Talin1 controls dendritic cell activation by regulating TLR complex assembly and signaling.

Talin critically controls integrin-dependent cell migration, but its regulatory role in skin dendritic cells (DCs) during inflammatory responses has not been investigated. Here, we show that talin1 regulates not only integrin-dependent Langerhans cell (LC) migration, but also MyD88-dependent Toll-like receptor (TLR)-stimulated DC activation. Talin1-deficient LCs failed to exit the epidermis, resulting in reduced LC migration to skin-draining lymph nodes (sdLNs) and defective skin tolerance induction, while talin1-deficient dermal DCs unexpectedly accumulated in the dermis despite their actomyosin-dependent migratory capabilities. Furthermore, talin1-deficient DCs exhibited compromised chemotaxis, NFκB activation, and proinflammatory cytokine production. Mechanistically, talin1 was required for the formation of preassembled TLR complexes in DCs at steady state via direct interaction with MyD88 and PIP5K. Local production of PIP2 by PIP5K then recruited TIRAP to the preassembled complexes, which were required for TLR signalosome assembly during DC activation. Thus, talin1 regulates MyD88-dependent TLR signaling pathways in DCs through a novel mechanism with implications for antimicrobial and inflammatory immune responses.

Coupling of ß2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis.

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells.

The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.

Critical role of integrin CD11c in splenic dendritic cell capture of missing-self CD47 cells to induce adaptive immunity.

CD11c, also known as integrin alpha X, is the most widely used defining marker for dendritic cells (DCs). CD11c can bind complement iC3b and mediate phagocytosis in vitro, for which it is also referred to as complement receptor 4. However, the functions of this prominent marker protein in DCs, especially in vivo, remain poorly defined. Here, in the process of studying DC activation and immune responses induced by cells lacking self-CD47, we found that DC capture of CD47-deficient cells and DC activation was dependent on the integrin-signaling adaptor Talin1. Specifically, CD11c and its partner Itgb2 were required for DC capture of CD47-deficient cells. CD11b was not necessary for this process but could partially compensate in the absence of CD11c. Mice with DCs lacking Talin1, Itgb2, or CD11c were defective in supporting T-cell proliferation and differentiation induced by CD47-deficient cell associated antigen. These findings establish a critical role for CD11c in DC antigen uptake and activation in vivo. They may also contribute to understanding the functional mechanism of CD47-blockade therapies.

Integrin Activation Controls Regulatory T Cell-Mediated Peripheral Tolerance.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and ß subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a ß integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4ß1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Talin Plays a Critical Role in the Maintenance of the Regulatory T Cell Pool.

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.

Serum soluble Talin-1 levels are elevated in patients with multiple sclerosis, reflecting its disease activity.

Previously, we identified anti-Talin-1 antibodies in the serum of MS. In this case, we measured the serum soluble Talin-1 (sTalin-1) levels by enzyme-linked immunosorbent assay. The serum sTalin-1 levels were significantly higher in 40 patients with MS than in 43 normal controls and in the acute phase of disease than in the remission phase. Interestingly, serum sTalin-1 levels were associated with a sustained increase in disability after MS attack but not with serum anti-Talin-1 antibody levels. sTalin-1 may be a biomarker for the acute phase of MS and may be used for the short-term prognosis of MS.

Novel serum autoantibodies against talin1 in multiple sclerosis: Possible pathogenetic roles of the antibodies.

In the pathogenesis of multiple sclerosis (MS), B cell/antibody-related mechanisms have recently received attention. To investigate the role of autoantibody in MS, we performed SEREX which can identify autoantibody cyclopedically. We identified serum antibodies against cytoskeletal protein talin1, and the levels of whom were remarkably higher in 39 MS than 43 normal controls (P < 0.01) and 35 disease controls (P = 0.06), and in MS patients without oligoclonal bands than ones with them. Moreover, we found negative-correlations between serum anti-talin1 antibody and IgG index in MS (P = 0.03). Anti-talin1 antibody exists in MS patients' sera, which may have some protective factor.

Expression changes and novel interaction partners of talin 1 in effector cells of autoimmune uveitis.

Autoimmune uveitis is characterized by crossing of blood-retinal barrier (BRB) by autoaggressive immune cells. Equine recurrent uveitis (ERU) is a valuable spontaneous model for autoimmune uveitis and analyses of differentially expressed proteins in ERU unraveled changed protein clusters in target tissues and immune system. Healthy eyes are devoid of leukocytes. In ERU, however, leukocytes enter the inner eye and subsequently destroy it. Molecular mechanisms enabling cell migration through BRB still remain elusive. Previously, we detected decreased talin 1 expression in blood-derived granulocytes of ERU cases, linking the innate immune system to ERU. Because changes in leukocyte protein expression pattern may play a role in pathological abnormalities leading to migration ability, we aimed at identifying interactors of talin 1 in leukocytes with immunoprecipitation, followed by LC-MS/MS for candidate identification. This enabled us to identify CD90 (Thy1) as novel interactor of talin 1 besides several other interactors. In blood-derived granulocytes from healthy individuals, CD90 was highly abundant and significantly reduced in ERU, especially in effector cells. Connection between talin 1 and CD90 and their expression differences in inflammation is an interesting novel finding allowing deeper insight into immune response of innate immune system and granulocyte migration ability in this organ-specific autoimmune disease.

Altered expression of talin 1 in peripheral immune cells points to a significant role of the innate immune system in spontaneous autoimmune uveitis.

The molecular mechanism which enables activated immune cells to cross the blood-retinal barrier in spontaneous autoimmune uveitis is yet to be unraveled. Equine recurrent uveitis is the only spontaneous animal model allowing us to investigate the autoimmune mediated transformation of leukocytes in the course of this sight threatening disease. Hypothesizing that peripheral blood immune cells change their protein expression pattern in spontaneous autoimmune uveitis, we used DIGE to detect proteins with altered abundance comparing peripheral immune cells of healthy and ERU diseased horses. Among others, we found a significant downregulation of talin 1 in peripheral blood granulocytes of ERU specimen, pointing to changes in ß integrin activation and indicating a significant role of the innate immune system in spontaneous autoimmune diseases.

Talin-1 and kindlin-3 regulate alpha4beta1 integrin-mediated adhesion stabilization, but not G protein-coupled receptor-induced affinity upregulation.

Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α(4)ß(1) integrin affinity and consequent adhesive events. Affinity upregulation of α(4)ß(1) integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α(4)ß(1) expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α(4)ß(1) and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α(4)ß(1) after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α(4)ß(1)-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.

Mechanisms and functions for the duration of intercellular contacts made by lymphocytes.

Communication across intercellular contacts is central to establishing appropriate innate and adaptive immune responses. Recent imaging of lymphocyte interactions suggests that a complex orchestration of cell-cell contact times is a key correlate to establishing appropriate immune responses. Here I review the molecular and cellular processes that influence the duration of intercellular contacts, including integrin activation and dynamic changes in membrane morphology. I discuss how these processes can be regulated, for example, by the balance of activating and inhibitory receptor signals, and how they can establish the appropriate outcome for individual cell-cell interactions.

Involvement of Rap-1 activation and early termination of immune synapse in CTLA-4-mediated negative signal.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell co-stimulation receptor that delivers inhibitory signals upon activation. This inhibitory effect by CTLA-4 requires activation of small GTPase Rap-1. However, the precise mechanism underlying these negative signals remains unclear. Here, we show that CTLA-4-induced suppression of IL-2 production correlates with rapid destabilization of immunological synapse (IS) formation in murine normal T cell clones. Overexpression of Spa-1, a Rap-1-specific GTPase activating protein (GAP), abolished both Rap-1 activation and IL-2 suppression induced by CTLA-4. Although we failed to find any specific inhibition of activation of early signals upon CTLA-4 engagement, we found that CTLA-4 specifically up-regulates cell motility and suppresses prolonged accumulation of Talin at the contact area with antigen presenting cells upon antigen stimulation. These results suggest that Rap-1 is activated upon CTLA-4 ligation and mediates inhibitory signals through prevention of IS formation.

Integrin-associated proteins as potential therapeutic targets.

SUMMARY: Integrins are adhesion receptors important for hematopoiesis, leukocyte trafficking, and formation of immunological synapses; hence, they may provide targets for therapeutic intervention in leukocyte-driven pathologies. Blocking integrin-ligand binding is one strategy for inhibiting integrins; however, a complete loss of integrin function can lead to mechanism-based toxicities. Because integrin alpha and beta subunits interact with a variety of other proteins to receive and transmit cellular signals, targeting these integrin-associated proteins may utilize alternative sites for intervention that lead to therapies with fewer side effects. This review summarizes integrin-associated proteins in leukocytes and focuses on four of these proteins with perceived therapeutic potential. Specific mutations in the alpha4 integrin cytoplasmic tail block or enforce binding to paxillin and thus modulate integrin signaling required for efficient cell migration. Similarly, the association of RAPL(NORE1B) with beta2 integrins may participate in adhesive and migratory events in leukocytes. The beta integrin cytoplasmic tail-binding protein talin is critical for increasing the affinity of integrins (activation), and blockade of talin binding can prevent leukocyte arrest on the endothelium. Finally, the membrane protein CD98 mediates beta1 and beta3 integrin signaling and may be involved in leukocyte functions. Identification of biologically important interactions of integrins and signaling proteins can thus pave the way to new strategies for manipulating leukocyte functions.

Talin1 regulates TCR-mediated LFA-1 function.

The leukocyte integrin LFA-1 plays a critical role in T cell trafficking and T cell adhesion to APCs. It is known that integrin-mediated adhesion is regulated by changes in integrin ligand-binding affinity and valency through inside-out signaling. However, the molecular mechanisms involved in TCR-mediated LFA-1 regulation are not well understood. In this study, we show that the cytoskeletal protein talin1 is required for TCR-mediated activation of LFA-1 through regulation of LFA-1 affinity and clustering. Depletion of talin1 from human T cells by small interfering RNAs impairs TCR-induced adhesion to ICAM-1 and T cell-APC conjugation. TCR-induced LFA-1 polarization, but not actin polarization, is defective in talin1-deficient T cells. Although LFA-1 affinity is also reduced in talin1-deficient T cells, rescue of LFA-1 affinity alone is not sufficient to restore LFA-1 adhesive function. Together, our findings indicate that TCR-induced up-regulation of LFA-1-dependent adhesiveness and resulting T cell-APC conjugation require talin1.

TA205, an anti-talin monoclonal antibody, inhibits integrin-talin interaction.

Talin mediates integrin signaling by binding to integrin cytoplasmic tails through its FERM domain which consists of F1, F2 and F3 subdomains. TA205, an anti-talin monoclonal antibody, disrupts actin stress fibers and focal adhesion when microinjected into fibroblasts. Here, we showed that TA205 caused an allosteric inhibition of integrin alphaIIb beta3 binding to the talin FERM domain and mapped the TA205 epitope to residues 131-150 in talin F1. Furthermore, binding of a talin rod fragment to talin head was partially inhibited by TA205. These findings suggest that talin F1 may be important in regulation of integrin binding and talin head-rod interaction.

Identification of talin head domain as an immunodominant epitope of the antiplatelet antibody response in patients with HIV-1-associated thrombocytopenia.

HIV-1-associated thrombocytopenia (HIV-1-ITP) is a common complication of HIV-1 infection, frequently caused by increased peripheral platelet destruction mediated by antiplatelet antibodies (Abs) and/or platelet-bound immune complexes. Little is known about the specificity of the antiplatelet Abs at a molecular level. Here, we used immunoglobulin G (IgG) phage-display libraries generated from 3 HIV-1-ITP patients to isolate a large panel of human monoclonal antiplatelet Abs by selection on unfixed platelets. The platelet antigen recognized by all the cloned Abs was identified to be the talin head domain (talin-H), a cleavage product of talin that can be generated by platelet activation or HIV-1 protease. Talin-H was found in HIV-1-ITP-circulating immune complexes, and antitalin Abs were detected in HIV-1-ITP sera but not in controls. The cloned anti-talin-H IgGs were highly somatically mutated, indicative of an antigen-driven, affinity-matured response. These findings suggest that talin-H Ab may be a marker of HIV-1-ITP elicited due to exposure of immunodominant epitopes on talin-H as a result of a disease-related process. Abs to talin-H and related immune complexes (ICs) may contribute to HIV-1-ITP.

Staging and resetting T cell activation in SMACs.

During the productive interaction of T cells with antigen-presenting cells (APCs), engaged receptors, including the T cell antigen receptors and their associated tyrosine kinases, assemble into spatially segregated supramolecular activation clusters (SMACs) at the area of cell contact. Here, we studied intracellular signaling in SMACs by three-dimensional immunofluorescence microscopic localization of CD3, CD45, talin, phosphotyrosine, Lck and phosphorylated ZAP-70 in T cell-APC conjugates. Two distinct phases of spatial-temporal activation, one before and one after SMAC formation, which were separated by a brief state of inactivation caused by CD45, were observed at the T cell-APC contact area. We propose that pre-SMAC signals are sufficient to activate cell adhesion, but not productive T cell responses, which require orchestrated signaling in SMACs.

Talin controls the exit of the integrin alpha 5 beta 1 from an early compartment of the secretory pathway.

Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, we characterized the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of beta1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin was found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains.

Thrombin causes pseudopod detachment via a pathway involving cytosolic phospholipase A2 and 12/15-lipoxygenase products.

Thrombin causes rapid pseudopod detachment and shortening in Dunning rat prostatic carcinoma (MAT-Lu) cells. As seen by interference reflection microscopy and by immunofluorescence analysis with antibodies to paxillin and talin, the primary event is disassembly of adhesion sites. Biochemically, thrombin is a potent activator of cytosolic phospholipase A2 and increases eicosanoid production in these cells. The pseudopod effects are blocked by lipoxygenase (but not cyclooxygenase) inhibitors. Arachidonic acid and 12(S)-hydroxyeicosatetraenoic acid or 15(S)-hydroxyeicosatetraenoic acid mimic the thrombin effect. We conclude that in certain cancer cells, thrombin is a pseudopod repellent that exerts its effect via a cascade involving cytosolic phospholipase A2, 12/15-lipoxygenase, and 12(S)- and/or 15(S)-hydroxyeicosatetraenoic acid.