Nitro-oleic acid regulates T cell activation through post-translational modification of calcineurin.

Nitro-fatty acids (NO2-FAs) are unsaturated fatty acid nitration products that exhibit anti-inflammatory actions in experimental mouse models of autoimmune and allergic diseases. These electrophilic molecules interfere with intracellular signaling pathways by reversible post-translational modification of nucleophilic amino-acid residues. Several regulatory proteins have been identified as targets of NO2-FAs, modifying their activity and promoting gene expression changes that result in anti-inflammatory effects. Herein, we report the effects of nitro-oleic acid (NO2-OA) on pro-inflammatory T cell functions, showing that 9- and 10-NOA, but not their oleic acid precursor, decrease T cell proliferation, expression of activation markers CD25 and CD71 on the plasma membrane, and IL-2, IL-4, and IFN-γ cytokine gene expressions. Moreover, we have found that NO2-OA inhibits the transcriptional activity of nuclear factor of activated T cells (NFAT) and that this inhibition takes place through the regulation of the phosphatase activity of calcineurin (CaN), hindering NFAT dephosphorylation, and nuclear translocation in activated T cells. Finally, using mass spectrometry-based approaches, we have found that NO2-OA nitroalkylates CaNA on four Cys (Cys129, 228, 266, and 372), of which only nitroalkylation on Cys372 was of importance for the regulation of CaN phosphatase activity in cells, disturbing functional CaNA/CaNB heterodimer formation. These results provide evidence for an additional mechanism by which NO2-FAs exert their anti-inflammatory actions, pointing to their potential as therapeutic bioactive lipids for the modulation of harmful T cell-mediated immune responses.

Regulation of Cyclooxygenase-2 Expression in Human T Cells by Glucocorticoid Receptor-Mediated Transrepression of Nuclear Factor of Activated T Cells.

Cyclooxygenase (COX) is the key enzyme in prostanoid synthesis from arachidonic acid (AA). Two isoforms, named COX-1 and COX-2, are expressed in mammalian tissues. The expression of COX-2 isoform is induced by several stimuli including cytokines and mitogens, and this induction is inhibited by glucocorticoids (GCs). We have previously shown that the transcriptional induction of COX-2 occurs early after T cell receptor (TCR) triggering, suggesting functional implications of this enzyme in T cell activation. Here, we show that dexamethasone (Dex) inhibits nuclear factor of activated T cells (NFAT)-mediated COX-2 transcriptional induction upon T cell activation. This effect is dependent on the presence of the GC receptor (GR), but independent of a functional DNA binding domain, as the activation-deficient GRLS7 mutant was as effective as the wild-type GR in the repression of NFAT-dependent transcription. Dex treatment did not disturb NFAT dephosphorylation, but interfered with activation mediated by the N-terminal transactivation domain (TAD) of NFAT, thus pointing to a negative cross-talk between GR and NFAT at the nuclear level. These results unveil the ability of GCs to interfere with NFAT activation and the induction of pro-inflammatory genes such as COX-2, and explain some of their immunomodulatory properties in activated human T cells.

Bringing up the rear: defining the roles of the uropod.

Renewed interest in cell shape has been prompted by a recent flood of evidence that indicates that cell polarity is essential for the biology of motile cells. The uropod, a protrusion at the rear of amoeboid motile cells such as leukocytes, exemplifies the importance of morphology in cell motility. Remodelling of cell shape by uropod-interfering agents disturbs cell migration. But even though the mechanisms by which uropods regulate cell migration are beginning to emerge, their functional significance remains enigmatic.

eNOS S-nitrosylates ß-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of ß-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated ß-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of ß-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse.

During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

ICAMs in Immunity, Intercellular Adhesion and Communication.

Interactions among leukocytes and leukocytes with immune-associated auxiliary cells represent an essential feature of the immune response that requires the involvement of cell adhesion molecules (CAMs). In the immune system, CAMs include a wide range of members pertaining to different structural and functional families involved in cell development, activation, differentiation and migration. Among them, ß2 integrins (LFA-1, Mac-1, p150,95 and αDß2) are predominantly involved in homotypic and heterotypic leukocyte adhesion. ß2 integrins bind to intercellular (I)CAMs, actin cytoskeleton-linked receptors belonging to immunoglobulin superfamily (IgSF)-CAMs expressed by leukocytes and vascular endothelial cells, enabling leukocyte activation and transendothelial migration. ß2 integrins have long been viewed as the most important ICAMs partners, propagating intracellular signalling from ß2 integrin-ICAM adhesion receptor interaction. In this review, we present previous evidence from pioneering studies and more recent findings supporting an important role for ICAMs in signal transduction. We also discuss the contribution of immune ICAMs (ICAM-1, -2, and -3) to reciprocal cell signalling and function in processes in which ß2 integrins supposedly take the lead, paying particular attention to T cell activation, differentiation and migration.

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.

Nitric Oxide and Electrophilic Cyclopentenone Prostaglandins in Redox signaling, Regulation of Cytoskeleton Dynamics and Intercellular Communication.

Nitric oxide (NO) and electrophilic cyclopentenone prostaglandins (CyPG) are local mediators that modulate cellular response to oxidative stress in different pathophysiological processes. In particular, there is increasing evidence about their functional role during inflammation and immune responses. Although the mechanistic details about their relationship and functional interactions are still far from resolved, NO and CyPG share the ability to promote redox-based post-translational modification (PTM) of proteins that play key roles in cellular homeostasis, signal transduction and transcription. NO-induced S-nitrosylation and S-glutathionylation as well as cyclopentenone-mediated adduct formation, are a few of the main PTMs by which intra- and inter-cellular signaling are regulated. There is a growing body of evidence indicating that actin and actin-binding proteins are susceptible to covalent PTM by these agents. It is well known that the actin cytoskeleton is key for the establishment of interactions among leukocytes, endothelial and muscle cells, enabling cellular activation and migration. In this review we analyze the current knowledge about the actions exerted by NO and CyPG electrophilic lipids on the regulation of actin dynamics and cytoskeleton organization, and discuss some open questions regarding their functional relevance in the regulation of intercellular communication.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis.

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes.

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as alpha-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

Lymphocyte chemotaxis is regulated by histone deacetylase 6, independently of its deacetylase activity.

In this work, the role of HDAC6, a type II histone deacetylase with tubulin deacetylase activity, in lymphocyte polarity, motility, and transmigration was explored. HDAC6 was localized at dynamic subcellular structures as leading lamellipodia and the uropod in migrating T-cells. However, HDAC6 activity did not appear to be involved in the polarity of migrating lymphocytes. Overexpression of HDAC6 in freshly isolated lymphocytes and T-cell lines increased the lymphocyte migration mediated by chemokines and their transendothelial migration under shear flow. Accordingly, the knockdown of HDAC6 expression in T-cells diminished their chemotactic capability. Additional experiments with HDAC6 inhibitors (trichostatin, tubacin), other structural related molecules (niltubacin, MAZ-1391), and HDAC6 dead mutants showed that the deacetylase activity of HDAC6 was not involved in the modulatory effect of this molecule on cell migration. Our results indicate that HDAC6 has an important role in the chemotaxis of T-lymphocytes, which is independent of its tubulin deacetylase activity.

Complex I dysfunction and tolerance to nitroglycerin: an approach based on mitochondrial-targeted antioxidants.

Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). Electrochemical detection revealed that the incubation dose of GTN (5x10(-6) mol/L) did not release NO or modify O(2) consumption when administered acutely. However, development of tolerance produced a decrease in both mitochondrial O(2) consumption and the K(m) for O(2) in animal and human vessels and endothelial cells in a noncompetitive action. GTN tolerance has been associated with impairment of GTN biotransformation through inhibition of aldehyde dehydrogenase (ALDH)-2, and with uncoupling of mitochondrial respiration. Feeding rats with mitochondrial-targeted antioxidants (mitoquinone [MQ]) and in vitro coincubation with MQ (10(-6) mol/L) or glutathione (GSH) ester (10(-4) mol/L) prevented tolerance and the effects of GTN on mitochondrial respiration and ALDH-2 activity. Biotransformation of GTN requires functionally active mitochondria and induces reactive oxygen species production and oxidative stress within this organelle, as it is inhibited by mitochondrial-targeted antioxidants and is absent in HUVECrho(0) cells. Experiments analyzing complex I-dependent respiration demonstrate that its inhibition by GTN is prevented by mitochondrial-targeted antioxidants. Furthermore, in presence of succinate (10x10(-3) mol/L), a complex II electron donor added to bypass complex I-dependent respiration, GTN-treated cells exhibited O(2) consumption rates similar to those of controls, thus suggesting that complex I was affected by GTN. We propose that, following prolonged treatment with GTN in addition to ALDH-2, complex I is a target for mitochondrially generated reactive oxygen species. Our data also suggest a role for mitochondrial-targeted antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.

Histone deacetylase 6 regulates human immunodeficiency virus type 1 infection.

Efficient human immunodeficiency virus (HIV)-1 infection depends on multiple interactions between the viral gp41/gp120 envelope (Env) proteins and cell surface receptors. However, cytoskeleton-associated proteins that modify membrane dynamics may also regulate the formation of the HIV-mediated fusion pore and hence viral infection. Because the effects of HDAC6-tubulin deacetylase on cortical alpha-tubulin regulate cell migration and immune synapse organization, we explored the possible role of HDAC6 in HIV-1-envelope-mediated cell fusion and infection. The binding of the gp120 protein to CD4+-permissive cells increased the level of acetylated alpha-tubulin in a CD4-dependent manner. Furthermore, overexpression of active HDAC6 inhibited the acetylation of alpha-tubulin, and remarkably, prevented HIV-1 envelope-dependent cell fusion and infection without affecting the expression and codistribution of HIV-1 receptors. In contrast, knockdown of HDAC6 expression or inhibition of its tubulin deacetylase activity strongly enhanced HIV-1 infection and syncytia formation. These results demonstrate that HDAC6 plays a significant role in regulating HIV-1 infection and Env-mediated syncytia formation.

Nitric Oxide Signaling in T Cell-Mediated Immunity.

Nitric oxide (NO) is a key messenger in the pathogenesis of inflammation, linking innate and adaptive immunity. By targeting signaling molecules, NO from inducible NO synthase (iNOS) and endothelial (e)NOS affects T helper cell differentiation and the effector functions of T lymphocytes, and is a potential target for therapeutic manipulation. In this review we discuss the regulatory actions exerted by NO on T cell functions, focusing on S-nitrosylation as an important post-translational modification by which NO acts as a signaling molecule during T cell-mediated immunity. We also present recent findings showing novel mechanisms through which NO regulates the activation of human T cells, and consider their potential in strategies to treat tumoral, allergic, and autoimmune diseases.

Nitrosothiols in the immune system: signaling and protection.

SIGNIFICANCE: In the immune system, nitric oxide (NO) has been mainly associated with antibacterial defenses exerted through oxidative, nitrosative, and nitrative stress and signal transduction through cyclic GMP-dependent mechanisms. However, S-nitrosylation is emerging as a post-translational modification (PTM) involved in NO-mediated cell signaling. RECENT ADVANCES: Precise roles for S-nitrosylation in signaling pathways have been described both for innate and adaptive immunity. Denitrosylation may protect macrophages from their own S-nitrosylation, while maintaining nitrosative stress compartmentalized in the phagosomes. Nitrosothiols have also been shown to be beneficial in experimental models of autoimmune diseases, mainly through their role in modulating T-cell differentiation and function. CRITICAL ISSUES: Relationship between S-nitrosylation, other thiol redox PTMs, and other NO-signaling pathways has not been always taken into account, particularly in the context of immune responses. Methods for assaying S-nitrosylation in individual proteins and proteomic approaches to study the S-nitrosoproteome are constantly being improved, which helps to move this field forward. FUTURE DIRECTIONS: Integrated studies of signaling pathways in the immune system should consider whether S-nitrosylation/denitrosylation processes are among the PTMs influencing the activity of key signaling and adaptor proteins. Studies in pathophysiological scenarios will also be of interest to put these mechanisms into broader contexts. Interventions modulating nitrosothiol levels in autoimmune disease could be investigated with a view to developing new therapies.

Specificity in S-nitrosylation: a short-range mechanism for NO signaling?

SIGNIFICANCE: Nitric oxide (NO) classical and less classical signaling mechanisms (through interaction with soluble guanylate cyclase and cytochrome c oxidase, respectively) operate through direct binding of NO to protein metal centers, and rely on diffusibility of the NO molecule. S-Nitrosylation, a covalent post-translational modification of protein cysteines, has emerged as a paradigm of nonclassical NO signaling. RECENT ADVANCES: Several nonenzymatic mechanisms for S-nitrosylation formation and destruction have been described. Enzymatic mechanisms for transnitrosylation and denitrosylation have been also studied as regulators of the modification of specific subsets of proteins. The advancement of modification-specific proteomic methodologies has allowed progress in the study of diverse S-nitrosoproteomes, raising clues and questions about the parameters for determining the protein specificity of the modification. CRITICAL ISSUES: We propose that S-nitrosylation is mainly a short-range mechanism of NO signaling, exerted in a relatively limited range of action around the NO sources, and tightly related to the very controlled regulation of subcellular localization of nitric oxide synthases. We review the nonenzymatic and enzymatic mechanisms that support this concept, as well as physiological examples of mammalian systems that illustrate well the precise compartmentalization of S-nitrosylation. FUTURE DIRECTIONS: Individual and proteomic studies of protein S-nitrosylation-based signaling should take into account the subcellular localization in order to gain further insight into the functional role of this modification in (patho)physiological settings.

HDAC6: a key regulator of cytoskeleton, cell migration and cell-cell interactions.

Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that regulates many important biological processes, including cell migration, immune synapse formation, viral infection, and the degradation of misfolded proteins. HDAC6 deacetylates tubulin, Hsp90 and cortactin, and forms complexes with other partner proteins. Although HDAC6 enzymatic activity seems to be required for the regulation of cell morphology, the role of HDAC6 in lymphocyte chemotaxis is independent of its tubulin deacetylase activity. The diverse functions of HDAC6 suggest that it is a potential therapeutic target for the treatment of a range of diseases. This review examines the biological actions of HDAC6, focusing on its deacetylase activity and its potential scaffold functions in the regulation of cell migration and other key biological processes in which the cytoskeleton plays an important role.

Mitochondrial redistribution: adding new players to the chemotaxis game.

Leukocyte polarization and chemotaxis have a key role in the homeostasis of the immune system and in inflammation. Recent work shows that chemoattractants induce the redistribution of mitochondria towards the uropod of polarized migrating leukocytes through a mechanism involving microtubules and mitochondrial fission. These findings underscore the key role this organelle can have in leukocyte chemotaxis by fuelling motor proteins at their trailing edge.

Endothelial nitric oxide synthase regulates T cell receptor signaling at the immunological synapse.

The role of nitric oxide (NO) in T cells remains controversial, and the origin and localization of endogenous NO and whether it regulates lymphocyte activation are unclear. We show here that, within minutes of binding to antigen, T cells produce NO via endothelial nitric oxide synthase (eNOS). This process required increased intracellular Ca2+ and phosphoinositide3-kinase activity. By using an eNOS-green fluorescent fusion protein and fluorescent probes to detect NO, we show that eNOS translocates with the Golgi apparatus to the immune synapse of T helper cells engaged with antigen-presenting cells (APC), where it was fully activated. Overexpression of eNOS prevented the central coalescence of CD3 at the T cell-APC contact site, which was accompanied by increased phosphorylation of CD3zeta chain, ZAP-70, and extracellular signal-regulated kinases and increased IFN-gamma synthesis, but reduced production of IL-2. Therefore, eNOS-derived NO selectively potentiates T cell receptor signaling to antigen at the immunological synapse.

HDAC6 deacetylase activity links the tubulin cytoskeleton with immune synapse organization.

We investigated the role of acetylated microtubules in the antigen-specific interaction of T helper and antigen-presenting cells (APCs). In T cells, acetylated microtubules concentrated at contact site with APCs, surrounding clusters of CD3 and LFA-1. TcR engagement induced a transient deacetylation of microtubules at early times and an enhanced acetylation at late times. Confocal videomicroscopy studies revealed that the HDAC6 tubulin deacetylase was translocated and concentrated at the contact site of T cells with APCs. Overexpression of HDAC6 but not a dead deacetylase mutant in T cells disorganized CD3 and LFA-1 at the immune synapse. This effect was reverted by treatment with the deacetylase inhibitor trichostatin A. The antigen-specific translocation of the microtubule organizing center (MTOC) and IL-2 production were also severely impaired by overexpression of HDAC6. Our results underscore the key role for HDAC6 in the organization of the immune synapse and the antigen-specific reorientation of the MTOC.