Gαi2 Signaling Regulates Inflammasome Priming and Cytokine Production by Biasing Macrophage Phenotype Determination

This information is current as Ali Vural, Neel R. Nabar, Il-Young Hwang, Silke Sohn, of September 26, 2021. Chung Park, Mikael C. I. Karlsson, Joe B. Blumer and John H. Kehrl J Immunol published online 25 January 2019 http://www.jimmunol.org/content/early/2019/01/24/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 25, 2019, doi:10.4049/jimmunol.1801145 The Journal of Immunology

Gai2 Signaling Regulates Inﬂammasome Priming and Cytokine Production by Biasing Macrophage Phenotype Determination

Ali Vural,*,1,2 Neel R. Nabar,*,†,1 Il-Young Hwang,* Silke Sohn,† Chung Park,* Mikael C. I. Karlsson,† Joe B. Blumer,‡ and John H. Kehrl*

Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein–coupled receptors are essential in transducing signals from

the microenvironment, and heterotrimeric Ga signaling links these receptors to downstream effectors. Several Gai-coupled G-protein–coupled receptors have been implicated in macrophage polarization. In this study, we use genetically modiﬁed mice

to investigate the role of Gai2 on inflammasome activity and macrophage polarization. We report that Gai2 in murine bone marrow– Downloaded from derived macrophages (BMDMs) regulates IL-1b release after activation of the NLRP3, AIM2, and NLRC4 inflammasomes. We 2/2 G184S/G184S show this regulation stems from the biased polarity of Gai2 deficient (Gnai2 ) and RGS-insensitive Gai2 (Gnai2 ) G184S/G184S BMDMs. We determined that although Gnai2 BMDMs (excess Gai2 signaling) have a tendency toward classically 2/2 activated proinflammatory (M1) phenotype, Gnai2 BMDMs (Gai2 deficient) are biased toward alternatively activated anti- inflammatory (M2) phenotype. Finally, we find that Gai2-deficient macrophages have increased Akt activation and IFN-b pro-

duction but defects in ERK1/2 and STAT3 activation after LPS stimulation. Gai2-deﬁcient macrophages also exhibit increased http://www.jimmunol.org/ STAT6 activation after IL-4 stimulation. In summary, our data indicates that excess Gai2 signaling promotes an M1 macrophage phenotype, whereas Gai2 signaling deﬁciency promotes an M2 phenotype. Understanding Gai2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics. The Journal of Immunology, 2019, 202: 000–000.

acrophages are critical mediators of innate immunity macrophages have heterogeneous and flexible phenotypes that are and participate in both the initial propagation of the dictated by signaling inputs from their microenvironment. Mac- M inflammatory response and later in the suppression of rophages are broadly classified as proinflammatory M1 macro- by guest on September 26, 2021 inflammation promoting a return to homeostasis (1). To do so, phages implicated in initiating and sustaining inflammation or anti-inflammatory M2 macrophages that produce high levels of *B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National IL-10 and promote tissue repair (2). Although progress has been Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, made, the signaling networks responsible for macrophage polari- † MD 20892; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, zation remain unclear. 171 77 Stockholm, Sweden; and ‡Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425 G-protein–coupled receptors (GPCRs) play a central role in reg- 1A.V. and N.R.N. contributed equally to this work. ulating macrophage function, as lipid-based inflammatory mediators, 2Current address: Department of Pharmacology, Wayne State University School of vasoactive amines, complement fragments, and other inflammatory/ Medicine, Detroit, MI. anti-inflammatory stimuli activate signaling by GPCRs linked to ORCIDs: 0000-0002-1269-6338 (A.V.); 0000-0002-9704-3570 (N.R.N.); 0000-0002- heterotrimeric G-proteins (3). Typically, GPCR activation leads 7051-0933 (S.S.); 0000-0002-7819-5333 (C.P.); 0000-0002-0020-3815 (J.B.B.); to GTP nucleotide exchange on Ga, causing its dissociation from 0000-0002-6526-159X (J.H.K.). the Gbg heterodimer; this allows both GTP-bound Ga and Gbg Received for publication August 17, 2018. Accepted for publication December 19, heterodimers to activate their corresponding downstream effec- 2018. tor molecules. Signaling is terminated by the inherent GTPase This work was supported by the intramural program of the National Institutes of Allergy and Infectious Diseases. N.R.N. is supported in part by the Thomas Jefferson activity of Ga subunits, which hydrolyze GTP to GDP and cause University M.D., Ph.D. training program. the reassociation of GDP-bound Ga and Gbg. This cycle highlights A.V., N.R.N., and S.S. did the experimental work; A.V., N.R.N., M.C.I.K, and J.H.K. the importance of heterotrimeric G-proteins in cell signaling net- designed and analyzed the experiments; I.-Y.H., C.P., and J.B.B. provided knockout works, as they transduce environmental stimuli from cell surface mouse strains. A.V., N.R.N., and J.H.K. wrote and edited the manuscript. receptors to generate a coordinated biological response (4). Address correspondence and reprint requests to Dr. Neel R. Nabar and Dr. John H. Kehrl, B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Insti- Apart from the classical GPCR/G-protein signaling paradigm, tute of Allergy and Infectious Diseases, National Institutes of Health, 10 Center Drive, there exist several regulatory proteins that modulate G-protein Room 11A01, Bethesda, MD 20892. E-mail addresses: [email protected] (N.R.N.) and signaling. The regulators of G-protein signaling (RGS) family [email protected] (J.H.K.) consists of more than 30 members and is defined by the presence of Abbreviations used in this article: AGS, activator of G-protein signaling; Arg1, Arginase 1; BMDM, bone marrow–derived macrophage; ChemR23, chemerin receptor 23; FPR2, an RGS domain (5, 6). RGS proteins act as GTPase accelerating formyl peptide receptor 2; GPCR, G-protein–coupled receptor; iNOS, inducible proteins (GAPs) for specific Ga subunits. They enhance the in- NO synthase; KI, knock-in; KO, knockout; PTX, pertussis toxin; RGS, regulator of trinsic GTPase activity of Ga by stabilizing the GTPase transition G-protein signaling; WT, wild-type. state, accelerating the intrinsic Ga GTPase activity by as much as Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 100-fold (6, 7). In this way, RGS proteins limit the duration of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801145 2 A CRITICAL ROLE FOR Gai2 IN MACROPHAGE POLARIZATION

GPCR-mediated G-protein signaling, and G-proteins genetically previously described (32–35). All strains were backcrossed a minimum of modified to be RGS-insensitive proteins show higher basal levels of 12 times to C57BL/6J and experiments were performed using 6- to 14-wk- signaling (6). Conversely, the activators of G-protein signaling old mice with littermate controls. (AGS) family broadly functions as receptor independent activators Cell isolation and culture of G-proteins (8, 9). They do so by serving as alternative binding Bone marrow–derived macrophages (BMDMs) were prepared as previ- partners to influence subunit interactions independent of nucleotide ously described (36). Gai2-deficient macrophages were derived from exchange. Group II AGS proteins, including AGS3 and AGS4, are Gnai22/2 or Gnai2fl/flvav1-cre mice; no differences were observed be- of interest because of their high expression levels in macrophages tween strains. In certain experiments, pertussis toxin (PTX) (Sigma- (10). They exert their effects on heterotrimeric G-proteins through Aldrich) was added either on day 0 (50 ng/ml for days 0–2; 100 ng/ml their G-protein regulatory (GPR) motif, which allows them to bind for days 3–7) or on day 7 (200 ng/ml) overnight. Macrophages were M1 polarized using 1 mg/ml LPS from Escherichia coli (Enzo Life Sciences) to GDP-bound Ga, thereby regulating Gbg signaling (8). for4handpolarizedtowardM2bytreatmentwith20ng/mlmouse Several studies have shown that GPCRs can drive both M1 and M2 rIL-4 (R&D Systems) for 48 h. CD4+CD252 splenic Th cells were macrophage polarization (11). Specifically, formyl peptide receptor isolated by negative depletion using biotinylated Abs to B220 (RA3-6B2; 2 (FPR2), chemokine receptor CXCR3, and chemerin receptor BioLegend), CD8a (53.67; BD Biosciences), CD11b (M1/70; BioLegend), CD11c (HL3; BD Biosciences), and CD25 (PC61; BioLegend) and 23 (ChemR23) have all have been implicated. FPR2 has multiple Dynabeads M-280 Streptavidin (Thermo Fisher Scientific). T cell purity cognate ligands that drive either M1 or M2 polarization in a ligand- was .95%. specific manner (12–15). CXCR3 exhibits isoform restricted expression on M1 and M2 subsets, with each isoform functioning to Inflammasome activation assays polarize macrophages in opposite directions (16, 17). ChemR23, in BMDMs were primed with 1 mg/ml LPS for 4 h (signal 1), then stimulated Downloaded from contrast, is only expressed on M1 macrophages and like FPR2 has with the following activators (signal 2): 4 mM nigericin (Sigma-Aldrich) multiple ligands with opposing proinflammatory/proresolving ef- for 1 h (NLRP3), 1 mg/ml poly(dA:dT) (Sigma-Aldrich) transfected with Lipofectamine 2000 for 4 h (AIM2), and 0.5 mg/ml flagellin from Sal- fects. Of note, all three of these receptors couple the inhibitory class monella typhimurium (tlrl-pstfla; InvivoGen) transfected with Profect-P1 of heterotrimeric G-proteins (Gai), although the isoform/ligand- (Targeting Systems) for 4 h (NLRC4). Untreated and LPS only treated specific effects on Gai signaling are still unclear (18–22). Given control groups were included for all inflammasome experiments. BMDM

that these receptors have pleiotropic isoform/ligand-specific effects, culture supernatant was collected after inflammasome activation, and http://www.jimmunol.org/ these observations raise the possibility that Ga signaling functions IL-1b levels were measured using a commercially available kit following i the manufacturer’s protocol (R&D Systems). as a molecular switch regulating macrophage polarization. Inflammasomes are multimeric complexes that generate IL-1b,an Cytokine production assays important early mediator of the inflammatory response (23, 24). BMDMs were stimulated with 1 mg/ml LPS for 4 h. In certain experi- Each inflammasome consists of a sensor molecular (NLRP3, AIM2, ments, BMDMs were concomitantly treated with 10 mg/ml of either an or NLRC4), the adaptor protein ASC, and caspase-1, which cleaves IL-10 neutralizing Ab (JES5-2A5; BioLegend) or a rat IgG1, k isotype pro–IL-1b and pro–IL-18 upon activation (25, 26). Inflammasome control Ab (RTK2071; BioLegend). Cytokines were measured from activation is a two-step process. First, an initial priming signal BMDM culture supernatant using the following commercial kits: TNF-a

(R&D Systems), IL-6 (BioLegend), IL-10 (BioLegend), and IFN-b (PBL by guest on September 26, 2021 (signal 1) is needed to license inflammasome assembly and mediate Assay Science). the transcriptional upregulation of pro–IL-1b.Next,asensor- specific stimulus (signal 2) initiates inflammasome assembly via RNA isolation and quantitative RT-PCR activation of the sensor molecule. Macrophage polarization regulates RNA was isolated from BMDMs using RNeasy Mini Kit (Qiagen), and the priming phase of inflammasome activation (27). cDNA was synthesized using Omniscript RT Kit (Qiagen). RT-PCR Gai signaling has previously been shown to modulate macrophage was performed using the StepOne Real Time PCR System (Applied chemotaxis, phagocytosis, and activation (6, 28, 29). Although several Biosytems) and the QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions. Primers for TNF-a, IL-12p40, in- Gai-coupled GPCRs show opposing isoform/ligand-specific effects on ducible NO synthase (iNOS), Arginase 1 (Arg1), Fizz1, Ym1, and macrophage polarization, the role of Gai in macrophage polarization HPRT are from Zhu et al. (37) and the GAPDH primer is from Qiagen is unknown. In this study, we use genetically modified mice to directly (QT01658692). The results were normalized to HPRT or GAPDH, and relative mRNA levels were calculated using the 22DDCt cycle threshold investigate the physiological role of Gai and its accessory RGS/AGS proteins on inflammasome activation and macrophage polarization. (Ct) method. We find that Gai2 regulates IL-1b release upon NLRP3, AIM2, and Immunoblotting NLRC4 inflammasome activation via modulation of the LPS-priming For signaling experiments, BMDMs were treated with LPS (250 ng/ml) or phase (signal 1). This regulation stems from the biased M1 and M2 rIL-4 (20 ng/ml) for the indicated time. BMDM cell lysates were analyzed polarity of macrophages with excess and deficient Gai2 signaling, as described (36). Proteins were transferred onto a nitrocellulose mem- respectively. We further establish that these phenotypic differences are brane and subject to immunoblotting using the following primary Abs: built into the macrophage life history, as persistent chemical inhibition IL-1b (D4T2D; Cell Signaling Technology), phospho-p65 (Ser536) (93H1; of Ga is required to recapitulate the macrophage polarization Cell Signaling Technology), total p65 (no. 06-418; Millipore Sigma), i2 phospho-ERK (Thr202/Tyr204) (E10; Cell Signaling Technology), total phenotype seen in genetically modified cells. ERK (137F5; Cell Signaling Technology), phospho-JNK (Thr183/Tyr185) (no. 9251; Cell Signaling Technology), total JNK (D-2; Santa Cruz Bio- Materials and Methods technology), phospho-Akt (Ser473) (193H2; Cell Signaling Technology), 727 Animals total Akt (40D4; Cell Signaling Technology), phospho-STAT3 (Ser ) (no. 9134; New England BioLabs), total STAT3 (no. 9132; New England All animal experiments and protocols used were approved by the National BioLabs), phospho-STAT6 (Tyr641) (D8S9Y; Cell Signaling Technology), Institute of Allergy and Infectious Diseases Animal Care and Use total STAT6 (D-1; Santa Cruz Biotechnology), GAPDH (HRP-60004; Committee at the National Institutes of Health. C57BL/6J and C57BL/6J Proteintech), and b-actin–peroxidase Ab (A3854; Sigma-Aldrich). vav1-cre mice are from Jackson Laboratory. Gnai22/2, Gnai32/2, and Membranes were washed and incubated with the appropriate HRP- Gnai2fl/fl mice were provided by Dr. L. Birnbaumer (30) (National Institute conjugated secondary Ab (Cell Signaling Technology) prior to detec- on Environmental Health Sciences, National Institutes of Health), and the tion using Amersham Hyperfilm (GE Healthcare) or the iBright Gnai2G184S/G184S mice were a gift from Dr. R. Neubig (31) (Michigan FL1000 (Thermo Fisher Scientific). Blots were scanned and imported State University). Rgs19-deficient GFP knock-in (KI) mice were generated into Photoshop as unmodified tagged image file format. Quantification by Ozgene. Ric82/2, Rgs102/2, Rgs12/2, and Gpsm12/2 mice have been of band intensity was performed using standard methods on ImageJ. The Journal of Immunology 3

Phosphorylated protein bands were normalized to their respective total Results protein bands. All Western blot quantifications are presented as fold Ga signaling regulates IL-1b release after change of the indicated control. i2 inflammasome activation Flow cytometry To delineate the role of Gai and related RGS/AGS proteins on For cell surface staining, cells were stained with fluorochrome-conjugated inflammasome activation, we activated the NLRP3, AIM2, and or biotinylated Abs against CD11b (M1/70; BioLegend), F4/80 (BM8; NLRC4 inflammasomes in BMDMs from various genetically modi- BioLegend), CD169 (3D6.112; BioLegend), SIGN-R1 (eBio22D1; eBioscience), CD53 (OX-79; BioLegend), CD9 (MZ3; BioLegend), fied mice (36). Gai2 deficient BMDMs show a severe (2.5-fold) re- CD37 (Duno85; BioLegend), Tim1 (RMT1-4; BioLegend), Tim 2 (F37-2C4; ductioninIL-1b release for all inflammasomes assayed (Fig. 1A). To BioLegend), Tim 3 (RMT3.23; BioLegend), Tim 4 (RMT4-54; BioLegend), confirm that Gai2-deficient cells have decreased IL-1b release after CD63 (NVG-2; BioLegend), CD81 (Eat-2; BioLegend), and ADAM10 inflammasome activation, we looked at Ric-8a–deficient BMDMs. (139,712; R&D Systems). Biotin-labeled Abs were visualized with fluorochrome-conjugated streptavidin (Thermo Fisher Scientific). LIVE/DEAD Ric-8a is a molecular chaperone critical in targeting Gai subunits to the Fixable Aqua Stain (Thermo Fisher Scientific) was used to exclude dead plasma membrane (6); thus, Ric-8a deficiency leads to a vital loss of cells. For phosphorylated protein expression assays, cells were analyzed Gai subunits. Like Gai2-deficient BMDMs, Ric-8a–deficient BMDMs 180 182 with Abs against phospho-p38 (Thr /Tyr ) (28B10; Cell Signal- demonstrated reduced IL-1b release for all inflammasomes tested ing Technology) and total p38 (no. 9212; Cell Signaling Technology) (Fig. 1B). Collectively, this suggests that Ga is critical for IL-1b and detected using mouse or rabbit Alexa Fluor 647, respectively. i2 Data acquisitions were done on a FACSCanto II (BD Biosciences) release in macrophages independent of the inflammasome activated. flow cytometer and analyzed with FlowJo software version 9 or 10 To determine whether excess Gai2 signaling reciprocally affects IL-1b (Tree Star). release after inflammasome activation, we used Gnai2G184S/G184S Downloaded from T cell suppression assay KI BMDMs that express the RGS-insensitive Gai2 G184S allele. The Gai2 G184S mutation does not affect GPCR-Gai2 interactions or Gai2 Ninety six-well round bottom plates were coated with 1 mg/ml CD3 (145- stability but results in higher basal levels of Ga signaling because of 2C11; BD Biosciences) and 0.5 mg/ml CD28 (37.5; BD Biosciences). i2 Macrophages were M1 (250 ng/ml LPS) or M2 (20 ng/ml IL-4) polarized the inability of the Gai2 G184S allele to interact with RGS proteins prior to the experiment for 16 h. CD4+CD252 T cells were stained with (6). Consistent with our previous results, G184S KI macrophages CFSE proliferation dye (Thermo Fisher Scientific) and cocultured with released significantly more IL-1b than wild-type (WT) macrophages macrophages at a 1:2 ratio for 4 d in RPMI 1640 supplemented with 20 ng/ml for all inflammasomes assayed (3-fold for NLRP3 and AIM2, 40% http://www.jimmunol.org/ rIL-2 (R&D Systems). Upon collection, samples were stained with LIVE/ DEAD Fixable Near-IR Stain Dye (Thermo Fisher Scientific) and analyzed for NLRC4; Fig. 1C). This phenomenon was confirmed in BMDMs by flow cytometry. Proliferation modeling was performed using FlowJo 10.1, from mice lacking RGS10, the most highly expressed macrophage and results were normalized to the T cell–only control group. RGS protein (38). These BMDMs displayed increased IL-1b release Statistical analysis following NLRP3 and NLRC4 activation (Fig. 1D). Next, we determined whether Gai3-deficient BMDMs also show Data are expressed as a mean value 6 SEM. All results were confirmed in differences in IL-1b release after inflammasome activation. Interest- at least three independent experiments. Data were analyzed using Student t test or ordinary one-way ANOVA with multiple comparisons on Prism ingly, no difference was observed in Gai3-deficient BMDMs (Fig. 2A). software (GraphPad), and p , 0.05 was considered statistically significant Although both Gai2 and Gai3 are expressed in C57BL/6J BMDMs, the by guest on September 26, 2021 (*p , 0.05, **p , 0.005, and ***p , 0.001). Gnai2 transcript is five times more abundant, which may explain the

2/2 FIGURE 1. Gai2 signaling regulates inflammasome activation in BMDMs. BMDMs were generated from WT and (A)Gai2 KO (Gnai2 ), (B) Ric-8 KO (Ric82/2), (C) G184S KI (Gnai2G184S/G184S), or (D) RGS10 KO (Rgs102/2) mice. BMDM inflammasomes were activated as indicated in the methods section, and supernatant IL-1b was assayed by ELISA. The graphs shown are representative of five separate experiments with three replicates. The p values were determined using Student t test, and p , 0.05 was considered statistically significant. *p , 0.05, **p , 0.005, ***p , 0.001. 4 A CRITICAL ROLE FOR Gai2 IN MACROPHAGE POLARIZATION

relative contributions of Gai2 and Gai3 to inflammasome activation (6). after LPS priming, as opposed to IL-1b–specific secretion defects, are Additionally, Gai3 may have properties different from those of Gai2, responsible for the differences in IL-1b release observed after although it is unclear at present which of these properties are important inflammasome activation in the Gai2 deficient BMDMs. Consistently, for regulating IL-1b release after inflammasome activation. Mice immunoblotting of pro–IL-1b after LPS stimulation showed decreased lacking genes for the less well expressed Gai2 accessory proteins pro–IL-1b in the Gai2-deficient BMDMs and increased pro–IL-1b in (RGS1, RGS19, and AGS3) show no phenotypic change after inflam- the G184S KI BMDMs (Fig. 3C, 3D). Finally, analysis of the anti- masome activation (Fig. 2B–D), likely because of compensatory inflammatory cytokine IL-10 after LPS stimulation showed an in- overlap from other family members. Untreated and LPS only treated crease in the Gai2-deficient BMDMs and a decrease in the G184S KI control groups were included for all inflammasome activation ex- BMDMs (Fig. 3A, 3B). IL-10 is produced after LPS stimulation in periments,andnoIL-1b release was detected for these groups in all macrophages as an autocrine feedback mechanism to curtail the in- cases (data not shown). In sum, our data indicates that deficiencies in flammatory response (41). Thus, we next investigated whether the Gai2 but not Gai3 signaling decrease IL-1b release after inflamma- increased IL-10 production in Gai2-deficient BMDMs contributes to some activation. In contrast, prolonged Gai2 signaling enhances it. the IL-6 and TNF-a production defects observed in Gai2-deficient BMDMs. Neutralization of IL-10 during LPS stimulation rescued both Ga -mediated signaling plays a role in the priming phase of i2 IL-6 and TNF-a production in Ga -deficient BMDMs (Fig. 3E), inflammasome activation i2 emphasizing the importance of the increased IL-10 production in The observation that Gai2-dependent signaling consistently affects Gai2-deficient BMDMs. IL-1b releaseacrossthreetypesofinflammasomesarguesagainsta a

G i2-mediated signaling is critical in macrophage polarization Downloaded from role for Gai2 in inflammasome assembly, as each inflammasome has a different assembly mechanism. Rather, this observation points to- Increased IL-10 despite decreases in TNF-a and IL-6 in Gai2- ward Gai2 modulating either the LPS priming or the IL-1b secretion deficient BMDMs with reciprocal changes in G184S KI BMDMs phase, which share mechanisms across inflammasomes (39). To de- suggests alterations in Gai2 signaling bias macrophage phenotypic termine the phase of Gai2 involvement, we mimicked inflammasome determination. To determine the M1/M2 phenotype propensity priming (signal 1) by treatment with LPS and monitored some key of Gai2-deficient and G184S KI BMDMs, we examined mRNA

downstream cytokines: TNF-a, IL-6, and IL-10 (40). IL-1b is se- levels of standard proinflammatory M1 markers (TNF-a, http://www.jimmunol.org/ creted via a noncanonical pathway that utilizes different machinery IL-12p40, and iNOS) after LPS stimulation and standard anti- from standard cytokines; thus, changes in supernatant IL-1b without inflammatory M2 markers (Arg1, Ym1, and Fizz1) after IL-4 changes in TNF-a andIL-6secretionsuggestIL-1b–specific secre- stimulation. LPS-stimulated Gai2-deficient BMDMs showed de- tion defects. Conversely, changes in IL-1b secretion with accompa- creased TNF-a, IL-12p40, and iNOS mRNA and increased Ym1 nying changes in TNF-a and IL-6 point toward global inflammatory and Fizz1 mRNA after IL-4 stimulation (Fig. 4A, 4B). In contrast, changes affecting the LPS priming phase. After LPS stimulation, G184S KI BMDMs had higher levels of proinflammatory markers Gai2-deficient BMDMs secrete less TNF-a andIL-6thanWTcon- (TNF-a, IL-12p40, and iNOS) after LPS and lower levels of anti- trols, whereas G184S KI BMDMs secrete more of these cytokines inflammatory markers (Arg1, Ym1, Fizz1) after IL-4 stimulation (Fig. 3A, 3B). These results suggest global inflammatory differences (Fig. 4C, 4D). These results are consistent with our observed by guest on September 26, 2021

2/2 FIGURE 2. Gai3 deficiency does not affect inflammasome activity in macrophages. BMDMs were generated from WT and (A)Gai3 KO (Gnai3 )mice, (B)RGS1KO(Rgs12/2), (C)RGS19KO(Rgs192/2), or (D)AGS3KO(Gpsm12/2) mice. BMDM inflammasomes were activated as indicated in the Materials and Methods section, and the supernatant IL-1b was assayed by ELISA. The graphs shown are representative of five separate experiments with three replicates. The p values were determined using Student t test, and p , 0.05 was considered statistically significant. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

2 2 / by guest on September 26, 2021 FIGURE 3. Gai2 deficiency causes global inflammatory changes. BMDMs generated from WT and (A)Gai2 KO (Gnai2 )or(B) G184S KI (Gnai2G184S/G184S) mice were stimulated with LPS (1 mg/ml) for 4 h. The supernatant was collected and assayed for TNF-a, IL-6, and IL-10 by ELISA. The bar graphs shown are representative of five separate experiments with three replicates. (C) Immunoblotting for pro–IL-1b after stimulation with LPS

(250 ng/ml) in WT and Gai2-deficient BMDM lysates. (D) Immunoblotting for pro–IL-1b after stimulation with LPS (250 ng/ml) in WT and G184S KI 2/2 BMDM lysates. (E) BMDMs generated from WT and Gai2 KO (Gnai2 ) mice were stimulated with LPS (1 mg/ml) for 4 h in the presence of either 10 mg/ml isotype control or IL-10 neutralizing Ab. The supernatant was collected and assayed for TNF-a and IL-6. The graphs shown are representative of five separate experiments with three replicates. The p values were determined using Student t test or ordinary one-way ANOVA followed by multiple comparisons, and p , 0.05 was considered statistically significant. **p , 0.005, ***p , 0.001.

cytokine and inflammasome changes, indicating that Gai2 deficiency thepresenceofPTXfromday0today7(42).Onday7,cells skews macrophages toward an M2 phenotype, whereas excess Gai2 were treated with either LPS or IL-4, followed by transcript signaling results in a tendency toward the proinflammatory M1 analysis of the pro- and anti-inflammatory markers to determine phenotype. Taken together, this suggests that Gai2 signaling is critical macrophage polarization. Interestingly, BMDMs differentiated in macrophage phenotype determination. in the presence of PTX (long-term) demonstrated a reduction in M1 genes upon LPS stimulation (Fig. 5B) and a corresponding Long-term, but not short-term, inhibition of Ga signaling i2 induction M2 genes upon IL-4 treatment (Fig. 5C). Together, recapitulates inflammatory changes these data indicate that prolonged Gai2 signaling plays a critical To investigate the mechanism through which Gai2 affects mac- role in the establishment of macrophage phenotype, potentially rophage phenotype, we assayed inflammasome activation in during growth and differentiation. BMDMs treated overnight with PTX. PTX catalyzes the ADP- Gai2 modulates signaling downstream of LPS and IL-4 ribosylation of Gai, which blocks receptor/G-protein interaction and inhibits Gai from undergoing GPCR-mediated nucleotide To investigate the mechanisms through which Gai2 alters mac- exchange. Unexpectedly, overnight treatment with PTX showed rophage polarization, we stimulated WT and Gai2-deficient no differences in IL-1b secretion after inflammasome activation BMDMs with LPS and monitored critical downstream signal- (Fig. 5A). It is unlikely that nucleotide exchange independent ing outputs. NF-kB signaling is a major inflammatory pathway effects of Gai2 are responsible for these changes, given the re- downstream of LPS (43), but our results show Gai2-deficient ciprocal phenotypes of Gai2-deficient and G184S KI BMDMs. BMDMs have no alterations in NF-kB signaling as assayed by p65 Thus, we investigated whether long-term inhibition of Gai2 phosphorylation (Fig. 6A). LPS/TLR4 signaling also results in during macrophage development affects phenotype determina- MyD88-dependent activation of the MAPK cascade, activating tion. To test this, we eliminated Gai signaling during the course ERK1/2 (p42/44), JNK1/2 (p46/54), and p38 (43). Activation of of macrophage development by growing bone marrow cells in MAPK cascade proteins is important in the cytokine production 6 A CRITICAL ROLE FOR Gai2 IN MACROPHAGE POLARIZATION Downloaded from

FIGURE 4. Gai2 signaling is a critical determinant of macrophage proinflammatory (M1) versus anti-inflammatory (M2) phenotype. (A) Expression of M1 genes in Gai2-deficient BMDMs after treatment with LPS (1 mg/ml) for 4 h. (B) Expression of M2 genes in Gai2-deficient BMDMs after treatment with IL-4 (20 ng/ml) for 48 h. (C) Expression of M1 genes in G184S KI BMDMs after treatment with LPS (1 mg/ml) for 4 h. (D) Expression of M2 genes in G184S KI BMDMs after treatment with IL-4 (20 ng/ml) for 48 h. Data are presented as fold change relative to the WT control. Experiments were conducted at least three times in triplicate. The p values were determined using Student t test, and p , 0.05 was considered statistically significant. *p , 0.05, **p , 0.005, ***p , 0.001. http://www.jimmunol.org/ downstream of LPS (44). Gai2-deficient BMDMs exhibit defects and monitored IFN-b.Gai2-deficient BMDMs release signifi- in ERK1/2 activation but no changes in JNK1/2 or p38 activation cantly more IFN-b after LPS stimulation, whereas G184S KI after LPS stimulation (Fig. 6B, 6C). LPS/TLR4 signaling also BMDMs release less (Fig. 6D). This result suggests that Gai2 activates IRF3 in a MyD88-independent manner, resulting in the restricts IRF3-mediated signaling downstream of LPS. Finally, production of type 1 IFNs (43). To monitor this pathway, we both STAT3 and the kinase Akt are activated after LPS stimula- stimulated WT, Gai2-deficient, and G184S KI BMDMs with LPS tion (45–47). Gai2-deficient BMDMs show significant STAT3 by guest on September 26, 2021

FIGURE 5. Long-term, but not short term, inhibition of Gai2 signaling recapitulates the phenotypes seen in genetically altered BMDMs. (A) BMDMs from WT mice treated at only day 7 with PTX (200 ng/ml) overnight before inflammasome activation. BMDM culture supernatants were collected and IL-1b was assayed by ELISA. The bar graphs shown are representative of five separate experiments with three replicates. (B) Expression of M1 genes in PTX-treated BMDMs (50 ng/ml for days 0–2; 100 ng/ml for days 3–7) after treatment with LPS (1 mg/ml) for 4 h. (C) Expression of M2 genes in PTX-treated BMDMs (50 ng/ml for days 0–2; 100 ng/ml for days 3–7) after treatment with IL-4 (20 ng/ml) for 48 h. RT-PCR experiments were conducted at least three times in triplicate. Data are presented as fold change relative to the WT control. The p values were determined using Student t test, and p , 0.05 was considered statistically significant. **p , 0.005, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

2/2 FIGURE 6. Gai2 modulates signaling downstream of LPS and IL-4. BMDMs generated from WT and Gai2 KO (Gnai2 ) mice were treated with LPS (250 ng/ml) for the indicated time. Whole cell lysates were collected and separated on an SDS-PAGE gel. Immunoblots are shown for (A) NF-kB–related protein p-65 (phosphorylated and total) and GAPDH and (B) MAPK cascade proteins ERK1/2 (phosphorylated and total) and JNK1/2 (phosphorylated and total). (C) Phosflow analysis of p38 phosphorylation after LPS stimulation (250 ng/ml) for the indicated time in BMDMs generated from WT and Gai2 KO (Gnai22/2) mice. Data are presented as the mean fluorescence intensity ratio of phospho-p38 to total p38 and normalized to the control. (D) BMDMs 2/2 generated from WT and Gai2 KO (Gnai2 ) mice were treated with LPS (1 mg/ml) for 4 h, and cell culture supernatant was collected and assayed for IFN-b.(E) Immunoblotting for STAT3 (phosphorylated and total) and Akt (phosphorylated and total) from lysates obtained from LPS-stimulated (250 ng/ml) 2/2 2/2 WT or Gai2 KO (Gnai2 )mice.(F) BMDMs generated from WT and Gai2 KO (Gnai2 ) mice were treated with IL-4 (20 ng/ml) for the indicated time. Immunoblotting for STAT6 (phosphorylated and total) and actin are shown. Phosphorylated protein bands are quantified relative to their respective total protein band. Band intensities are normalized to the indicated control. Experiments were performed a minimum of three times. The p values were determined using Student t test, and p . 0.05 was considered statistically significant. **p , 0.005.

activation defects after LPS stimulation and enhanced Akt acti- inflammatory enhancers (48). Gai2-deficient BMDMs show ele- vation both basally and after LPS stimulation (Fig. 6E). Col- vated levels of STAT6 activation after IL-4 stimulation, consistent lectively, these data indicate that Gai2 modulates signaling with the M2 skewed phenotype seen in Gai2-deficient BMDMs downstream of TLR4/LPS, which may play a role in the observed (Fig. 6F). macrophage polarization phenotypes observed. To determine if Gai2-deficient macrophages have both functional and Gai2 affects signaling downstream of IL-4, we stimulated WT and qualitative changes Gai2-deficient BMDMs with IL-4–assayed STAT6 activation. STAT6 is an essential transcription factor downstream of IL-4 Whereas M1 type macrophages promote T cell proliferation, M2 that functions as a transcriptional repressor to limit activation of macrophages are suppressive and inhibit T cell proliferation (49). 8 A CRITICAL ROLE FOR Gai2 IN MACROPHAGE POLARIZATION

To test whether Gai2-deficient macrophages have altered T cell LPS priming (27). Specifically, M1-polarized macrophages have suppression characteristics, we cocultured naive Th cells higher basal levels and more robustly upregulate pro–IL-1b + 2 (CD4 CD25 ) with either M1 or M2 polarized WT or Gai2- mRNA after LPS stimulation compared with their M2 counter- deficient BMDMs while stimulating with IL-2 and plate-bound parts. These transcriptional differences in pro–IL-1b levels affect anti-CD3/CD28. As expected, coculture with M1-polarized WT IL-1b release after inflammasome activation, as the same study macrophages induced significant proliferation, whereas M1-polarized showed M1 macrophages release more IL-1b than M2 macro- Gai2-deficient macrophages induced significantly less prolifer- phages after NLRP3 activation (27). ation (Fig. 7A). M2 polarization of WT macrophages signifi- Apart from changes in inflammasome activation, our data show cantly suppressed T cell proliferation compared with the M1 phenotypic differences in cytokine release after LPS stimulation in macrophages, and this suppression was augmented in M2- Gai2-deficient and G184S KI BMDMs. Of note, we find that the polarized Gai2-deficient BMDMs (Fig. 7A). Together, these increased IL-10 production in Gai2-deficient macrophages is im- data suggests that Gai2-deficient BMDMs have functional portant in limiting production of IL-6 and TNF-a in these mac- changes that fit the M1/M2 paradigm. Finally, we investigated if rophages. With regard to the mechanism through which Gai2 Gai2-deficient BMDMs express a variety of macrophages regulates macrophage polarization and cytokine production, many markers at different levels than WT BMDMs. Interestingly, Gai2 lines of evidence suggest that Gai signaling plays a direct role in knockout (KO) BMDMs had decreased levels of F4/80 and LPS-induced TLR4 signal transduction (50–52). For example, CD169 and increased levels of SIGN-R1 and Tim2 (Fig. 7B). Gai2 is phosphorylated in response to LPS and coimmunopreci- Together, these data suggest that Gai2 KO BMDMs have func- pitates with the LPS coreceptor CD14 (53, 54), and genetic tional changes affecting macrophage/T cell interactions and deletion of Gai2 decreases the activation of signaling pathways Downloaded from qualitative phenotypic differences after in vitro culture. downstream of TLR4 (51, 55, 56). Although NF-kB is an essential transcription factor downstream of LPS responsible for pro– Discussion IL-1b, IL-6, and TNF-a transcription (43, 57), our data are in Our study identifies that Gai2 signaling regulates IL-1b release consensus with the literature in indicating that Gai2-deficient after inflammasome activation, likely because of differences in macrophages exhibit no changes in NF-kB signaling after TLR4

inflammasome priming related to biased M1 and M2 polarization activation (51, 56). http://www.jimmunol.org/ in G184S KI and Gai2-deficient BMDMs, respectively. Previous We observe increased IFN-b production and Akt activa- studies have established that macrophage polarization affects tion in Gai2-deficient macrophages after LPS stimulation, and transcriptional upregulation of inflammasome-related genes upon both IFN-b and Akt have previously been implicated in IL-10 by guest on September 26, 2021

FIGURE 7. Gai2-deﬁcient macrophages have functional and qualitative defects. (A) CSFE proliferation peaks of puriﬁed T cells stimulated with IL-2 (20 ng/ml), anti-CD3 (1 mg/ml), and anti-CD28 (0.5 mg/ml) and cocultured with LPS- or IL-4–treated

WT or Gai2-deficient BMDMs. Proliferation index and division index are normalized to the T cell–only control and shown for each group. (B) Mean fluorescence intensity of several cell surface markers in day 7 BMDMs from WT or Gai2 KO mice. Experi- ments were performed a minimum of three times. Significance was measured by ANOVAwith multiple comparisons, and p , 0.05 was considered statistically significant. *p , 0.05, ***p , 0.001. The Journal of Immunology 9 production downstream of LPS (41, 47). Increased signaling this study are consistent with our previous findings that unprimed through these pathways may be responsible for the observed in- G184S KI macrophages have enhanced phagocytic capacity crease in IL-10 in Gai2-deficient macrophages, which is important (29, 66). Our data are also consistent with a study on RGS10- in limiting IL-6 and TNF-a production. We also find defects in deficient macrophages (38). RGS10 is the best expressed RGS ERK1/2 and STAT3 activation in Gai2-deficient macrophages protein in macrophages, and absence of RGS10 decelerates after LPS stimulation. ERK1/2 activation downstream of LPS has GTPase activity of the Gai subunit (like the Gai2 G184S KI previously been shown to contribute to pro–IL-1b, IL-6, and TNF-a allele). Lee et al. (38) showed that RGS10-deficient peritoneal production (44, 58), and acute STAT3 activation downstream of and BMDMs are proinflammatory and M1 skewed. TLR4 has been shown to play a role in IL-6 and pro–IL-1b pro- The existing data on Gai-coupled GPCR signaling remains duction (46). The altered signaling downstream of TLR4 likely difficult to interpret, as the functional consequences of specific plays an important role in the observed phenotypes after LPS ligands on Gai signaling is not well established. FPR2 promotes stimulation. In addition to alterations after LPS stimulation, we M1 polarization upon stimulation with the cathelicidin-associated see greater M2 (alternative) activation after IL-4 signaling in microbial peptide LL-37 or serum amyloid protein A (12). In Gai2-deficient macrophages and less M2 activation in G184S KI contrast, this same receptor promotes M2 polarization when macrophages. Signaling studies upon IL-4 stimulation show bound by annexin A1, lipid lipoxin A4, or the anti-inflammatory greater STAT6 activation in Gai2-deficient macrophages. STAT6 molecule resolvin D1 (12, 15). Studies in FPR2 KO mice show is a critical transcription factor activated by IL-4 that acts as a that loss of FPR2 promotes an M2 macrophage phenotype and transcriptional repressor to limit inflammatory enhancer activation tumor progression (13, 14), suggesting the physiological role of Downloaded from in macrophages (48). Differences in M2 activation in Gai2 defi- FPR2 is to promote M1 polarization. As FPR2 couples Gai cient and G184S KI macrophages after IL-4 stimulation may be proteins and is known to be constitutively active (18, 67), it is related to signaling downstream of IL-4. possible that the M2 polarization seen in FPR2 KO cells stems

We note that in addition to Gai2 modulating signaling down- from a reduction of Gai2 signaling. Of note, FPR2 activation stream of LPS and IL-4, other mechanisms may exist that con- of Gai2 results in concurrent Gbg dissociation, which may tribute to the observed macrophage polarization phenotypes. For play a role in the observed macrophage polarization phenotype. example, M1-polarized macrophages are characterized in part http://www.jimmunol.org/ CXCR3, which also couples Gai, is similarly implicated in M1 by their dependence on glycolysis, whereas M2-polarized mac- polarization, as CXCR3 KO macrophages are M2 biased and rophages are reliant on oxidative metabolism (59). The tran- promote tumorigenesis (17). Although CXCR3 ligands (CXCL9–11) scriptional upregulation of pro–IL-1b after LPS stimulation is have similar effects on polarization, recent studies show that dependent on this glycolytic switch, which activates the tran- CXCR3 has multiple isoforms that are differentially expressed on scription factor HIF-1a, enabling its direct binding to the Il1b M1 (CXCR3.1) and M2 (CXCR3.2) macrophages. These isoforms promoter (60). Our findings warrant further investigation into have opposing effects on macrophage polarization, as stimulation of whether metabolic changes in Gai2-deficient and G184S KI CXCR3.1 promotes an M1 phenotype, whereas CXCR3.2 promotes BMDMs are responsible for alterations in pro–IL-1b upregulation an M2 phenotype (16). These CXCR3-related phenomena may be after LPS stimulation. Additionally, epigenetic changes have been by guest on September 26, 2021 related to CXCR3 isoform-specific differences in Gai coupling. implicated in regulating macrophage polarization (61, 62). An in- Finally, the ChemR23 receptor is expressed only on M1 macro- teresting observation in our study pointing to the existence of phages; it too couples Ga and has ligand-specific proinflammatory a i mechanisms in addition to G i2 regulating signaling downstream of (chemerin) and proresolving (resolvin E1) affects (68). LPS and IL-4 is that short-term inhibition of Ga using PTX (prior i2 Importantly, the question remains as to whether prolonged Ga to TLR4 stimulation with LPS) does not recapitulate the LPS- i2 inhibition itself reprograms macrophages toward the M2 phe- a dependent inflammasome-related changes seen in G i2-deficient notype or whether this inhibition must occur during macrophage macrophages, whereas 7-d inhibition of Ga during differentia- i2 differentiation. The former scenario has major therapeutic im- tion from bone marrow recapitulates the anti-inflammatory phe- plications, as prolonged stimulation or inhibition of macrophage- notype. The mechanisms responsible for this phenomenon remain specific Ga -coupled GPCRs could lay the foundation for unclear, but these finding raise the possibility that changes re- i2 the treatment of many inflammatory diseases via macrophage sponsible for the macrophage polarization phenotype are built into reprograming. Finally, although the M1/M2 paradigm is useful the macrophage life history. for in vitro evaluation of macrophage phenotype, several mac- Our studies generally exhibit a consensus with regard to cy- rophage subsets exist in vivo. Notably, Ga -deficient BMDMs tokine response between Ga -deficient BMDMs investigated in i2 i2 have alterations in SIGN-R1 and CD169 expression, both of this study and Ga -deficient peritoneal macrophages previously i2 which define specific splenic and lymph node macrophage sub- studied (52, 55). However, G184S KI peritoneal macrophages sets with unique functions (69). Moving forward, further inves- were previously reported to have decreased cytokine release tigation into the role of Ga in these macrophage subsets and after LPS stimulation, whereas our study found increased TNF-a i2 their role in disease pathogenesis is warranted. and IL-6 (63). This discrepancy may be explained by develop- mental differences between peritoneal and BMDMs. Peritoneal macrophages are resident tissue macrophages, which are typi- Acknowledgments cally derived prenatally from embryonic precursors as opposed We thank Dr. Anthony Fauci for long-standing support. to adult hematopoietic stem cells (64). BMDMs generated in vitro likely share more similarities with in vivo monocyte- Disclosures derived macrophages, which are of hematopoietic origin (64). The authors have no financial conflicts of interest. 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