Gabriel Criado

Zap-70-independent Ca(2+) Mobilization and Erk Activation in Jurkat T cells in Response to T-cell Antigen Receptor Ligation

ABSTRACT The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1) and increased the intracellular Ca2+ concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCγ1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.

Macrophages from the synovium of active rheumatoid arthritis exhibit an activin A-dependent pro-inflammatory profile.

Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathogenesis and severity correlates with the presence of macrophage‐derived pro‐inflammatory cytokines within the inflamed synovium. Macrophage‐derived cytokines fuel the pathological processes in RA and are targets of clinically successful therapies. However, although macrophage polarization determines cytokine production, the polarization state of macrophages in RA joints remains poorly defined. To dissect the molecular basis for the tissue‐damaging effects of macrophages in RA joints, we undertook the phenotypic and transcriptomic characterization of ex vivo isolated CD14+ RA synovial fluid (RA‐SF) macrophages. Flow cytometry and gene profiling indicated that RA‐SF macrophages express pro‐inflammatory polarization markers (MMP12, EGLN3, CCR2), lack expression of markers associated with homeostatic and anti‐inflammatory polarization (IGF1, HTR2B) and exhibit a transcriptomic profile that resembles the activin A‐dependent gene signature of pro‐inflammatory in vitro‐generated macrophages. In fact, high levels of Smad‐activating activin A were found in RA‐SF and, accordingly, the Smad signalling pathway was activated in ex vivo‐isolated RA‐SF macrophages. In vitro experiments on monocytes and macrophages indicated that RA‐SF promoted the acquisition of pro‐inflammatory markers (INHBA, MMP12, EGLN3, CCR2) but led to a significant reduction in the expression of genes associated with homeostasis and inflammation resolution (FOLR2, SERPINB2, IGF1, CD36), thus confirming the pro‐inflammatory polarization ability of RA‐SF. Importantly, the macrophage‐polarizing ability of RA‐SF was inhibited by an anti‐activin A‐neutralizing antibody, thus demonstrating that activin A mediates the pro‐inflammatory macrophage‐polarizing ability of RA‐SF. Moreover, and in line with these findings, multicolour immunofluorescence evidenced that macrophages within RA synovial membranes (RA‐SM) also express pro‐inflammatory polarization markers whose expression is activin A‐dependent. Altogether, our results demonstrate that macrophages from RA synovial fluids and membranes exhibit an MMP12+ EGLN3+ CCR2+ pro‐inflammatory polarization state whose acquisition is partly dependent on activin A from the synovial fluid. Copyright

Peritoneal dialysis solutions inhibit the differentiation and maturation of human monocyte-derived dendritic cells: effect of lactate and glucose-degradation products

Peritoneal dialysis (PD) is a well‐established therapy for end‐stage renal failure, but its efficiency is limited by recurrent peritonitis. As PD solutions impair local inflammatory responses within the peritoneal cavity, we have analyzed their influence on the in vitro maturation of human monocyte‐derived dendritic cells (MDDC). Evaluation of MDDC maturation parameters [expression of adhesion and costimulatory molecules, receptor‐mediated endocytosis, allogeneic T cell activation, production of tumor necrosis factor α, interleukin (IL)‐6 and IL‐12 p70, and nuclear factor (NF)‐κB activation] revealed that currently used PD solutions differentially inhibit the lipopolysaccharide (LPS)‐induced maturation of MDDC, an inhibition that correlated with their ability to impair the LPS‐stimulated NF‐κB activation. Evaluation of PD components revealed that sodium lactate and glucose‐degradation products impaired the acquisition of maturation parameters and NF‐κB activation in a dose‐dependent manner. Moreover, PD solutions impaired monocyte‐MDDC differentiation, inhibiting the acquisition of DC markers such as CD1a and DC‐specific intercellular adhesion molecule‐3 grabbing nonintegrin (CD209). These findings have important implications for the initiation of immune responses under high lactate conditions, such as those occurring within tumor tissues or after macrophage activation.

T Cell Signalling Induced by Bacterial Superantigens

Bacterial superantigens (SAgs) constitute a large family of bacterial toxins that share the capacity to induce massive activation of the human immune system. Such a feature is based on the ability of these toxins to activate T cells that express Beta-chains of the T cell antigen receptor (TCR) containing variable regions (V) coded by specific families of VBeta genes. In addition, bacterial SAgs bypass the need for processing by antigen-presenting cells by directly binding to major histocompatibility complex class II molecules on the surface of these cells. Emerging work indicates that bacterial SAgs utilize not only the canonical pathways of TCR-mediated T cell activation but also other pathways. Here, we review the structural information on recognition of bacterial SAgs by T cells, the TCR signalling induced by this recognition event, and the effector functions that this recognition triggers. We analyze experimental evidence suggesting the existence of alternative receptors and coreceptors for bacterial SAgs, and outline future challenges in the research with these toxins.

Superantigen Stimulation Reveals the Contribution of Lck to Negative Regulation of T Cell Activation

The conventional paradigm of T cell activation through the TCR states that Lck plays a critical activating role in this signaling process. However, the T cell response to bacterial superantigens does not require Lck. In this study we report that not only is Lck dispensable for T cell activation by superantigens, but it actively inhibits this signaling pathway. Disruption of Lck function, either by repression of Lck gene expression or by selective pharmacologic inhibitors of Lck, led to increased IL-2 production in response to superantigen stimulation. This negative regulatory effect of Lck on superantigen-induced T cell responses required the kinase activity of Lck and correlated with early TCR signaling, but was independent of immunological synapse formation and TCR internalization. Our data demonstrate that the multistage role of Lck in T cell signaling includes the activation of a negative regulatory pathway of T cell activation.

Hierarchical Regulation of CTLA-4 Dimer-Based Lattice Formation and Its Biological Relevance for T Cell Inactivation

CTLA-4 is an activation-induced, homodimeric inhibitory receptor in T cells. Recent crystallographic reports have suggested that it may form lattice-like arrays on the cell surface upon binding B7.1/B7.2 (CD80, CD86) molecules. To test the biological relevance of these CTLA-4-B7 lattices, we introduced a C122A point mutation in human CTLA-4, because this residue was shown to be essential for dimerization in solution. Surprisingly, we found that up to 35% of C122A CTLA-4 dimerized in human T lymphocytes. Moreover, C122A CTLA-4 partitioned within lipid rafts, colocalized with the TCR in the immunological synapse, and inhibited T cell activation. C122-independent dimerization of CTLA-4 involved N-glycosylation, because further mutation of the N78 and N110 glycosylation sites abrogated dimerization. Despite being monomeric, the N78A/N110A/C122A triple mutant CTLA-4 localized in the immunological synapse and inhibited T cell activation. Such functionality correlated with B7-induced dimerization of these mutant molecules. Based on these data, we propose a model of hierarchical regulation of CTLA-4 oligomerization by which B7 binding ultimately determines the formation of dimer-dependent CTLA-4 lattices that may be necessary for triggering B7-dependent T cell inactivation.

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I‐related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR‐dependent activation signals, including p56lck‐, ζ‐associated protein‐70 (ZAP‐70), Vav‐1, Akt, and extracellular signal‐regulated kinase (ERK) phosphorylation but also costimulation‐dependent mitogen‐activated protein kinases (MAPK), such as the stress‐activated c‐Jun N‐terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent‐insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3ɛ, and p59‐60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol‐3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin‐4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP‐70, Akt, Vav‐1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.

Human embryonic stem cell-derived mesenchymal stromal cells ameliorate collagen-induced arthritis by inducing host-derived indoleamine 2,3 dioxygenase

BackgroundThe immunosuppressive and anti-inflammatory properties of mesenchymal stromal cells (MSC) have prompted their therapeutic application in several autoimmune diseases, including rheumatoid arthritis. Adult MSC are finite and their clinical use is restricted by the need for long-term expansion protocols that can lead to genomic instability. Inhibition of Smad2/3 signaling in human pluripotent stem cells (hPSC) provides an infinite source of MSC that match the phenotype and functional properties of adult MSC. Here, we test the therapeutic potential of hPSC-MSC of embryonic origin (embryonic stem cell-derived mesenchymal stromal cells, hESC-MSC) in the experimental model of collagen-induced arthritis (CIA).MethodsCIA was induced in DBA/1 mice by immunization with type II collagen (CII) in Complete Freund’s Adjuvant (CFA). Mice were treated with either a single dose (106 cells/mouse) of hESC-MSC on the day of immunization (prophylaxis) or with three doses of hESC-MSC every other day starting on the day of arthritis onset (therapy). Arthritis severity was evaluated daily for six weeks and ten days, respectively. Frequency of Treg (FoxP3+), Th1 (IFNγ+) and Th17 (IL17+) CD4+ T cells in inguinal lymph nodes (ILN) was quantified by flow cytometry. Serum levels of anti-CII antibodies were determined by ELISA. Detection of hESC-MSC and quantification of murine and human indoleamine 2,3 dioxygenase (IDO1) expression was performed by quantitative real-time PCR. Statistical differences were analyzed by ANOVA and the Mann-Whitney U test.ResultsAdministration of hESC-MSC to mice with established arthritis reduced disease severity compared to control-treated mice. Analysis of CD4 T cell populations in treated mice showed an increase in FoxP3+ Treg and IFNγ+ Th1 cells but not in Th17 cells in the ILN. Anti-CII antibody levels were not affected by treatment. Migration of hESC-MSC to the ILN in treated mice was associated with the induction of murine IDO1.ConclusionTreatment with hESC-MSC ameliorates CIA by inducing IFNγ+ Th1 cells and IDO1 in the host. Thus, hESC-MSC can provide an infinite cellular source for treatment of rheumatoid arthritis.

Hif-1α Knockdown Reduces Glycolytic Metabolism and Induces Cell Death of Human Synovial Fibroblasts Under Normoxic Conditions

Increased glycolysis and HIF-1α activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O2 environments, elevated rates of glycolysis support critical cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1α to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1α was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplexes. Expression analysis by qRT-PCR and western blot of known HIF-1α target genes in hypoxia demonstrated the presence of functional HIF-1α in normoxic SF and confirmed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a HIF-1α target even in normoxia. HIF-1α silencing induced apoptotic cell death in cultured SF and, similarly, treatment with glycolytic, but not with OXPHOS inhibitors, induced SF death. Finally, in vivo HIF-1α targeting by siRNA showed a significant reduction in the viability of human SF engrafted into a murine air pouch. Our results demonstrate that SF are highly dependent on glycolytic metabolism and that HIF-1α plays a regulatory role in glycolysis even under aerobic conditions. Local targeting of HIF-1α provides a feasible strategy to reduce SF hyperplasia in chronic arthritic diseases.

Therapeutic effect of the immunomodulatory drug lenalidomide, but not pomalidomide, in experimental models of rheumatoid arthritis and inflammatory bowel disease

Thalidomide is an immunomodulatory drug (IMiD) with proven therapeutic action in several autoimmune/inflammatory diseases; however, its inherent high toxicity has led to the development of more powerful and safer thalidomide analogs, including lenalidomide and pomalidomide. These are new generation IMiDs that exhibit direct antitumor activity as well as anti-inflammatory/immunomodulatory properties, and are FDA-approved for the treatment of several hematological malignances. Here we investigated the potential therapeutic effects of lenalidomide and pomalidomide in several experimental murine models of autoimmune/inflammatory diseases: 2,4,6-trinitrobenzene sulfonic acid- and dextran sulfate sodium-induced inflammatory bowel disease and type II collagen-induced arthritis. Lenalidomide displayed a strong therapeutic effect in all these models of autoimmune/inflammatory diseases, while the effect of pomalidomide was less pronounced. In vitro experiments confirmed the immunosuppressive effect of both IMiDs on the proliferative response of stimulated human lymphocytes and on the balance of secreted cytokines toward an anti-inflammatory profile. We conclude that lenalidomide may offer a therapeutic opportunity against autoimmune/inflammatory diseases.