Karim J. Brandt

Glatiramer acetate increases IL-1 receptor antagonist but decreases T cell-induced IL-1β in human monocytes and multiple sclerosis

Mechanisms of action as well as cellular targets of glatiramer acetate (GA) in multiple sclerosis (MS) are still not entirely understood. IL-1β is present in CNS-infiltrating macrophages and microglial cells and is an important mediator of inflammation in experimental autoimmune encephalitis (EAE), the MS animal model. A natural inhibitor of IL-1β, the secreted form of IL-1 receptor antagonist (sIL-1Ra) improves EAE disease course. In this study we examined the effects of GA on the IL-1 system. In vivo, GA treatment enhanced sIL-1Ra blood levels in both EAE mice and patients with MS, whereas IL-1β levels remained undetectable. In vitro, GA per se induced the transcription and production of sIL-1Ra in isolated human monocytes. Furthermore, in T cell contact-activated monocytes, a mechanism relevant to chronic inflammation, GA strongly diminished the expression of IL-1β and enhanced that of sIL-1Ra. This contrasts with the effect of GA in monocytes activated upon acute inflammatory conditions. Indeed, in LPS-activated monocytes, IL-1β and sIL-1Ra production were increased in the presence of GA. These results demonstrate that, in chronic inflammatory conditions, GA enhances circulating sIL-1Ra levels and directly affects monocytes by triggering a bias toward a less inflammatory profile, increasing sIL-1Ra while diminishing IL-1β production. This study sheds light on a mechanism that is likely to participate in the therapeutic effects of GA in MS.

Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins.

Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with human monocytes/macrophages, inducing a massive up‐regulation of the prototypical proinflammatory cytokines IL‐1β and TNF. This mechanism that might be implicated in chronic inflammation is specifically inhibited by high‐density lipoproteins (HDL). Like many other stimuli, besides proinflammatory cytokines, the contact‐mediated activation of monocytes induces the production of cytokine inhibitors such as the secreted form of the IL‐1 receptor antagonist (sIL‐1Ra). The present study demonstrates that stimulated T cells generate microparticles (MP) that induce the production of TNF, IL‐1β, and sIL‐1Ra in human monocytes; the production of TNF and IL‐1β but not that of sIL‐1Ra is inhibited in the presence of HDL. The results were similar when monocytes were stimulated by whole membranes of T cells or soluble extracts of the latter. This suggests that MP carry similar monocyte‐activating factors to cells from which they originate. Thus, by releasing MP, T cells might convey surface molecules similar to those involved in the activation of monocytes by cellular contact. By extension, MP might affect the activity of cells, which are usually not in direct contact with T cells at the inflammatory site. Furthermore, this study demonstrates that HDL exert an anti‐inflammatory effect in nonseptic activation of human monocytes, not only by inhibiting the production of IL‐1β and TNF but also, by leaving sIL‐1Ra production unchanged.

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-1β 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-1β 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-1β 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-1β, 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.

Glatiramer acetate triggers PI3Kδ/Akt and MEK/ERK pathways to induce IL-1 receptor antagonist in human monocytes.

Glatiramer acetate (GA), an immunomodulator used in multiple sclerosis (MS) therapy, induces the production of secreted IL-1 receptor antagonist (sIL-1Ra), a natural inhibitor of IL-1β, in human monocytes, and in turn enhances sIL-1Ra circulating levels in MS patients. GA is a mixture of peptides with random Glu, Lys, Ala, and Tyr sequences of high polarity and hydrophilic nature that is unlikely to cross the blood–brain barrier. In contrast, sIL-1Ra crosses the blood–brain barrier and, in turn, may mediate GA anti-inflammatory activities within the CNS by counteracting IL-1β activities. Here we identify intracellular signaling pathways induced by GA that control sIL-1Ra expression in human monocytes. By using kinase knockdown and specific inhibitors, we demonstrate that GA induces sIL-1Ra production via the activation of PI3Kδ, Akt, MEK1/2, and ERK1/2, demonstrating that both PI3Kδ/Akt and MEK/ERK pathways rule sIL-1Ra expression in human monocytes. The pathways act in parallel upstream glycogen synthase kinase-3α/β (GSK3α/β), the knockdown of which enhances sIL-1Ra production. Together, our findings demonstrate the existence of signal transduction triggered by GA, further highlighting the mechanisms of action of this drug in MS.

NF‐κB is activated from endosomal compartments in antiphospholipid antibodies‐treated human monocytes

The antiphospholipid antibody syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated that Toll‐like receptor 2 (TLR2) and CD14 contribute to monocyte activation of aPLA.

TLR2 Ligands Induce NF-κB Activation from Endosomal Compartments of Human Monocytes

Localization of Toll-like receptors (TLR) in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

A novel MEK2/PI3Kδ pathway controls the expression of IL‐1 receptor antagonist in IFN‐β‐activated human monocytes

IFN‐β and sIL‐1Ra play crucial roles in the regulation of innate immunity and inflammation. IFN‐β, which is widely used to improve the course of relapsing, remitting multiple sclerosis, induces the production of sIL‐1Ra in human monocytes through mechanisms that remain largely unknown. In this study, we identified PI3Kδ and MEK2 as key elements that control sIL‐1Ra production in isolated human monocytes activated by IFN‐β. Blockade of MEK2, but not of MEK1, by inhibitors and siRNA prevented IFN‐β‐induced PI3Kδ recruitment to the membrane, Akt phosphorylation, and sIL‐1Ra production, suggesting that MEK2 acted upstream of PI3Kδ. Furthermore, ERK1/2, the only identified substrates of MEK1/2 to date, are dispensable for sIL‐1Ra production in response to IFN‐β stimulation. Upon IFN‐β activation, MEK2 and PI3Kδ are translocated to monocyte membranes. These data suggest that MEK1 and MEK2 display different, nonredundant functions in IFN‐β signaling. That neither MEK1 nor ERK1/2 play a part in this mechanism is also an unexpected finding that gives rise to a better understanding of the MAPK signaling network. Together, these findings demonstrate that IFN‐β triggers an atypical MEK2/PI3Kδ signaling cascade to regulate sIL‐1Ra expression in monocytes. The premise that MEK1 and MEK2 play a part in the induction of the proinflammatory cytokine, IL‐1β in human monocytes provides a rationale for an alternative, IFN‐β‐mediated pathway to induce/enhance sIL‐1Ra production and thus, to dampen inflammation.

Differential regulation of cytokine production by PI3Kδ in human monocytes upon acute and chronic inflammatory conditions

Deregulated production of cytokines, including IL-1beta, IL-6 and TNF plays an important role in chronic inflammation. Relevant to this condition, direct cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes/macrophages. We previously demonstrated that PI3Ks regulate differential production of IL-1beta and its specific inhibitor secreted IL-1 receptor antagonist (sIL-1Ra) by human monocytes. Here we show that in contrast with PI3Kalpha, beta and gamma, PI3Kdelta accounts for most of the PI3K-dependent signaling ruling the production of IL-1beta, IL-6, TNF and sIL-1Ra in monocytes activated by cellular contact with stimulated T cells (mimicked by CHAPS-solubilized membranes of stimulated T cells, CE sHUT) and lipopolysaccharides (LPS); the latter stimuli being relevant to chronic/sterile and acute/infectious inflammation, respectively. Interestingly, PI3Kdelta activity dampened the production of pro-inflammatory cytokines in LPS-activated monocytes, but induced it in CE sHUT-activated cells. In both CE sHUT- and LPS-activated monocytes PI3Kdelta regulated cytokine transcript expression through the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK3beta). The blockade of GSK3beta displayed inverse effects to those of PI3Kdelta blockade. Thus, by displaying opposite functions in conditions mimicking chronic/sterile and acute/infectious inflammation, i.e., by repressing pro-inflammatory cytokine expression in LPS-activated monocytes but inducing such mediators in T cell contact-activated monocytes, PI3Kdelta represents a potential therapeutic target specific to chronic/sterile inflammatory conditions.

GPR55 agonist lysophosphatidylinositol and lysophosphatidylcholine inhibit endothelial cell hyperpolarization via GPR-independent suppression of Na+-Ca2 + exchanger and endoplasmic reticulum Ca2 + refilling

Lysophosphatidylinositol (LPI) and lysophosphatidylcholine (LPC) are lipid signaling molecules that induce endothelium-dependent vasodilation. In addition, LPC suppresses acetylcholine (Ach)-induced responses. We aimed to determine the influence of LPC and LPI on hyperpolarizing responses in vitro and in situ endothelial cells (EC) and identify the underlying mechanisms. Using patch-clamp method, we show that LPI and LPC inhibit EC hyperpolarization to histamine and suppress Na+/Ca2+ exchanged (NCX) currents in a concentration-dependent manner. The inhibition is non-mode-specific and unaffected by intracellular GDPβS infusion and tempol, a superoxide dismutase mimetic. In excised mouse aorta, LPI strongly inhibits the sustained and the peak endothelial hyperpolarization induced by Ach, but not by SKA-31, an opener of Ca2+-dependent K+ channels of intermediate and small conductance. The hyperpolarizing responses to consecutive histamine applications are strongly reduced by NCX inhibition. In a Ca2+-re-addition protocol, bepridil, a NCX inhibitor, and KB-R7943, a blocker of reversed NCX, inhibit the hyperpolarizing responses to Ca2+-re-addition following Ca2+ stores depletion. These finding indicate that LPC and LPI inhibit endothelial hyperpolarization to Ach and histamine independently of G-protein coupled receptors and superoxide anions. Reversed NCX is critical for ER Ca2+ refilling in EC. The inhibition of NCX by LPI and LPC underlies diminished endothelium-dependent responses and endothelial dysfunction accompanied by increased levels of these lipids in the blood.

F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2.

NLRP3 and ASC are able to form a large multimeric complex called inflammasome in response to a number danger signals. The NLRP3 inflammasome is required for the activation of caspase-1 and subsequent maturation of pro-IL-1β into active IL-1β. Although the mechanisms regulating the formation and activity of NLRP3 inflammasome are yet not fully elucidated, data suggest that the assembly of NLRP3 inflammasome requires microtubules to induce the proximity of ASC and NLRP3. In this study we show that microfilaments (F-actin) inhibit NLRP3 inflammasome activity and interact with NLRP3 and ASC. We demonstrate that the inhibition depends on the actin polymerization state but not on the active polymerization process. In ATP- or nigericin-activated macrophages, our data further indicate that Flightless-I (FliI) and leucine-rich repeat FliI-interaction protein 2 (LRRFIP2) are required for the co-localization of NLRP3, ASC and F-actin. We also established that the ability of Ca2+ to accentuate the activity of NLRP3 inflammasome is abrogated in FliI- and LRRFIP2-knockdown macrophages, suggesting that Ca2+ signaling requires the presence of FliI and LRRFIP2. Accordingly, we observed that Ca2+/FliI-dependent severing of F-actin suppresses F-actin/FliI/LRRFIP2-dependent NLRP3 inflammasome inhibition leading to increase IL-1β production. Altogether, our results unveil a new function of F-actin in the regulation of NLRP3 inflammasome activity strengthening the importance of cytoskeleton in the regulation of inflammation.