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

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 Gα signaling links these receptors to downstream effectors. Several Gαi-coupled G-protein-coupled receptors have been implicated in macrophage polarization. In this study, we use genetically modified mice to investigate the role of Gαi2 on inflammasome activity and macrophage polarization. We report that Gαi2 in murine bone marrow-derived macrophages (BMDMs) regulates IL-1ß release after activation of the NLRP3, AIM2, and NLRC4 inflammasomes. We show this regulation stems from the biased polarity of Gαi2 deficient (Gnai2 -/-) and RGS-insensitive Gαi2 (Gnai2 G184S/G184S) BMDMs. We determined that although Gnai2 G184S/G184S BMDMs (excess Gαi2 signaling) have a tendency toward classically activated proinflammatory (M1) phenotype, Gnai2-/- BMDMs (Gαi2 deficient) are biased toward alternatively activated anti-inflammatory (M2) phenotype. Finally, we find that Gαi2-deficient macrophages have increased Akt activation and IFN-ß production but defects in ERK1/2 and STAT3 activation after LPS stimulation. Gαi2-deficient macrophages also exhibit increased STAT6 activation after IL-4 stimulation. In summary, our data indicates that excess Gαi2 signaling promotes an M1 macrophage phenotype, whereas Gαi2 signaling deficiency promotes an M2 phenotype. Understanding Gαi2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.

Normal Thymocyte Egress, T Cell Trafficking, and CD4+ T Cell Homeostasis Require Interactions between RGS Proteins and Gαi2.

Adaptive immunity depends on mature thymocytes leaving the thymus to enter the bloodstream and the trafficking of T cells through lymphoid organs. Both of these require heterotrimeric Gαi protein signaling, whose intensity and duration are controlled by the regulator of G protein signaling (RGS) proteins. In this study, we show that RGS protein/Gαi2 interactions are essential for normal thymocyte egress, T cell trafficking, and homeostasis. Mature thymocytes with a Gαi2 mutation that disables RGS protein binding accumulated in the perivascular channels of thymic corticomedullary venules. Severe reductions in peripheral naive CD4+ T cells and regulatory T cells occurred. The mutant CD4+ T cells adhered poorly to high endothelial venules and exhibited defects in lymph node entrance and egress. The kinetics of chemokine receptor signaling were disturbed, including chemokine- induced integrin activation. Despite the thymic and lymph node egress defects, sphingosine-1-phosphate signaling was not obviously perturbed. This study reveals how RGS proteins modulate Gαi2 signaling to facilitate thymocyte egress and T cell trafficking.

B Lymphocyte-Specific Loss of Ric-8A Results in a Gα Protein Deficit and Severe Humoral Immunodeficiency.

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a highly evolutionarily conserved cytosolic protein initially identified in Caenorhabditis elegans, where it was assigned a regulatory role in asymmetric cell divisions. It functions as a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13 and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes in embryonic stem cell lines. To test its role in hematopoiesis and B lymphocytes specifically, we generated ric8 (fl/fl) vav1-cre and ric8 (fl/fl) mb1-cre mice. The major hematopoietic cell lineages developed in the ric8 (fl/fl) vav1-cre mice, notwithstanding severe reduction in Gαi2/3, Gαq, and Gα13 proteins. B lymphocyte-specific loss of Ric-8A did not compromise bone marrow B lymphopoiesis, but splenic marginal zone B cell development failed, and B cells underpopulated lymphoid organs. The ric8 (fl/fl) mb1-cre B cells exhibited poor responses to chemokines, abnormal trafficking, improper in situ positioning, and loss of polarity components during B cell differentiation. The ric8 (fl/fl) mb1-cre mice had a severely disrupted lymphoid architecture and poor primary and secondary Ab responses. In B lymphocytes, Ric-8A is essential for normal Gα protein levels and is required for B cell differentiation, trafficking, and Ab responses.

An essential role for RGS protein/Gαi2 interactions in B lymphocyte-directed cell migration and trafficking.

Chemokines engage B lymphocyte surface receptors, triggering heterotrimeric G protein Gαi subunit guanine nucleotide exchange. RGS proteins limit the duration that Gαi subunits remain GTP bound, and the loss of an individual RGS protein typically enhances chemokine receptor signaling. In this study, we show that B cells carrying a Gαi2 (G184S/G184S) mutation that disables all RGS protein/Gαi2 interactions exhibit an unexpectedly severe reduction in chemokine receptor signaling. The Gαi2 (G184S/G184S) B cells have markedly elevated basal calcium levels, but poor chemokine-induced increases, enhanced nonspecific migration, but extremely poor chemotaxis. In striking contrast, the Gαi2 (G184S/G184S) B cells exhibited enhanced sensitivity to sphingosine 1-phosphate (S1P). S1P elicited heightened intracellular calcium responses and enhanced S1P-triggered cell migration. Mice with the Gαi2 (G184S/G184S) mutation displayed excessive numbers of germinal center-like structures; abnormal serum Ig profiles; and aberrant B lymphocyte trafficking. These findings establish an essential role for RGS proteins in B cell chemoattractant signaling and for the proper position of B lymphocytes in lymphoid organs.

Canonical and noncanonical g-protein signaling helps coordinate actin dynamics to promote macrophage phagocytosis of zymosan.

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gαi nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gαi proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca(2+) that was partially sensitive to Gαi nucleotide exchange inhibition and expression of GTP-bound Gαi recruited Elmo1 to the plasma membrane. Reducing GDP-Gαi nucleotide exchange, decreasing Gαi expression, pharmacologically interrupting Gßγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gαi remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.

Interplay between T(h)1 and T(h)17 effector T-cell pathways in the pathogenesis of spontaneous colitis and colon cancer in the Gαi2-deficient mouse.

Gαi2-deficient mice spontaneously develop colitis. Using xMAP technology and RT-PCR, we investigated cytokine/chemokine profiles during histologically defined phases of disease: (i) no/mild, (ii) moderate, (iii) severe colitis without dysplasia/cancer and (iv) severe colitis with dysplasia/cancer, compared with age-matched wild-type (WT) littermates. Colonic dysplasia was observed in 4/11 mice and cancer in 1/11 mice with severe colitis. The histology correlated with progressive increases in colon weight/cm and spleen weight, and decreased thymus weight, all more advanced in mice with dysplasia/cancer. IL-1ß, IL-6, IL-12p40, IL-17, TNF-α, CCL2 and CXCL1 protein levels in colons, but not small intestines increased with colitis progression and were significantly increased in mice with moderate and severe colitis compared with WT mice, irrespective of the absence/presence of dysplasia/cancer. CCL5 did not change during colitis progression. Colonic IL-17 transcription increased 40- to 70-fold in all stages of colitis, whereas IFN-γ mRNA was gradually up-regulated 12- to 55-fold with colitis progression, and further to 62-fold in mice with dysplasia/cancer. IL-27 mRNA increased 4- to 15-fold during the course of colitis, and colonic IL-21 transcription increased 3-fold in mice with severe colitis, both irrespective of the absence/presence of dysplasia/cancer. FoxP3 transcription was significantly enhanced (3.5-fold) in mice with moderate and severe colitis, but not in mice with dysplasia/cancer, compared with WT mice. Constrained correspondence analysis demonstrated an association between increased protein levels of TNF-α, CCL2, IL-1ß, IL-6 and CXCL1 and dysplasia/cancer. In conclusion, colonic responses are dominated by a mixed T(h)1/T(h)17 phenotype, with increasing T(h)1 cytokine transcription with progression of colitis in Gαi2(-/-) mice.

The loss of RGS protein-Gα(i2) interactions results in markedly impaired mouse neutrophil trafficking to inflammatory sites.

Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gα(i) subunits to exchange GDP for GTP. By limiting the duration that Gα(i) subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gα(i2) interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gα(i) activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.

IEX-1 deficiency protects against colonic cancer.

The immediate early response gene X-1 (IEX-1) is involved in regulation of various cellular processes including proliferation, apoptosis in part by controlling homeostasis of reactive oxygen species (ROS) at mitochondria. The present study shows reduced inflammatory responses and colorectal cancer in IEX-1 knockout (KO) mice treated with azoxymethane/dextran sulfate sodium (DSS). However, DSS induced worse colitis in RAG(-/-)IEX-1(-/-) double KO mice than in RAG and IEX-1 single KO mice, underscoring an importance of T cells in IEX-1 deficiency-induced protection against colon inflammation. Lack of IEX-1 promoted the differentiation of interleukin (IL)-17-producing T cells, concomitant with upregulation of Gαi2 expression, a gene that is well-documented for its role in the control of inflammation in the colon. In accordance with this, T-helper 17 (T(H)17) cell differentiation was compromised in the absence of Gαi2, and deletion of Gαi2 in T cells alone aggravated colon inflammation and colorectal cancer development after azoxymethane/DSS treatment. Null mutation of IEX-1 also enhanced both proliferation and apoptosis of intestinal epithelial cells (IEC) after injury. A potential impact of this altered IEC turnover on colon inflammation and cancer development is discussed. These observations provide a linkage of IEX-1 and Gαi2 expression in the regulation of T(H)17 cell differentiation and suggest a previously unappreciated role for IEX-1 in the control of colon epithelial homeostasis.

G alpha i2 and ZAP-70 mediate RasGRP1 membrane localization and activation of SDF-1-induced T cell functions.

RasGRP1, a Ras guanine-nucleotide exchange factor, critically mediates T cell development and function and controls immunodeficiency and autoimmunity. In this study, we describe a unique mechanism of mobilization and activation of RasGRP1 in response to SDF-1, a chemokine that signals via the G protein-coupled receptor CXCR4. Depletion of RasGRP1 impaired SDF-1-stimulated human T cell migration, expression of the activation marker CD69, and activation of the ERK MAPK pathway, indicating that RasGRP1 mediates SDF-1 functions. SDF-1 treatment caused RasGRP1 to localize to the plasma membrane to activate K-Ras and to the Golgi to activate N-Ras. These events were required for cellular migration and for ERK activation that mediates downstream transcriptional events in response to SDF-1. SDF-1-dependent localization of RasGRP1 did not require its diacylglycerol-binding domain, even though diacyglycerol was previously shown to mediate localization of RasGRP1 in response to Ag stimulation. This domain was, however, required for activity of RasGRP1 after its localization. Intriguingly, SDF-1 treatment of T cells induced the formation of a novel molecular signaling complex containing RasGRP1, Gαi2, and ZAP-70. Moreover, SDF-1-mediated signaling by both Gi proteins and ZAP-70 was required for RasGRP1 mobilization. In addition, RasGRP1 mobilization and activation in response to SDF-1 was dependent on TCR expression, suggesting that CXCR4 heterodimerizes with the TCR to couple to ZAP-70 and mobilize RasGRP1. These results increase understanding of the molecular mechanisms that mediate SDF-1 effects on T cells and reveal a novel mechanism of RasGRP1 regulation. Other G protein-coupled receptors may similarly contribute to regulation of RasGRP1.

FTY720 blocks egress of T cells in part by abrogation of their adhesion on the lymph node sinus.

Egress of lymphocytes from lymphoid tissues is a complex process in which Gαi-mediated signals play a decisive role. We show here that although FTY720, an agonist of the sphingosine 1-phosphate (S1P)(1) receptor, induces S1P(1) receptor internalization sufficiently in the presence or absence of Gαi2 or Gαi3, the drug blocks egress of wild-type (WT) and Gαi3-deficent T cells, but not Gαi2-deficient T cells, in both WT and Gαi2-deficient hosts. Intravital imaging of lymph nodes revealed that all three groups of T cells approached and engaged cortical sinusoids similarly in the presence or absence of FTY720. The cells also entered and departed the sinus at an almost identical frequency in the absence of the drug. However, after engagement of the sinus, most WT and Gαi3-deficient T cells retracted and migrated back into the parenchyma in FTY720-treated animals, due to a failure of the cells to establish adhesion on the sinus, whereas Gαi2-deficient T cells adhered firmly on the sinus, which prevented their retraction, facilitating their transmigration of the lymphatic endothelial barrier. These data confirm egress of Gαi2(-/-) T cells independent of S1P-mediated chemotaxis and failure of FTY720 to close lymphatic stromal channels and argue for the first time, to our knowledge, that FTY720 induces lymphopenia in part by impairing T cell adhesion to the sinus in a manner dependent on Gαi2.

Potential role and mechanism of IFN-gamma inducible protein-10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in rheumatoid arthritis.

INTRODUCTION: IFN-gamma inducible protein-10 (CXCL10), a member of the CXC chemokine family, and its receptor CXCR3 contribute to the recruitment of T cells from the blood stream into the inflamed joints and have a crucial role in perpetuating inflammation in rheumatoid arthritis (RA) synovial joints. Recently we showed the role of CXCL10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in an animal model of RA and suggested the contribution to osteoclastogenesis. We tested the effects of CXCL10 on the expression of RANKL in RA synoviocytes and T cells, and we investigated which subunit of CXCR3 contributes to RANKL expression by CXCL10. METHODS: Synoviocytes derived from RA patients were kept in culture for 24 hours in the presence or absence of TNF-α. CXCL10 expression was measured by reverse transcriptase polymerase chain reaction (RT-PCR) of cultured synoviocytes. Expression of RANKL was measured by RT-PCR and western blot in cultured synoviocytes with or without CXCL10 and also measured in Jurkat/Hut 78 T cells and CD4+ T cells in the presence of CXCL10 or dexamethasone. CXCL10 induced RANKL expression in Jurkat T cells was tested upon the pertussis toxin (PTX), an inhibitor of Gi subunit of G protein coupled receptor (GPCR). The synthetic siRNA for Gαi(2) was used to knock down gene expression of respective proteins. RESULTS: CXCL10 expression in RA synoviocytes was increased by TNF-α. CXCL10 slightly increased RANKL expression in RA synoviocytes, but markedly increased RANKL expression in Jurkat/Hut 78 T cell or CD4+ T cell. CXCL10 augmented the expression of RANKL by 62.6%, and PTX inhibited both basal level of RANKL (from 37.4 ± 16.0 to 18.9 ± 13.0%) and CXCL10-induced RANKL expression in Jurkat T cells (from 100% to 48.6 ± 27.3%). Knock down of Gα(i2) by siRNA transfection, which suppressed the basal level of RANKL (from 61.8 ± 17.9% to 31.1 ± 15.9%) and CXCL10-induced RANKL expression (from 100% to 53.1 ± 27.1%) in Jurkat T cells, is consistent with PTX, which inhibited RANKL expression. CONCLUSIONS: CXCL10 increased RANKL expression in CD4+ T cells and it was mediated by Gα(i) subunits of CXCR3. These results indicate that CXCL10 may have a potential role in osteoclastogenesis of RA synovial tissue and subsequent joint erosion.

Heterotrimeric Gα(i) proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis.

Previous studies have implicated a role of heterotrimeric Gα(i) proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gα(i) proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gα(i)-GTP protein complex was immunoprecipitated with a Gα(i) protein activation assay. In subsequent in vivo studies, the Gα(i) protein inhibitor pertussis toxin (PTx) or G(i) protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gα(i2) in sepsis, Gα(i2) (-/-) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gα(i) proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα(i) proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gα(i2) (-/-) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gα(i2) (-/-) compared to WT mice. Gα(i2) (-/-) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα(i) proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gα(i2) protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.

Interplay between the heterotrimeric G-protein subunits Galphaq and Galphai2 sets the threshold for chemotaxis and TCR activation.

BACKGROUND: TCR and CXCR4-mediated signaling appears to be reciprocally regulated pathways. TCR activation dampens the chemotactic response towards the CXCR4 ligand CXCL12, while T cells exposed to CXCL12 are less prone to subsequent TCR-activation. The heterotrimeric G proteins Galphaq and Galphai2 have been implicated in CXCR4-signaling and we have recently also reported the possible involvement of Galphaq in TCR-dependent activation of Lck (Ngai et al., Eur. J. Immunol., 2008, 38: 32083218). Here we examined the role of Galphaq in migration and TCR activation. RESULTS: Pre-treatment of T cells with CXCL12 led to significantly reduced Lck Y394 phosphorylation upon TCR triggering indicating heterologous desensitization. We show that knockdown of Galphaq significantly enhanced basal migration in T cells and reduced CXCL12-induced SHP-1 phosphorylation whereas Galphai2 knockdown inhibited CXCL12-induced migration. CONCLUSION: Our data suggest that Galphai2 confers migration signals in the presence of CXCL12 whereas Galphaq exerts a tonic inhibition on both basal and stimulated migrational responses. This is compatible with the notion that the level of Galphaq activation contributes to determining the commitment of the T cell either to migration or activation through the TCR.

S1P1 receptor signaling overrides retention mediated by G alpha i-coupled receptors to promote T cell egress.

The mechanism by which sphingosine-1-phosphate receptor-1 (S1P1) acts to promote lymphocyte egress from lymphoid organs is not defined. Here, we showed that CCR7-deficient T cells left lymph nodes more rapidly than wild-type cells did, whereas CCR7-overexpressing cells were retained for longer. After treatment with FTY720, an agonist that causes downmodulation of lymphocyte S1P1, CCR7-deficient T cells were less effectively retained than wild-type T cells. Moreover, treatment with pertussis toxin to inactivate signaling via G alpha i-protein-coupled receptors restored egress competence to S1P1-deficient lymphocytes. We also found that T cell accumulation in lymph node cortical sinusoids required intrinsic S1P1 expression and was antagonized by CCR7. These findings suggest a model where S1P1 acts in the lymphocyte to promote lymph node egress by overcoming retention signals mediated by CCR7 and additional G alpha i-coupled receptors. Furthermore, by simultaneously upregulating S1P1 and downregulating CCR7, T cells that have divided multiple times switch to a state favoring egress over retention.