formation of inflammasome, or by caspase-4, -5,and -11. Alternatively, activation of the cGAS-cGAMP-STING pathway may promote apoptosis via both tran-scriptional and nontranscriptional mechanisms (84,85). Ithas been shown that STING activation in T cells inducesapoptosis through an IRF3- and p53-mediated transcrip-tional proapoptotic program (86). A proapoptotic buttranscription-independent role of STING is observed inhepatocytes that is mediated by the association of IRF3withthe proapoptotic molecule Bax/Bak, which contributes toalcoholic liver disease (55). The proapoptotic role of STINGwas also found in other cells such as B cells and endothelialcells (87,88). Collectively, these findings demonstrate

a complicated cross talk between cGAS-cGAMP-STINGsignaling and apoptosis. Given the importance of apopto-sis in regulating metabolic homeostasis, it would be ofgreat interest to determine the functional roles of the crosstalk between cGAS-cGAMP-STING signaling and apoptosisin metabolic tissues.

Concluding RemarksInflammation is now clearly recognized as a major riskfactor for obesity-induced metabolic disorders. Suppressinginflammatory pathways, therefore, holds promise for de-veloping effective therapeutic treatment of obesity-relateddiseases. However, identification of pharmacological targetsto suppress inflammation is usually a challenge, requiringbetter understanding of the mechanisms underlyingobesity-induced inflammation. The identification of thecGAS-cGAMP-STING pathway as a key player in mediatingobesity-induced chronic low-grade inflammation haspointed out an exciting new direction to elucidate themechanism underlying obesity-induced metabolic diseasesand to develop potential therapeutic strategies to improvemetabolic homeostasis. However, a number of importantquestions remain to be answered.

First, while the pivotal roles of the cGAS-cGAMP-STINGpathway in immune defense against various microbial patho-gens have been extensively studied (3–5), its function in non-immune cells remains largely unexplored. The findings thatthe cGAS-cGAMP-STING pathway components such as cGAS,STING, and TBK1 are highly expressed in adipocytes and thatthe expression levels of these molecules are stimulated underobesity conditions (24) raise a possibility that activation of thispathway may play an important role in metabolic diseases.However, it should be acknowledged thatHFD feeding activatesthe cGAS-cGAMP-STING pathway not only in adipocytes but

Figure 5—The association of cGAS-cGAMP-STING signaling with apoptosis. The activation of prodeath proteins such as Bax/Bak promotesmitochondrial outer-membrane permeabilization and the release of cytochrome c to the cytosol, leading to activation of intrinsic apoptosisthrough initiator and executioner caspases cascade. Defects in apoptosis by knocking out caspase-9 or caspase-3/-7 lead to constitutiveactivation of the cGAS-cGAMP-STING pathway; however, the precise mechanisms by which caspase-induced apoptosis inhibits innateimmune remain unknown. Inflammatory caspases including caspase-1, which can be activated by mtDNA release–induced formation ofinflammasome, or caspase-4, -5, and -11 cleave cGAS, thus preventing the activation of immune response. Alternatively, activation of thecGAS-cGAMP-STING pathway promotes apoptosis via both transcriptional and nontranscriptional programs in a TBK1-IRF3–dependentmanner.

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also in macrophages (24,42), the predominant proinflamma-tory immune cell type in obese adipose tissue (89), suggestingthat activation of the cGAS-cGAMP-STING pathway inadipose tissue–resident macrophages may make a signifi-cant contribution to the deteriorated metabolic phenotypesof the obese mice. Further studies will be warranted to dissectthe relative contribution of the cGAS-cGAMP-STING path-way in metabolic relevant cells, such as such hepatocytesand adipocytes, and tissue-resident immune cells and thepotential cross talk between these cells triggered by cGAS-cGAMP-STING pathway activation.

It is interesting to note that the function of cGAS andSTING is regulated by mTOR complexes and vice versa(63,74) (Fig. 3), suggesting that activation of the cGAS-cGAMP-STING pathway may be modulated by environ-mental inputs such as cellular nutrient status and/orgrowth factors’ stimulation. However, the underlyingmechanism by which DNA-induced activation of thecGAS-cGAMP-STING pathway is moderated by environ-mental changes remains unexplored. In addition, giventhat mTORC1 plays a pivotal role in regulating cell me-tabolism and energy homeostasis, it is possible that acti-vation of the cGAS-cGAMP-STING pathway may mediatesome of the multifaceted roles of mTORC1 signalingpathway. Interestingly, activation of TBK1 has been shownto inhibit mTORC1 activity (58), suggesting a potentialnegative regulation of mTORC1 signaling by activation ofthe cGAS-cGAMP-STING pathway. Further studies willbe needed to elucidate the precise mechanism underlyingthe cross talk between these two signaling pathways andthe physiological roles of the interaction. In addition to themTORC1 signaling pathway, available evidence suggeststhat the cGAS-cGAMP-STING pathway also cross talks toboth autophagy and apoptosis (Figs. 4 and 5). Autophagyand apoptosis regulate the turnover of cellular organellesand cells within organisms, and their interaction is highlycontext dependent and in most of cases mutually inhib-itory (90). The identification of the cGAS-cGAMP-STINGpathway linking to both autophagy and apoptosis suggestsan important new layer of regulation of these distinctmechanisms for the control of cell homeostasis. However,an integrated understanding of the interplay between thecGAS-cGAMP-STING and these cellular pathways remainslargely unclear. It is also unknown when and which inputsto the network are dominant and how this depends on thephysiological or pathophysiological context. Answers tothese questions will likely require both deeper biochemicaland physiological studies of the interaction both in vitroand with tissue-specific transgenic and/or knockout mousemodels that enable more specific perturbation and mon-itoring of these pathways in vivo.

Lastly, it remains to be established whether and howtargeting the cGAS-cGAMP-STING pathway is a suitablestrategy to treat metabolic diseases. Given that the cGAS-cGAMP-STING pathway is activated by mitochondrialstress under obesity conditions, it is tempting to speculatethat at least a part of the effects of increased inflammation

may be due to activation of the cGAS-cGAMP-STINGpathway. In fact, much progress has been made on thedevelopment of small-molecule inhibitors targeting com-ponents in the cGAS-cGAMP-STING pathway over thepast several years. As of now, several cGAS inhibitorshave been reported. By in silico screening of drug librariesusing mouse cGAS/DNA target (PDB 4LEZ), An et al. (91)recently identified hydroxychloroquine, quinacrine, and9-amino-6-chloro-2-methoxyacridine as potential cGASinhibitors. These molecules do not bind to the active siteof cGAS but are instead found to localize to the minorgroove of DNA between the cGAS/DNA interface (91).RU.521 and RU.365, however, are found to inhibit cGASby binding at the cGAS active site (92). Wang et al. (93)showed that suramin, a drug used clinically for the treat-ment of African sleeping sickness (94), binds to the DNAbinding site of cGAS and inhibits cGAS activity by disruptingdsDNA/cGAS binding. In addition to targeting cGAS, severalsmall-molecule inhibitors of STING have also been reported.By a cell-based chemical screen, Haag et al. (95) recentlyidentified two nitrofuran derivatives—C-178 and C-176—that strongly reduced STING-mediated, but not RIG-I– orTBK1-mediated, IFNb reporter activity. Excitingly, theyfound that these derivatives reduce STING-mediated in-flammatory cytokine production in both human and mousecells and attenuate pathological features of autoinflam-matory disease in mice (95). Very recently, Dai et al. (96)reported that aspirin, a nonsteroidal anti-inflammatorydrug, can directly bind and enforce the acetylation ofcGAS, leading to a robust inhibition of cGAS enzymaticactivity and self-DNA–induced immune response in bothAicardi-Goutières syndrome patient cells and a mousemodel of Aicardi-Goutières syndrome. These results provideproof of concept that targeting the cGAS-cGAMP-STINGpathway may be efficacious in the treatment of inflam-matory diseases, which opens new avenues for developingnovel therapies for inflammation-related metabolic dis-eases. Future work on themolecular details of the regulationand network of the cGAS-cGAMP-STING pathway and itstissue-specific function should enable the rational targetingof this signaling to unravel the full therapeutic potentialof this extraordinary pathway in metabolism and relevantbiological functions.

Funding. This work was supported in part by grants from the Foundation for theNational Institutes of Health (DK114479 to F.L.), the National Natural ScienceFoundation of China (81730022 to F.L. and 81870601 to J.B.), and InnovativeBasic Science Awards of the American Diabetes Association (1-18-IBS-344 to F.L.and 1-19-IBS-147 to J.B.).Duality of Interest. No potential conflicts of interest relevant to this articlewas reported.Data Availability. No applicable resources were generated or analyzedduring the current study.

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