Abstract The activity of cofilin, an actin-remodeling protein, is required for T lymphocyte activation with regard to formation of the immunological synapse, cytokine production, and proliferation. In unstimulated T PBL (PB-T), cofilin is present in its Ser3-phosphorylated inactive form. Costimulation of TCR/CD3 and CD28 induces dephosphorylation and, thus, activation of cofilin. In this study we characterized the signaling cascades leading to cofilin activation in untransformed human PB-T. We show that a Ras-PI3K cascade regulates dephosphorylation of cofilin in PB-T. The GTPase Ras is a central mediator of this pathway; transient expression of an activated form of H-Ras in PB-T triggered the dephosphorylation of cofilin. Inhibition of either MAPK/ERK kinase or PI3K blocked both Ras-induced and costimulation-induced cofilin dephosphorylation in PB-T, showing that the combined activities of both signaling proteins are required to activate cofilin. That Ras functions as a central regulator of cofilin dephosphorylation after costimulation through CD3 × CD28 was finally proven by transient expression of a dominant negative form of H-Ras in primary human PB-T. It clearly inhibited costimulation-induced cofilin dephosphorylation, and likewise, activation of PI3K was diminished. Our data, in addition, demonstrate that regarding the downstream effectors of Ras, a clear difference exists between untransformed human PB-T and the T lymphoma line Jurkat. Thus, in PB-T the Ras signaling cascade is able to activate PI3K, whereas in Jurkat cells this is not the case. In addition to the insights into the regulation of cofilin, this finding discloses a to date unrecognized possibility of PI3K activation in T lymphocytes.
Sulforaphan (SFN), ein natürlicher Nahrungsbestandteil in Kreuzblütengewächsen (z.B. Brokkoli), findet im Kontext möglicher Therapien bei Tumorerkrankungen zunehmend Beachtung. Mehrere Studien berichten über einen hemmenden Effekt auf das Wachstum von Tumorzellen und eine stark antioxidative Wirkung [1 – 4]. Krebsabwehr und Immunsystem sind eng miteinander verzahnt. Eine intakte Immunantwort erkennt entartete Tumorzellen und eliminiert sie. Inwieweit SFN auch immunmodulierend wirkt, ist bisher wenig bekannt.
Dendritic cells (DCs) are key components of the adaptive immune system contributing to initiation and regulation of T cell responses. T cells continuously scan DCs in lymphoid organs for the presence of foreign antigen. However, little is known about the functional consequences of these frequent T cell–DC interactions without cognate antigen. Here we demonstrate that these contacts in the absence of foreign antigen serve an important function, namely, induction of a basal activation level in T cells required for responsiveness to subsequent encounters with foreign antigens. This basal activation is provided by self-recognition of MHC molecules on DCs. Following DC depletion in mice, T cells became impaired in TCR signaling and immune synapse formation, and consequently were hyporesponsive to antigen. This process was reversible, as T cells quickly recovered when the number of DCs returned to a normal level. The extent of T cell reactivity correlated with the degree of DC depletion in lymphoid organs, suggesting that a full DC compartment guarantees optimal T cell responsiveness. These findings indicate that DCs are specialized cells that not only present foreign antigen, but also promote a “tonic” state in T cells for antigen responsiveness.
Plasma fibronectin is a circulating protein that facilitates phagocytosis by connecting bacteria to immune cells. A fibronectin isoform, which includes a sequence of 90 AA called extra-domain B (EDB), is synthesized de novo at the messenger RNA (mRNA) level in immune cells, but the reason for its expression remains elusive. We detected an 80-fold increase in EDB-containing fibronectin in the cerebrospinal fluid of patients with bacterial meningitis that was most pronounced in staphylococcal infections. A role for this isoform in phagocytosis was further suggested by enhanced EDB fibronectin release after internalization of Staphylococcus aureus in vitro. Using transgenic mouse models, we established that immune cell production of fibronectin contributes to phagocytosis, more so than circulating plasma fibronectin, and that accentuated release of EDB-containing fibronectin by immune cells improved phagocytosis. In line with this, administration of EDB fibronectin enhanced in vitro phagocytosis to a larger extent than plasma fibronectin. This enhancement was mediated by αvβ3 integrin as shown using inhibitors or cells from β3 integrin knockout mice. Thus, we identified both a novel function for EDB fibronectin in augmenting phagocytosis over circulating plasma fibronectin, as well as the mediating receptor. Our data also establish for the first time, a direct role for β3 integrin in bacterial phagocytosis in mammals.• Fibronectin containing an extra domain called EDB is released in bacterial meningitis. • EDB-containing fibronectin enhances phagocytosis more than plasma fibronectin. • The enhancement is mediated by activation of αvβ3 integrin in the presence of EDB.
Objective Neutrophils are typically the most abundant leucocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. Methods We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. Results Blood neutrophils from healthy donors and patients with active arthritis showed largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN-γ), as well as to tumour necrosis factor, interleukin-6 and hypoxia, in both humans and mice. Mass cytometry confirmed that healthy and arthritic donor blood neutrophils are largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN-γ and prolonged culture. Conclusions Circulating neutrophils from patients with arthritis resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN-γ response and ageing as complementary drivers of the synovial fluid neutrophil phenotype.
Piperlongumine (PL), a natural small molecule derived from the Piper longum Linn plant, has received growing interest as a prooxidative drug with promising anticancer properties. Yet, the influence of PL on primary human T cells remained elusive. Knowledge of this is of crucial importance, however, since T cells in particular play a critical role in tumor control. Therefore, we investigated the effects of PL on the survival and function of primary human peripheral blood T cells (PBTs). While PL was not cytotoxic to PBTs, it interfered with several stages of T cell activation as it inhibited T cell/APC immune synapse formation, co-stimulation-induced upregulation of CD69 and CD25, T cell proliferation and the secretion of proinflammatory cytokines. PL-induced immune suppression was prevented in the presence of thiol-containing antioxidants. In line with this finding, PL increased the levels of intracellular reactive oxygen species and decreased glutathione in PBTs. Diminished intracellular glutathione was accompanied by a decrease in S-glutathionylation on actin suggesting a global alteration of the antioxidant response. Gene expression analysis demonstrated that TH17-related genes were predominantly inhibited by PL. Consistently, the polarization of primary human naïve CD4+ T cells into TH17 subsets was significantly diminished while differentiation into Treg cells was substantially increased upon PL treatment. This opposed consequence for TH17 and Treg cells was again abolished by thiol-containing antioxidants. Taken together, PL may act as a promising agent for therapeutic immunosuppression by exerting prooxidative effects in human T cells resulting in a diminished TH17 but enhanced Treg cell differentiation.
Summary Cofilin is an actin‐binding protein that depolymerizes and/or severs actin filaments. This dual function of cofilin makes it one of the major regulators of actin dynamics important for T‐cell activation and migration. The activity of cofilin is spatio‐temporally regulated. Its main control mechanisms comprise a molecular toolbox of phospho‐, phospholipid, and redox regulation. Phosphorylated cofilin is inactive and represents the dominant cofilin fraction in the cytoplasm of resting human T cells. A fraction of dephosphorylated cofilin is kept inactive at the plasma membrane by binding to phosphatidylinositol 4,5‐bisphosphate. Costimulation via the T‐cell receptor/ CD 3 complex (signal 1) together with accessory receptors (signal 2) or triggering through the chemokine SDF 1α (stromal cell‐derived factor 1α) induce Ras‐dependent dephosphorylation of cofilin, which is important for immune synapse formation, T‐cell activation, and T‐cell migration. Recently, it became evident that cofilin is also highly sensitive for microenvironmental changes, particularly for alterations in the redox milieu. Cofilin is inactivated by oxidation, provoking T‐cell hyporesponsiveness or necrotic‐like programmed cell death. In contrast, in a reducing environment, even phosphatidylinositol 4,5‐bisphosphate ‐bound cofilin becomes active, leading to actin dynamics in the vicinity of the plasma membrane. In addition to the well‐established three signals for T‐cell activation, this microenvironmental control of cofilin delivers a modulating signal for T‐cell‐dependent immune reactions. This fourth modulating signal highly impacts both initial T‐cell activation and the effector phase of T‐cell‐mediated immune responses.
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