G-protein-coupled receptor P2Y10 facilitates chemokine-induced CD4 T cell migration through autocrine/paracrine mediators.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, play a central role in the regulation of T cell migration. Various GPCRs are upregulated in activated CD4 T cells, including P2Y10, a putative lysophospholipid receptor that is officially still considered an orphan GPCR, i.e., a receptor with unknown endogenous ligand. Here we show that in mice lacking P2Y10 in the CD4 T cell compartment, the severity of experimental autoimmune encephalomyelitis and cutaneous contact hypersensitivity is reduced. P2Y10-deficient CD4 T cells show normal activation, proliferation and differentiation, but reduced chemokine-induced migration, polarization, and RhoA activation upon in vitro stimulation. Mechanistically, CD4 T cells release the putative P2Y10 ligands lysophosphatidylserine and ATP upon chemokine exposure, and these mediators induce P2Y10-dependent RhoA activation in an autocrine/paracrine fashion. ATP degradation impairs RhoA activation and migration in control CD4 T cells, but not in P2Y10-deficient CD4 T cells. Importantly, the P2Y10 pathway appears to be conserved in human T cells. Taken together, P2Y10 mediates RhoA activation in CD4 T cells in response to auto-/paracrine-acting mediators such as LysoPS and ATP, thereby facilitating chemokine-induced migration and, consecutively, T cell-mediated diseases.

S1P2/G12/13 Signaling Negatively Regulates Macrophage Activation and Indirectly Shapes the Atheroprotective B1-Cell Population.

OBJECTIVES: Monocyte/macrophage recruitment and activation at vascular predilection sites plays a central role in the pathogenesis of atherosclerosis. Heterotrimeric G proteins of the G12/13 family have been implicated in the control of migration and inflammatory gene expression, but their function in myeloid cells, especially during atherogenesis, is unknown. APPROACH AND RESULTS: Mice with myeloid-specific deficiency for G12/13 show reduced atherosclerosis with a clear shift to anti-inflammatory gene expression in aortal macrophages. These changes are because of neither altered monocyte/macrophage migration nor reduced activation of inflammatory gene expression; on the contrary, G12/13-deficient macrophages show an increased nuclear factor-κB-dependent gene expression in the resting state. Chronically increased inflammatory gene expression in resident peritoneal macrophages results in myeloid-specific G12/13-deficient mice in an altered peritoneal micromilieu with secondary expansion of peritoneal B1 cells. Titers of B1-derived atheroprotective antibodies are increased, and adoptive transfer of peritoneal cells from mutant mice conveys atheroprotection to wild-type mice. With respect to the mechanism of G12/13-mediated transcriptional control, we identify an autocrine feedback loop that suppresses nuclear factor-κB-dependent gene expression through a signaling cascade involving sphingosine 1-phosphate receptor subtype 2, G12/13, and RhoA. CONCLUSIONS: Together, these data show that selective inhibition of G12/13 signaling in macrophages can augment atheroprotective B-cell populations and ameliorate atherosclerosis.

Endothelial Gαq/11 is required for VEGF-induced vascular permeability and angiogenesis.

AIMS: VEGF A (VEGF-A) is a central regulator of pre- and postnatal vascular development. In vitro studies suggested that heterotrimeric G-proteins of the Gq/11 family contribute to VEGF receptor 2 (VEGFR2) signalling, but the mechanism and physiological relevance of this finding is unknown. The aim of this study is to understand the role of endothelial Gαq/11 in VEGF-dependent regulation of vascular permeability and angiogenesis. METHODS AND RESULTS: We show here that VEGF-A-induced signalling events, such as VEGFR2 autophosphorylation, calcium mobilization, or phosphorylation of Src and Cdh5, were reduced in Gαq/11-deficient endothelial cells (ECs), resulting in impaired VEGF-dependent barrier opening, tube formation, and proliferation. Agonists at Gq/11-coupled receptors facilitated VEGF-A-induced VEGFR2 autophosphorylation in a Gαq/11-dependent manner, thereby enhancing downstream VEGFR2 signalling. In vivo, EC-specific Gαq/11- and Gαq-deficient mice showed reduced VEGF-induced fluid extravasation, and retinal angiogenesis was significantly impaired. Gαq-deficient ECs showed reduced proliferation, Cdh5 phosphorylation, and fluid extravasation, whereas apoptosis was increased. CONCLUSION: Gαq/11 critically contributes to VEGF-A-dependent permeability control and angiogenic behaviour in vitro and in vivo.

Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

Anti-apoptotic Bcl-2 family members are critical for the regulation of haematopoietic stem and progenitor cell (HSPC) survival. Little is known about the role of their pro-apoptotic antagonists, i.e. 'BH3-only' proteins, in this cell compartment. Based on the analysis of cytokine deprivation-induced changes in mRNA expression levels of Bcl-2 family proteins, we determined the consequences of BH3-only protein depletion on HSPC survival in culture and, for selected candidates, on engraftment in vivo. Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution. HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment. Moreover, in the absence of Bim, significantly lower numbers of transplanted HSPCs were able to fully engraft radio-depleted recipients. Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

PINCH-1 promotes Bcl-2-dependent survival signalling and inhibits JNK-mediated apoptosis in the primitive endoderm.

The focal adhesion (FA) protein PINCH-1 is required for the survival of primitive endoderm (PrE) cells. How PINCH-1 regulates this fundamental process is not known. Here, we use embryoid bodies (EBs) and isolated EB-derived PrE cells to investigate the mechanisms by which PINCH-1 promotes PrE survival. We report that loss of PINCH-1 in PrE cells leads to a sustained activity of JNK and the pro-apoptotic factor Bax. Mechanistically, the sustained JNK activation was due to diminished levels of the JNK inhibitory factor Ras suppressor protein-1 (RSU-1), whose stability was severely reduced upon loss of PINCH-1. Chemical inhibition of JNK attenuated apoptosis of PrE cells but failed to reduce Bax activity. The increased Bax activity was associated with reduced integrin signalling and diminished Bcl-2 levels, which were shown to inhibit Bax. Altogether our findings show that PINCH-1 is a pro-survival factor that prevents apoptosis of PrE cells by modulating two independent signalling pathways; PINCH-1 inhibits JNK-mediated apoptosis by stabilising the PINCH-1 binding protein RSU-1 and promotes Bcl-2-dependent pro-survival signalling downstream of integrins.

Necrosis-like death can engage multiple pro-apoptotic Bcl-2 protein family members.

Necroptosis is a physiologically relevant mode of cell death with some well-described initiating events, but largely unknown executioners. Here we investigated necrostatin-1 (Nec-1) sensitive death elicited by different necroptosis stimuli in L929 mouse fibrosarcoma cells, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages. We found that TNFα- or zVAD-induced necroptosis occurs independently of the recently implicated executioners Bmf or PARP-2, but can involve the Bcl-2 family proteins Bid and Bak. Furthermore, this type of necroptosis is associated with mitochondrial cytochrome c release and partly sensitive to cyclosporine A inhibition, suggesting a cross talk with the mitochondrial permeability transition pore. Necroptosis triggered by cadmium (Cd) exposure caused fully Nec-1-sensitive and caspase-independent death in L929 cells that was associated with autocrine TNFα-mediated feed-forward signalling. In MEF Cd-exposure elicited a mixed mode of cell death that was to some extent Nec-1-sensitive but also displayed features of apoptosis. It was partly dependent on Bmf and Bax/Bak, proteins typically considered to act pro-apoptotic, but ultimately insensitive to caspase inhibition. Overall, our study indicates that inducers of "extrinsic" and "intrinsic" necroptosis can both trigger TNF-receptor signalling. Further, necroptosis may depend on mitochondrial changes engaging proteins considered critical for MOMP during apoptosis that ultimately contribute to caspase-independent necrotic cell death.

Targeting antiapoptotic A1/Bfl-1 by in vivo RNAi reveals multiple roles in leukocyte development in mice.

Gene-targeting studies in mice have identified the essential roles of most prosurvival Bcl-2 family members in normal physiology and under conditions of stress. The function of one member, Bcl2a1/Bfl-1/A1, is only poorly understood because of quadruplication of its gene locus in mice, hindering conventional knockout studies. To overcome this problem, we generated mouse models allowing traceable constitutive or reversible ablation of A1 in the hematopoietic system by RNA interference. Knockdown of A1 impaired early stages of T-cell differentiation, B-cell homeostasis, and sensitized transitional as well as follicular B cells to apoptosis induced by ligation of the B-cell receptor. As a consequence, B-cell proliferation in response to mitogens was severely impaired, whereas that of T cells appeared unaffected. Furthermore, depending on the extent of A1 knockdown, granulocytes showed increased spontaneous death in culture or failed to accumulate in significant numbers in vivo. These models highlight the critical role of A1 in leukocyte development and homeostasis, constituting valuable tools for investigating presumed roles of this Bcl-2 family member in immunity, tumorigenesis, and drug resistance.

A1/Bfl-1 in leukocyte development and cell death.

The function of the anti-apoptotic Bcl-2 family member Bcl2a1/Bfl-1/A1 is poorly understood due to the lack of appropriate loss-of-function mouse models and redundant effects with other Bcl-2 pro-survival proteins upon overexpression. Expression analysis of A1 suggests predominant roles in leukocyte development, their survival upon viral or bacterial infection, as well as during allergic reactions. In addition, A1 has been implicated in autoimmunity and the pathology and therapy resistance of hematological as well as solid tumors that may aberrantly express this protein. In this review, we aim to summarize current knowledge on A1 biology, focusing on its role in the immune system and compare it to that of other pro-survival Bcl-2 proteins.

Defective cell death signalling along the Bcl-2 regulated apoptosis pathway compromises Treg cell development and limits their functionality in mice.

The Bcl-2 regulated apoptosis pathway is critical for the elimination of autoreactive lymphocytes, thereby precluding autoimmunity. T cells escaping this process can be kept in check by regulatory T (Treg) cells expressing the transcription and lineage commitment factor Foxp3. Despite the well-established role of Bcl-2 family proteins in shaping the immune system and their frequent deregulation in autoimmune pathologies, it is poorly understood how these proteins affect Treg cell development and function. Here we compared the relative expression of a panel of 40 apoptosis-associated genes in Treg vs. conventional CD4(+) T cells. Physiological significance of key-changes was validated using gene-modified mice lacking or overexpressing pro- or anti-apoptotic Bcl-2 family members. We define a key role for the Bim/Bcl-2 axis in Treg cell development, homeostasis and function but exclude a role for apoptosis induction in responder T cells as relevant suppression mechanism. Notably, only lack of the pro-apoptotic BH3-only protein Bim or Bcl-2 overexpression led to accumulation of Treg cells while loss of pro-apoptotic Bad, Bmf, Puma or Noxa had no effect. Remarkably, apoptosis resistant Treg cells showed reduced suppressive capacity in a model of T cell-driven colitis, posing a caveat for the use of such long-lived cells in possible therapeutic settings.

Stable silencing of the glucocorticoid receptor in myelin-specific T effector cells by retroviral delivery of shRNA: insight into neuroinflammatory disease.

Autoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factors remain partially elusive due to the lack of appropriate methods to achieve gene inactivation. Here we describe a protocol for stable gene silencing in differentiated rat T cells by retroviral transfer of small hairpin RNAs. Through the combination of an expression cassette containing the green fluorescent protein with a puromycin selection cassette this allows for the generation of pure knockdown cell lines suitable for tracking in animals. Exemplified for the glucocorticoid receptor, we demonstrate that gene silencing renders T effector cells unresponsive to ligand-induced apoptosis and gene regulation without affecting their ability to induce EAE in rats. Interestingly, glucocorticoid administration remains effective in the treatment of EAE despite strongly diminished glucocorticoid receptor expression in antigen-specific T cells. This highlights an important role of other cell types and bystander T cells as targets of glucocorticoid therapy. Collectively, our approach provides a simple tool for stable and efficient gene silencing in T effector cells, which should help to better understand brain autoimmune pathophysiology.

Peripheral T cells are the therapeutic targets of glucocorticoids in experimental autoimmune encephalomyelitis.

High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy, indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion molecules in peripheral T(H)17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition, dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalomyelitis and presumably multiple sclerosis.