Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide.

Naїve CD4+ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4+CD45RA+ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFß mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC2 receptor increased significantly with respect to the VPAC1 receptor from day 4 of CD4+CD45RA+ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4+CD45RA+ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.

Proteomic Analysis of Synovial Fibroblasts and Articular Chondrocytes Co-Cultures Reveals Valuable VIP-Modulated Inflammatory and Degradative Proteins in Osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.

A Clinical Approach for the Use of VIP Axis in Inflammatory and Autoimmune Diseases.

The neuroendocrine and immune systems are coordinated to maintain the homeostasis of the organism, generating bidirectional communication through shared mediators and receptors. Vasoactive intestinal peptide (VIP) is the paradigm of an endogenous neuropeptide produced by neurons and endocrine and immune cells, involved in the control of both innate and adaptive immune responses. Exogenous administration of VIP exerts therapeutic effects in models of autoimmune/inflammatory diseases mediated by G-protein-coupled receptors (VPAC1 and VPAC2). Currently, there are no curative therapies for inflammatory and autoimmune diseases, and patients present complex diagnostic, therapeutic, and prognostic problems in daily clinical practice due to their heterogeneous nature. This review focuses on the biology of VIP and VIP receptor signaling, as well as its protective effects as an immunomodulatory factor. Recent progress in improving the stability, selectivity, and effectiveness of VIP/receptors analogues and new routes of administration are highlighted, as well as important advances in their use as biomarkers, contributing to their potential application in precision medicine. On the 50th anniversary of VIP's discovery, this review presents a spectrum of potential clinical benefits applied to inflammatory and autoimmune diseases.

Healthy and Osteoarthritic Synovial Fibroblasts Produce a Disintegrin and Metalloproteinase with Thrombospondin Motifs 4, 5, 7, and 12: Induction by IL-1ß and Fibronectin and Contribution to Cartilage Damage.

Current description of osteoarthritis includes the involvement of synovial inflammation. Studies contributing to understanding the mechanisms of cross-talk and feedback among the joint tissues could be relevant to the development of therapies that block disease progression. During osteoarthritis, synovial fibroblasts exposed to anomalous mechanical forces and an inflammatory microenvironment release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) metalloproteinases that mediate tissue damage and perpetuate inflammation. We therefore studied the production of ADAMTS by synovial fibroblasts and their contribution to cartilage degradation. Moreover, we analyzed the implication of two mediators present in the osteoarthritis joint, IL-1ß as proinflammatory cytokine, and 45-kDa fibronectin fragments as products of matrix degradation. We reported that synovial fibroblasts constitutively express and release ADAMTS 4, 5, 7, and 12. Despite the contribution of both mediators to the stimulation of Runx2 and Wnt/ß-catenin signaling pathways, as well as to ADAMTS expression, promoting the degradation of aggrecan and cartilage oligomeric matrix protein from cartilage, fibronectin fragments rather than IL-1ß played the major pathological role in osteoarthritis, contributing to the maintenance of the disease. Moreover, higher levels of ADAMTS 4 and 7 and a specific regulation of ADAMTS-12 were observed in osteoarthritis, suggesting them as new potential therapeutic targets. Therefore, synovial fibroblasts provide the biochemical tools to the chronicity and destruction of the osteoarthritic joints.

VIP and CRF reduce ADAMTS expression and function in osteoarthritis synovial fibroblasts.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family is known to play an important role in the pathogenesis of osteoarthritis (OA), working on aggrecan degradation or altering the integrity of extracellular matrix (ECM). Thus, the main purpose of our study was to define the role of vasoactive intestinal peptide (VIP) and corticotrophin-releasing factor (CRF), as immunoregulatory neuropeptides, on ADAMTS production in synovial fibroblasts (SF) from OA patients and healthy donors (HD). OA- and HD-SF were stimulated with pro-inflammatory mediators and treated with VIP or CRF. Both neuropeptides decreased ADAMTS-4, -5, -7 and -12 expressions, aggrecanase activity, glycosaminoglycans (GAG), and cartilage oligomeric matrix protein (COMP) degradation after stimulation with fibronectin fragments (Fn-fs) in OA-SF. After stimulation with interleukin-1ß, VIP reduced ADAMTS-4 and -5, and both neuropeptides decreased ADAMTS-7 production and COMP degradation. Moreover, VIP and CRF reduced Runx2 and ß-catenin activation in OA-SF. Our data suggest that the role of VIP and CRF on ADAMTS expression and cartilage degradation could be related to the OA pathology since scarce effects were produced in HD-SF. In addition, their effects might be greater when a degradation loop has been established, given that they were higher after stimulation with Fn-fs. Our results point to novel OA therapies based on the use of neuropeptides, since VIP and CRF are able to stop the first critical step, the loss of cartilage aggrecan and the ECM destabilization during joint degradation.

Vasoactive intestinal peptide exerts an osteoinductive effect in human mesenchymal stem cells.

Several neuropeptides present in bone tissues, produced by nerve fibers and bone cells, have been reported to play a role in regulating the fine-tuning of osteoblast and osteoclast functions to maintain bone homeostasis. This study aims to characterize the influence of the neuropeptide vasoactive intestinal peptide (VIP) on the differentiation process of human mesenchymal stem cells (MSCs) into osteoblasts and on their anabolic function. We describe the mRNA and protein expression profile of VIP and its receptors in MSCs as they differentiate into osteoblasts, suggesting the presence of an autocrine signaling pathway in these cells. Our findings reveal that VIP enhances the expression of early osteoblast markers in MSCs under osteogenic differentiation and favors both bone matrix formation and proper cytoskeletal reorganization. Finally, our data suggest that VIP could be exerting a direct modulatory role on the osteoblast to osteoclast signaling by downregulating the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio. These results highlight the potential of VIP as an osteoinductive differentiation factor, emerging as a key molecule in the maintenance of human bone homeostasis.

IL-22/IL-22R1 axis and S100A8/A9 alarmins in human osteoarthritic and rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: Fibroblast-like synoviocytes (FLSs) are crucial players in the pathogenesis of synovitis in rheumatic diseases. Targeting FLS activation represents an approach to the development of therapeutic strategies. Our aim was to investigate whether the microenvironment of inflamed joints could modulate the expression of IL-22 and IL-22R1 on OA and RA FLSs. We also examined the effect of IL-22 on FLS activation as well as on their IL-17-related responses. METHODS: IL-22 and IL-22R1 expression was studied by RT-PCR and immunoblotting. Proliferation was measured by an ELISA kit. IL-17 receptors, p19IL-23 and alarmins were analysed by RT-PCR. IL-17 receptor expression was evaluated by flow cytometry. MMP1 and IL-23 were measured by ELISA. S100A8/A9 expression was detected by immunofluorescence and ELISA. Signal transducer and activator of transcription 3 (STAT3) phosphorylation was quantified using a cell-based ELISA kit. RESULTS: IL-22 and IL-22R1 were expressed constitutively in FLSs. We demonstrated that S100A8 and S100A9 were synthesized in FLSs. We reported that inflammatory mediators increased the expression of the IL-22/IL-22R1 axis, amplifying FLS activation. IL-22 enhanced FLS proliferation and up-regulated MMP1 and S100A8/A9 production. STAT3 phosphorylation was induced after IL-22 treatment and the stimulatory effect of IL-22 on S100A8/A9 was reduced after the activities of Janus kinase 2 (JAK2) and JAK3 were blocked. We showed an inhibitory action of IL-22 on IL-23 and IL-17RC expression in RA FLSs and on IL-17RA in OA FLSs. CONCLUSION: Therapies based on the pharmacological disruption signalling of IL-22 could be beneficial for the treatment of rheumatic diseases. The restricted expression of IL-22R1 to non-lymphoid cells could lead to a reduction of side effects mediated by immune responses.

Inflammatory mediators alter interleukin-17 receptor, interleukin-12 and -23 expression in human osteoarthritic and rheumatoid arthritis synovial fibroblasts: immunomodulation by vasoactive intestinal Peptide.

AIMS: To assess the contribution of fibroblast-like synoviocytes (FLS) to the inflammatory joint microenvironment under different pathogenic stimuli and their potential to respond to interleukin (IL)-17 and to determine whether the neuroimmunomodulatory vasoactive intestinal peptide (VIP) is able to modulate IL-17 receptor (IL-17R) and related cytokines. METHODS: The effect of proinflammatory cytokines [tumor necrosis factor α (TNFα) and IL-17] and Toll-like receptor (TLR) ligands [poly(I:C) and lipopolysaccharide (LPS)] on IL-17R expression and IL-12 and IL-23 production was studied in osteoarthritis (OA)- and rheumatoid arthritis (RA)-FLS, involved in Th1/Th17 differentiation. The effect of VIP was also determined. IL-17RA, IL-17RC, IL-12p35 and IL-23p19 expression was measured by real-time polymerase chain reaction. IL-12 and IL-23 protein levels were measured by ELISA in supernatant cultures. RESULTS: TNFα, LPS and poly(I:C) induced an increase in IL-17RA in RA-FLS, whereas TNFα, TNFα plus IL-17 and poly(I:C) enhanced IL-17RC transcripts in FLS. VIP diminished the upregulated expression of IL-17RA in RA-FLS following TNFα and poly(I:C). TNFα, LPS and poly(I:C) increased IL-12 and IL-23 levels in cells derived from patients presenting both pathologies. However, IL-17A DECREASED IL-12 AND AUGMENTED IL-23. VIP DECREASED IL-12P35 MRNA UPREGULATION BY POLY(I:C) AND IL-23P19 TRANSCRIPTS IN LPS-TREATED FLS. CONCLUSIONS: Inflammatory cytokines and TLR ligands modulate IL-17R, IL-12 and IL-23 possibly favoring the cross talk between FLS and Th1/Th17 cells. The ability of VIP to counteract the enhancing effect of proinflammatory molecules on IL-17R and the IL-12 family of cytokines corroborates and amplifies the beneficial effect of this endogenous neuroimmunopeptide in rheumatic diseases.

Effect of VIP on the balance between cytokines and master regulators of activated helper T cells.

CD4T helper cells are decisive in the struggle against pathogens and in maintaining immune homeostasis. Nevertheless, they also drive immune-mediated disease. Recently, emerging evidence suggests that seemingly committed Th cells possess plasticity and may convert into other types of effector cells. Vasoactive Intestinal Peptide (VIP) is an immunomodulator neuropeptide, which is able to promote or inhibit individually the differentiation or function of some T-helper subsets. We conducted ex vivo study with erythrocyte-depleted spleen cells from healthy mice to check the balance between cytokines and master regulators of different T-helper subsets. This neuropeptide adversely affected the differentiation and functionality phases of Th17 cells and had a negative influence on cytokines related to Th1 function, increasing Th17 cells over those of the Th1 cell subset. With respect to Th2 subsets, VIP augmented the interleukin (IL)-4/IL-9 mRNA ratio, and a negative correlation between IL-4 and IL-9 was observed in culture supernatants. VIP augmented Th2 relative to Th1 in cell subsets. VIP decreased the iTreg/Th17 balance. Regarding the induced T-regulatory (iTreg)/Th1 balance, VIP increased the presence of immunoregulatory cytokines in relation to IFNγ. Although additional studies are needed to clarify the role of VIP on the balance between cytokines and master regulators during T-helper differentiation, our data show that VIP reduces Th17/Th1 and Th1/Th2 ratios. However, the iTreg/Th17 ratio was differently counterbalanced, probably because of culture conditions. Finally, this is the first study showing that VIP also modulates Th2/Th9 and iTreg/Th1 ratios.

RNA sensors in human osteoarthritis and rheumatoid arthritis synovial fibroblasts: immune regulation by vasoactive intestinal peptide.

OBJECTIVE: The aim of this study was to analyze both the constitutive and induced expression and function of double-stranded RNA (dsRNA; Toll-like receptor 3 [TLR-3], retinoic acid-inducible gene I [RIG-I], and melanoma differentiation-associated gene 5 [MDA5]) and single-stranded RNA (ssRNA; TLR-7) receptors in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), by studying the transcription factors involved and the subsequent effects on antiviral interferon-ß (IFNß), the proinflammatory CXCL8 chemokine, and matrix metalloproteinase 3 (MMP-3). An additional goal was to study the effect of vasoactive intestinal peptide (VIP). METHODS: The expression of TLR-3, TLR-7, RIG-I, and MDA5 in cultured FLS was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and Western blotting. Transcription factors were studied using the ELISA-based TransAM transcription factor kit. The expression of IFNß, CXCL8 (interleukin-8), and MMP-3 was analyzed by RT-PCR and ELISA. RESULTS: FLS expressed TLR-3, TLR-7, RIG-I, and MDA5. The expression of TLR-3 and RIG-I was higher in RA FLS, while the expression of TLR-7 and MDA5 was higher in OA FLS. Stimulation with poly(I-C) induced the activation of IFN regulatory factor 3 (IRF-3), NF-κB, and activator protein 1 (AP-1) c-Jun as well as the subsequent production of IFNß, CXCL8, and MMP-3. VIP reduced the activation of IRF-3 and the production of IFNß in both OA and RA FLS. Imiquimod induced the activation of NF-κB, AP-1 c-Fos, and AP-1 c-Jun and the synthesis of CXCL8 and MMP-3. VIP significantly diminished MMP-3 production only in imiquimod-treated RA FLS. CONCLUSION: The results of this study revealed a prominent function of FLS in the recognition of both dsRNA and ssRNA, which may be present in the joint microenvironment. This study also advances the healing function of the endogenous neuroimmune peptide VIP, which inhibited TLR-3-, RIG-I-, MDA5-, and TLR-7-mediated stimulation of antiviral, proinflammatory, and joint destruction mediators.

Mapping the CRF-urocortins system in human osteoarthritic and rheumatoid synovial fibroblasts: effect of vasoactive intestinal peptide.

In addition to the brain and pituitary gland, the corticotrophin-releasing factor (CRF) system is expressed in peripheral tissues. In this study we characterize the expression of CRF, urocortins (UCN1, UCN2, and UCN3), and their receptors (CRFR1 and CRFR2) in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Moreover, we analyze the vasoactive intestinal peptide (VIP) effect on the CRF system, as well as its physiological consequences on mediators of inflammatory/destructive processes. CRF and UCNs exhibit differential pattern in OA and RA-FLS. By real-time PCR we detected more expression of CRF and UCN1 in RA, and UCN2 and UCN3 in OA, while the CRFR2 expression was similar. In RA-FLS VIP treatment resulted in a significant decrease of the proinflammatory peptides, CRF and UCN1, and a significant increase of the potential anti-inflammatory agents, UCN3 and CRFR2. Using Western blot assays, we showed that the ratio between phospho-CREB (p-CREB) and c-AMP response element-binding (CREB) is higher in OA and significantly lower in RA-FLS after VIP treatment, with consequences upon cAMP response element in CRF and UCN1 genes. Real-time PCR and EIA proved that VIP significantly inhibits cycloxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in RA-FLS. In all cases, we consider significant data when P < 0.05. These data indicate a role of endogenous CRF, UCNs, and CRFR2 in the OA and RA joint microenvironment. We confirm the anti-inflammatory function of VIP, through the modulation of the expression of CRF system that impacts in a reduction of mediators with inflammatory/destructive functions, supporting its therapeutic potential in rheumatic diseases.

CXCL12 gene expression is upregulated by hypoxia and growth arrest but not by inflammatory cytokines in rheumatoid synovial fibroblasts.

OBJECTIVES: CXCL12 is a constitutively expressed chemokine with important homeostatic functions. Increased CXCL12 expression has been observed in several inflammatory conditions, including rheumatoid arthritis (RA). This study was undertaken to identify potential mechanisms of regulation of CXCL12 gene expression by human fibroblasts under normal or inflammatory conditions. METHODS: Synovial fibroblasts (SF) were cultured from RA and osteoarthritis (OA) synovial tissues. CXCL12 mRNA expression was analysed by real time quantitative RT-PCR in RA-SF under different growth conditions, and exposed to hypoxia or to different pro-inflammatory factors. A 5'CXCL12 -1.4 kb promoter region fragment was cloned in a luciferase reporter plasmid and its activity analysed in human fibroblasts. RESULTS: CXCL12 mRNA expression was not constitutively increased in RA- compared to OA-SF. LPS, pro-inflammatory cytokines or growth factors did not induce CXCL12 mRNA expression in SF. Hypoxia and growth arrest by either serum starvation or confluent growth induced CXCL12 mRNA and protein expression in SF. Constitutive and induced expression of CXCL12 in fibroblasts was regulated at the transcriptional level by specific regions of the -1.4 kb promoter. CONCLUSIONS: Pro-inflammatory factors and cytokines do not up-regulate CXCL12 gene expression in SF. Growth arrest and hypoxia are potentially important inducers of CXCL12 expression in human fibroblasts and operate by regulating transcriptional activity of the promoter.

Enhanced Susceptibility of Galectin-1 Deficient Mice to Experimental Colitis.

Galectin-1 is a ß-galactoside-binding lectin, ubiquitously expressed in stromal, epithelial, and different subsets of immune cells. Galectin-1 is the prototype member of the galectin family which shares specificity with ß-galactoside containing proteins and lipids. Immunomodulatory functions have been ascribed to endogenous galectin-1 due to its induction of T cell apoptosis, inhibitory effects of neutrophils and T cell trafficking. Several studies have demonstrated that administration of recombinant galectin-1 suppressed experimental colitis by modulating adaptive immune responses altering the fate and phenotype of T cells. However, the role of endogenous galectin-1 in intestinal inflammation is poorly defined. In the present study, the well-characterized acute dextran sulfate sodium (DSS)-induced model of ulcerative colitis was used to study the function of endogenous galectin-1 during the development of intestinal inflammation. We found that galectin-1 deficient mice (Lgals1-/- mice) displayed a more severe intestinal inflammation, characterized by significantly elevated clinical scores, than their wild type counterparts. The mechanisms underlying the enhanced inflammatory response in colitic Lgals1-/- mice involved an altered Th17/Th1 profile of effector CD4+ T cells. Furthermore, increased frequencies of Foxp3+CD4+ regulatory T cells in colon lamina propria in Lgals1-/- mice were found. Strikingly, the exacerbated intestinal inflammatory response observed in Lgals1-/- mice was alleviated by adoptive transfer of wild type Foxp3+CD4+ regulatory T cells at induction of colitis. Altogether, these data highlight the importance of endogenous galectin-1 as a novel determinant in regulating T cell reactivity during the development of intestinal inflammation.

The Neuropeptide VIP Limits Human Osteoclastogenesis: Clinical Associations with Bone Metabolism Markers in Patients with Early Arthritis.

We aimed to evaluate the direct action of VIP on crucial molecules involved in human osteoclast differentiation and function. We also investigated the relationship between VIP serum levels and bone remodeling mediators in early arthritis patients. The expression of VIP receptors and osteoclast gene markers in monocytes and in vitro differentiated osteoclasts was studied by real-time PCR. NFATc1 activity was measured using a TransAM® kit. Osteoclastogenesis was confirmed by quantification of tartrate-resistant acid phosphatase positive multinucleated cells. OsteoAssay® Surface Multiple Well Plate was used to evaluate bone-resorbing activity. The ring-shaped actin cytoskeleton and the VPAC1 and VPAC2 expression were analyzed by immunofluorescence. We described the presence of VIP receptors in monocytes and mature osteoclasts. Osteoclasts that formed in the presence of VIP showed a decreased expression of osteoclast differentiation gene markers and proteolytic enzymes involved in bone resorption. VIP reduced the resorption activity and decreased both ß3 integrin expression and actin ring formation. Elevated serum VIP levels in early arthritis patients were associated with lower BMD loss and higher serum OPG concentration. These results demonstrate that VIP exerts an anti-osteoclastogenic action impairing both differentiation and resorption activity mainly through the negative regulation of NFATc1, evidencing its bone-protective effects in humans.

New insights into the role of VIP on the ratio of T-cell subsets during the development of autoimmune diabetes.

Type I diabetes is an autoimmune T-cell-mediated disease associated with overexpression of inflammatory mediators and the disturbance of different T-cell subsets. Vasoactive intestinal peptide (VIP) is a potent anti-inflammatory agent with regulatory effects on activated T cells. As the equilibrium between different T-cell subsets is involved in the final outcome, leading to tolerance or autoimmunity, we studied the evolution of markers for T cells in nonobese diabetic (NOD) mice. The study of different transcription factors, cytokines or cytokine receptors, shows that VIP interferes with functional phase of T helper 17 (Th17) cells and prevents the increase in the proportion of Th1 to Th17 cells. On the other hand, VIP-treated NOD mice show an increase in the proportion of CD4(+)CD25(+) cells in the spleen. Thus, VIP switches the Tregs/Th17 ratio leading to tolerance in NOD mice. Similarly, VIP reverses the ratio of Th1-/Th2-cell subsets associated with autoimmune pathology. All these effects on the ratio of T-cell subsets and the anti-inflammatory effect of VIP in decreasing proinflammatory mediators result in a reduction of ß-cell destruction in pancreas. Taken together, these results show that VIP provides significant protection against spontaneous diabetes by modulating T-cell subsets and counterbalancing tolerance and immunity.

Comparative Study of Senescent Th Biomarkers in Healthy Donors and Early Arthritis Patients. Analysis of VPAC Receptors and Their Influence.

Pro-inflammatory CD4+CD28- T cells are characteristic of immunosenescence, but also of several autoimmune/inflammatory diseases. Vasoactive intestinal peptide (VIP) acts as an anti-inflammatory and immunomodulatory mediator on these cells. Our objective was to study the mutual influence between senescent Th cells and VIP axis in early arthritis (EA), comparing with non-EA donors. We characterized the correlation between senescent Th cells and clinic parameters of EA as well as the behavior of senescent Th biomarkers by real-time PCR. Clinical data were systematically recorded at baseline and after 6 months of follow-up. The number of CD4+CD28- T cells measured by sorting is higher in patients who initially meet ACR classification criteria for rheumatoid arthritis (RA) compared to those who were classified as undifferentiated arthritis (UA). A slight positive correlation between EA CD4+CD28- T cells and CRP or ESR and a negative correlation with bone mineral density were found. Th senescent biomarkers in EA CD4+CD28- T cells were similar to donors, however some of them increased after 6 months of follow-up. VPAC receptors were analyzed by real-time PCR and immunofluorescence, and CD4+CD28- T cells showed higher expression of VPAC2 and lower of VPAC1, VPAC2 showing a significant increased expression in EA cells. Sorted CD4+CD28- T cells were in vitro expanded in presence of VIP, wherein VIP increased senescent biomarker CD27, while it diminished CD57 or NKG2 senescent biomarkers. Our study demonstrates for the first time the existence of a link between senescent Th cells and the VIP axis.

VIP reverses the expression profiling of TLR4-stimulated signaling pathway in rheumatoid arthritis synovial fibroblasts.

Since recent evidences point out the potential involvement of Toll-like receptors (TLRs) in the therapeutic effect of vasoactive intestinal peptide (VIP), the purpose of this study is to elucidate the role of VIP as a negative regulator of TLR-signaling. To this aim, we analyzed in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), the expression profile of TLR-pathway related molecules, as well as the alterations induced by LPS stimulation in RA-FLS and the effect of VIP treatment. Cultured FLS were obtained from patients with RA or OA. RA-FLS were next stimulated with lipopolysaccharide (LPS) in presence or absence of VIP. The gene expression profiling of molecules involved in LPS-mediated TLR4-signaling was studied by cRNA microarray analysis. Twenty three molecules involved in TLR signaling resulted over-expressed at mRNA level in basal RA-FLS compared to OA-FLS. Moreover, in RA-FLS, 23 of the analyzed genes were found to be up-regulated by LPS stimulation whereas 30 were not affected. VIP down-regulated the LPS-induced RNA expression of molecules involved in TLR signaling pathway. Up-regulation of RNA expression of CD14, MD2, TRAM, TRIF, IRAK4, TAB2, TRAF6 and TBK1 was corroborated by RT-PCR as well as the VIP regulatory effect. Increased protein levels of TRAF6, TBK1 and pIRAK1 after exposure to LPS, and the inhibitory effect of VIP, were described by Western blotting. As functional consequences, it was observed the VIP-induced impaired production of IL-6 and RANTES/CCL5 after LPS stimulation. In conclusion, VIP acts as a negative modulator of the TLR4-signaling by overturning the production of several checkpoints molecules of the cascade and thus, widening its potential therapeutic effects.

Profile of Matrix-Remodeling Proteinases in Osteoarthritis: Impact of Fibronectin.

The extracellular matrix (ECM) is a complex and specialized three-dimensional macromolecular network, present in nearly all tissues, that also interacts with cell surface receptors on joint resident cells. Changes in the composition and physical properties of the ECM lead to the development of many diseases, including osteoarthritis (OA). OA is a chronic degenerative rheumatic disease characterized by a progressive loss of synovial joint function as a consequence of the degradation of articular cartilage, also associated with alterations in the synovial membrane and subchondral bone. During OA, ECM-degrading enzymes, including urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), cleave ECM components, such as fibronectin (Fn), generating fibronectin fragments (Fn-fs) with catabolic properties. In turn, Fn-fs promote activation of these proteinases, establishing a degradative and inflammatory feedback loop. Thus, the aim of this review is to update the contribution of ECM-degrading proteinases to the physiopathology of OA as well as their modulation by Fn-fs.

An Overview of VPAC Receptors in Rheumatoid Arthritis: Biological Role and Clinical Significance.

The axis comprised by the Vasoactive Intestinal Peptide (VIP) and its G protein-coupled receptors (GPCRs), VPAC1, and VPAC2, belong to the B1 family and signal through Gs or Gq proteins. VPAC receptors seem to preferentially interact with Gs in inflammatory cells, rather than Gq, thereby stimulating adenylate cyclase activity. cAMP is able to trigger various downstream pathways, mainly the canonical PKA pathway and the non-canonical cAMP-activated guanine nucleotide exchange factor (EPAC) pathway. Classically, the presence of VPACs has been confined to the plasma membrane; however, VPAC1 location has been described in the nuclear membrane in several cell types such as activated Th cells, where they are also functional. VPAC receptor signaling modulates a number of biological processes by tipping the balance of inflammatory mediators in macrophages and other innate immune cells, modifying the expression of TLRs, and inhibiting MMPs and the expression of adhesion molecules. Receptor signaling also downregulates coagulation factors and acute-phase proteins, promotes Th2 over Th1, stimulates Treg abundance, and finally inhibits a pathogenic Th17 profile. Thus, the VIP axis signaling regulates both the innate and adaptive immune responses in several inflammatory/autoimmune diseases. Rheumatoid arthritis (RA) is a complex autoimmune disease that develops on a substrate of genetically susceptible individuals and under the influence of environmental factors, as well as epigenetic mechanisms. It is a heterogeneous disease with different pathogenic mechanisms and variable clinical forms between patients with the same diagnosis. The knowledge of VIP signaling generated in both animal models and human ex vivo studies can potentially be translated to clinical reality. Most recently, the beneficial effects of nanoparticles of VIP self-associated with sterically stabilized micelles have been reported in a murine model of RA. Another novel research area is beginning to define the receptors as biomarkers in RA, with their expression levels shown to be associated with the activity of the disease and patients-reported impairment. Therefore, VPAC expression together VIP genetic variants could allow patients to be stratified at the beginning of the disease with the purpose of guiding personalized treatment decisions.

Immunoregulatory properties of vasoactive intestinal peptide in human T cell subsets: implications for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease whose pathogenesis is not completely understood. Unbalanced Th1/Th2 T-cell polarization has been suggested to play a pathogenetic role and therefore, modulation of T-cell polarization is a potential therapeutic target. Vasoactive intestinal peptide (VIP) is a broadly distributed peptide that exerts anti-inflammatory and immunomodulatory effects, in the collagen-induced arthritis (CIA) murine model of RA, and ex vivo, in synovial cells from RA patients. In the present study, we have found that polyclonal stimulation of peripheral blood lymphocytes (PBL) from RA patients produces higher levels of inflammatory mediators and lower levels of Th1 cytokines than PBL from healthy controls; moreover, VIP has negligible effects on inflammatory mediators and Th1 cytokines produced by PBL from healthy controls but favours Th2 profile and enhanced IL-10 production after stimulation. VIP increases the levels of IL-10 and IL-4 in the supernatant of human CD4(+)CD45RA(+) cells cultured in a non-conditioned or a Th2-conditioned situation. In contrast, VIP does not modify the production of these cytokines in a Th1-conditioned medium. In summary, VIP can differentially modify the functional capacity of human lymphocytes by inducing Th2/Treg differentiation depending on their previous phenotype.