Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Activated Human CD4+CD45RO+ Memory T-Cells Indirectly Inhibit NLRP3 Inflammasome Activation through Downregulation of P2X7R Signalling

Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1β. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1β production induced by activated memory T-cells concealed this effect. Priming with IFNβ decreased pro-IL-1β production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFNβ suppresses NLRP3 inflammasome activation through an indirect mechanism involving decreased P2X7R signaling. The inhibition of pro-IL-1β production and suppression of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFNβ. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFNβ in MS

HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

BACKGROUND: Direct cellular contact with stimulated T cells is a potent mechanism that induces cytokine production in human monocytes in the absence of an infectious agent. This mechanism is likely to be relevant to T cell-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Microparticles (MP) generated by stimulated T cells (MPT) display similar monocyte activating ability to whole T cells, isolated T cell membranes, or solubilized T cell membranes. We previously demonstrated that high-density lipoproteins (HDL) inhibited T cell contact- and MPT-induced production of IL-1beta but not of its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra). METHODOLOGY/PRINCIPAL FINDINGS: Labeled MPT were used to assess their interaction with monocytes and T lymphocytes by flow cytometry. Similarly, interactions of labeled HDL with monocytes and MPT were assessed by flow cytometry. In parallel, the MPT-induction of IL-1beta and sIL-1Ra production in human monocytes and the effect of HDL were assessed in cell cultures. The results show that MPT, but not MP generated by activated endothelial cells, bond monocytes to trigger cytokine production. MPT did not bind T cells. The inhibition of IL-1beta production by HDL correlated with the inhibition of MPT binding to monocytes. HDL interacted with MPT rather than with monocytes suggesting that they bound the activating factor(s) of T cell surface. Furthermore, prototypical pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL3 and CCL4 displayed a pattern of production induced by MPT and inhibition by HDL similar to IL-1beta, whereas the production of CCL2, like that of sIL-1Ra, was not inhibited by HDL. CONCLUSIONS/SIGNIFICANCE: HDL inhibit both MPT binding to monocytes and the MPT-induced production of some but not all cytokines, shedding new light on the mechanism by which HDL display their anti-inflammatory functions

Type I IFN and TNFα cross-regulation in immune-mediated inflammatory disease: basic concepts and clinical relevance

A cross-regulation between type I IFN and TNFα has been proposed recently, where both cytokines are hypothesized to counteract each other. According to this model, different autoimmune diseases can be viewed as disequilibrium between both cytokines. As this model may have important clinical implications, the present review summarizes and discusses the currently available clinical evidence arguing for or against the proposed cross-regulation between TNFα and type I IFN. In addition, we review how this cross-regulation works at the cellular and molecular levels. Finally, we discuss the clinical relevance of this proposed cross-regulation for biological therapies such as type I IFN or anti-TNFα treatment

IFNβ and glatiramer acetate trigger different signaling pathways to regulate the IL-1 system in multiple sclerosis

Imbalance in cytokine homeostasis plays an important part in the pathogenesis of various chronic inflammatory diseases. In multiple sclerosis (MS), the pro-inflammatory cytokine interleukin-1β (IL-1β) is present in the central nervous system, being expressed mainly in infiltrating macrophages and microglial cells. IL-1β activity is inhibited by the secreted form of IL-1 receptor antagonist (sIL-1Ra) whose production is increased in patients' blood and induced in human monocytes by IFNβ and glatiramer acetate (GA)—both immunomodulators displaying similar therapeutic efficacy in MS. Because intracellular pathways are currently considered as potential therapeutic targets, identification of specific kinases used by both immunomodulators might lead to more specific therapeutic targeting. We addressed the question of intracellular pathways used by IFNβ and GA to induce sIL-1Ra in human monocytes in two recent studies. This addendum to these studies aims at discussing common pathways and different elements used by IFNβ and GA to induce sIL-1Ra in human monocytes. This pinpoints PI3Kδ activation as a requirement to induce sIL-1Ra production downstream monocyte stimulation by either IFNβ or GA. However, the immunomodulators differentially use MEK/ERK pathway to induce sIL-1Ra production in human monocytes. Together, our current studies suggest that PI3Kδ and MEK2 might represent new targets in MS therapy