Valentina Salvi

Gremlin is a novel agonist of the major proangiogenic receptor VEGFR2.

The bone morphogenic protein antagonist gremlin is expressed during embryonic development and under different pathologic conditions, including cancer. Gremlin is a proangiogenic protein belonging to the cystine-knot superfamily that includes transforming growth factor-β proteins and the angiogenic vascular endothelial growth factors (VEGFs). Here, we demonstrate that gremlin binds VEGF receptor-2 (VEGFR2), the main transducer of VEGF-mediated angiogenic signals, in a bone morphogenic protein-independent manner. Similar to VEGF-A, gremlin activates VEGFR2 in endothelial cells, leading to VEGFR2-dependent angiogenic responses in vitro and in vivo. Gremlin thus represents a novel proangiogenic VEGFR2 agonist distinct from the VEGF family ligands with implications in vascular development, angiogenesis-dependent diseases, and tumor neovascularization.

Blocking TH17-polarizing cytokines by histone deacetylase inhibitors in vitro and in vivo

Histone deacetylase (HDAC) inhibitors are small molecules inducing cell‐cycle arrest, differentiation, and apoptosis, currently undergoing clinical trials as anticancer drugs. In addition, emerging evidence suggests HDAC inhibitors may have anti‐inflammatory and immunomodulatory properties as well, although the molecular mechanisms remain poorly defined. Given the central role of dendritic cells (DC) in the induction and maintenance of the inflammatory and immune response, we investigated the effects of HDAC inhibitors on the maturation and activation of human monocyte‐derived DC in the presence of LPS and IFN‐γ. Our results show that the production of TH1‐ and TH17‐inducing cytokines, namely IL‐12 and IL‐23, was inhibited by trichostatin A (72% and 52%, respectively) and suberoylanilide hydroxamic acid (86% and 83%). Strikingly, HDAC inhibitors were effective if added simultaneously as well as after the proinflammatory challenge, and their effect was not associated to a reduction of expression or function of LPS/IFN‐γ receptors. These findings were confirmed in two different murine models. In addition, HDAC inhibitors selectively blocked the production of TH1‐attracting chemokines CXCL9, CXCL10, and CXCL11. The reduction of TH1‐ and TH17‐inducing cytokines as well as TH1‐attracting chemokines may represent relevant mechanisms through which HDAC inhibitors at nonproapoptotic doses exert their immunomodulatory properties.

Endothelial Cell–Derived Chemerin Promotes Dendritic Cell Transmigration

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-α, IFN-γ, and LPS, or calcitriol, are not effective. Retinoic acid–stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23+ dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1–dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid–activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of chemerin, the upregulation of CCRL2, and the activation of dendritic cell β1 integrin affinity.

Trichostatin A blocks type I interferon production by activated plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDC) represent the main type I interferon (IFN-I) producing cells. Emerging evidence supports a role for IFN-I in autoimmune diseases. Given the central role of PDC in the pathogenesis of systemic lupus erythematosus (SLE), we investigated the effect of Trichostatin A (TSA), a prototypic histone deacetylase inhibitor, on PDC activation. TSA inhibited the production of IFN-I, TRAIL and of the pro-inflammatory cytokines TNFalpha and IL-6 by CpG-activated PDC. These effects were associated with the inhibition of IFN Regulatory Factor (IRF)-7 nuclear translocation. Furthermore, TSA was also effective in inhibiting the production of IFNalpha by PDC cultured in vitro in the presence of serum obtained from SLE patients. This study describes a new level of regulation of immune responses by histone deacetylase inhibitors and defines the molecular basis for new strategies to be exploited in the treatment of autoimmune diseases.

Selective Activation of Human Dendritic Cells by OM-85 through a NF-kB and MAPK Dependent Pathway

OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), a product made of the water soluble fractions of 21 inactivated bacterial strain patterns responsible for respiratory tract infections, is used for the prevention of recurrent upper respiratory tract infections and acute exacerbations in chronic obstructive pulmonary disease patients. OM-85 is able to potentiate both innate and adaptive immune responses. However, the molecular mechanisms responsible for OM-85 activation are still largely unknown. Purpose of this study was to investigate the impact of OM-85 stimulation on human dendritic cell functions. We show that OM-85 selectively induced NF-kB and MAPK activation in human DC with no detectable action on the interferon regulatory factor (IRF) pathway. As a consequence, chemokines (i.e. CXCL8, CXCL6, CCL3, CCL20, CCL22) and B-cell activating cytokines (i.e. IL-6, BAFF and IL-10) were strongly upregulated. OM-85 also synergized with the action of classical pro-inflammatory stimuli used at suboptimal concentrations. Peripheral blood mononuclear cells from patients with COPD, a pathological condition often associated with altered PRR expression pattern, fully retained the capability to respond to OM-85. These results provide new insights on the molecular mechanisms of OM-85 activation of the immune response and strengthen the rational for its use in clinical settings.

Angiogenic and antiangiogenic chemokines

Chemokines are a family of vertebrate-specific, small-secreted molecules that were originally identified as mediators of leukocyte migration and tissue positioning during the immune response. Subsequently, chemokines were discovered to control movement also of endothelial cells and other cell types in many different contexts. The human chemokine system comprises about 50 chemokines and more than 20 receptors belonging to the seven-transmembrane receptor family. In the present chapter, we review the literature supporting a role for chemokines in angiogenesis and lymphangiogenesis. We highlight that chemokines exert both pro- and antiangiogenic roles either by acting directly on endothelial cells or by recruiting leukocytes that, in turn, secrete angiogenic mediators. This latter mode of action is possibly the most relevant in tumor angiogenesis. Finally, we explore the angiogenic properties of nonchemokine chemoattractant molecules.

Dual regulation of osteopontin production by TLR stimulation in dendritic cells

OPN, a cytokine produced, among others, by DCs, is involved in inflammation and defense against pathogens. Here, we report that the activation of the MyD88 pathway by TLR2, TLR5, and TLR7/8 agonists or IL‐1β induces high levels of OPN in human DCs. Conversely, LPS and Poly I:C, two TLR3 and TLR4 agonists that engage the TRIF pathway, were ineffective. TLR2 agonists were the strongest OPN inducers, and OPN production was highly stimulated by TLR2‐triggering bacteria (Staphylococcus aureus) but not by TLR4‐triggering Escherichia coli. Costimulation experiments revealed that TLR3 and TLR4 agonists, beyond being inactive by themselves, sharply limited TLR2‐dependent OPN production by activating a TRIF‐dependent inhibition of the MyD88‐dependent OPN production. MyD88 silencing impaired TLR2‐dependent OPN induction, whereas TRIF pathway blockage by chloroquine, dynasore, or TRIF knockdown prevented the TLR3/4 agonist‐mediated inhibition, which was independent from the endogenous production of type I IFN, IL‐29, IL‐10, or TGF‐β. LPS and Poly I:C inhibitory activity was associated with the release of a >10‐kDa protein factor(s). We also demonstrated that the higher OPN levels produced by S. aureus‐treated DCs compared with E. coli‐treated DCs were responsible for a markedly increased production of IL‐17 by CD4+ T cells. These results highlight the biological relevance of the differential OPN induction by TLR2 and TLR4 agonists and emphasize the importance of TLR cross‐talk in OPN induction. This implies that OPN regulation by TLR signaling is critical in shaping inflammatory responses and may modulate IL‐17 production in response to pathogens.

Adipokines as Potential Biomarkers in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease characterized by severe joint injury. Recently, research has been focusing on the possible identification of predictor markers of disease onset and/or progression, of joint damage, and of therapeutic response. Recent findings have uncovered the role of white adipose tissue as a pleiotropic organ not only specialized in endocrine functions but also able to control multiple physiopathological processes, including inflammation. Adipokines are a family of soluble mediators secreted by white adipose tissue endowed with a wide spectrum of actions. This review will focus on the recent advances on the role of the adipokine network in the pathogenesis of RA. A particular attention will be devoted to the action of these proteins on RA effector cells, and on the possibility to use circulating levels of adipokines as potential biomarkers of disease activity and therapeutic response.

CCRL2 regulates M1/M2 polarization during EAE recovery phase

Chemokine (CC motif) receptor‐like 2 is a 7‐transmembrane protein related to the family of the atypical chemokine receptors, which are proteins devoid of chemotactic activity and involved in the control of inflammation. Experimental autoimmune encephalitis is an autoimmune disorder that replicates the inflammatory aspects of multiple sclerosis. Chemokine (CC motif) receptor‐like 2–deficient mice developed exacerbated, nonresolving disease with protracted inflammatory response and increased demyelination. The increased severity of the disease was associated with higher levels of microglia/macrophage activation markers and imbalanced M1/M2 polarization. Thus, chemokine (CC motif) receptor‐like 2 is involved in the downregulation of central nervous system–associated experimental autoimmune encephalitis inflammation in the recovery phase of the disease. Therefore chemokine (CC motif) receptor‐like 2 should be considered to be a molecule involved in the regulation of the inflammatory response associated with multiple sclerosis.

Role of Atypical Chemokine Receptors in Microglial Activation and Polarization

Inflammatory reactions occurring in the central nervous system (CNS), known as neuroinflammation, are key components of the pathogenic mechanisms underlying several neurological diseases. The chemokine system plays a crucial role in the recruitment and activation of immune and non-immune cells in the brain, as well as in the regulation of microglia phenotype and function. Chemokines belong to a heterogeneous family of chemotactic agonists that signal through the interaction with G protein-coupled receptors (GPCRs). Recently, a small subset of chemokine receptors, now identified as “atypical chemokine receptors” (ACKRs), has been described. These receptors lack classic GPCR signaling and chemotactic activity and are believed to limit inflammation through their ability to scavenge chemokines at the inflammatory sites. Recent studies have highlighted a role for ACKRs in neuroinflammation. However, in the CNS, the role of ACKRs seems to be more complex than the simple control of inflammation. For instance, CXCR7/ACKR3 was shown to control T cell trafficking through the regulation of CXCL12 internalization at CNS endothelial barriers. Furthermore, D6/ACKR2 KO mice were protected in a model of experimental autoimmune encephalomyelitis (EAE). D6/ACKR2 KO showed an abnormal accumulation of dendritic cells at the immunization and a subsequent impairment in T cell priming. Finally, CCRL2, an ACKR-related protein, was shown to play a role in the control of the resolution phase of EAE. Indeed, CCRL2 KO mice showed exacerbated, non-resolving disease with protracted inflammation and increased demyelination. This phenotype was associated with increased microglia and macrophage activation markers and imbalanced M1 vs. M2 polarization. This review will summarize the current knowledge on the role of the ACKRs in neuroinflammation with a particular attention to their role in microglial polarization and function.