STING Agonist Mitigates Experimental Autoimmune Encephalomyelitis by Stimulating Type I IFN-Dependent and -Independent Immune-Regulatory Pathways.

The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of IFN genes (STING) pathway induces a powerful type I IFN (IFN-I) response and is a prime candidate for augmenting immunity in cancer immunotherapy and vaccines. IFN-I also has immune-regulatory functions manifested in several autoimmune diseases and is a first-line therapy for relapsing-remitting multiple sclerosis. However, it is only moderately effective and can induce adverse effects and neutralizing Abs in recipients. Targeting cGAMP in autoimmunity is unexplored and represents a challenge because of the intracellular location of its receptor, STING. We used microparticle (MP)-encapsulated cGAMP to increase cellular delivery, achieve dose sparing, and reduce potential toxicity. In the C57BL/6 experimental allergic encephalomyelitis (EAE) model, cGAMP encapsulated in MPs (cGAMP MPs) administered therapeutically protected mice from EAE in a STING-dependent fashion, whereas soluble cGAMP was ineffective. Protection was also observed in a relapsing-remitting model. Importantly, cGAMP MPs protected against EAE at the peak of disease and were more effective than rIFN-ß. Mechanistically, cGAMP MPs showed both IFN-I-dependent and -independent immunosuppressive effects. Furthermore, it induced the immunosuppressive cytokine IL-27 without requiring IFN-I. This augmented IL-10 expression through activated ERK and CREB. IL-27 and subsequent IL-10 were the most important cytokines to mitigate autoreactivity. Critically, cGAMP MPs promoted IFN-I as well as the immunoregulatory cytokines IL-27 and IL-10 in PBMCs from relapsing-remitting multiple sclerosis patients. Collectively, this study reveals a previously unappreciated immune-regulatory effect of cGAMP that can be harnessed to restrain T cell autoreactivity.

IL-11 Induces Encephalitogenic Th17 Cells in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

IL-11+CD4+ cells accumulate in the cerebrospinal fluid of patients with early relapsing-remitting multiple sclerosis (MS) and in active brain MS lesions. Mouse studies have confirmed a causal role of IL-11 in the exacerbation of relapsing-remitting experimental autoimmune encephalomyelitis (RREAE). Administration of IL-11 at the time of clinical onset of RREAE induced an acute exacerbation and increased clinical scores, which persisted during the entire course of the disease. IL-11 increased the numbers of spinal cord inflammatory foci, as well as the numbers of peripheral and CNS-infiltrating IL-17+CD4+ cells and IL-17A serum levels. Ag recall assays revealed that IL-11 induces IL-17A+, GM-CSF+, and IL-21+CD4+ myelin Ag-reactive cells. Passive transfer of these encephalitogenic CD4+ T cells induced severe RREAE with IL-17A+CCR6+ CD4+ and B cell accumulation within the CNS. Furthermore, passive transfer of nonmanipulated CNS-derived mononuclear cells from mice with RREAE after a single dose of IL-11 induced severe RREAE with increased accumulation of IL-17A+ and CCR6+ CD4+ cells within the CNS. These results suggest that IL-11 might serve as a biomarker of early autoimmune response and a selective therapeutic target for patients with early relapsing-remitting MS.

Glatiramer acetate treatment effects on gene expression in monocytes of multiple sclerosis patients.

BACKGROUND: Glatiramer acetate (GA) is a mixture of synthetic peptides used in the treatment of patients with relapsing-remitting multiple sclerosis (RRMS). The aim of this study was to investigate the effects of GA therapy on the gene expression of monocytes. METHODS: Monocytes were isolated from the peripheral blood of eight RRMS patients. The blood was obtained longitudinally before the start of GA therapy as well as after one day, one week, one month and two months. Gene expression was measured at the mRNA level by microarrays. RESULTS: More than 400 genes were identified as up-regulated or down-regulated in the course of therapy, and we analyzed their biological functions and regulatory interactions. Many of those genes are known to regulate lymphocyte activation and proliferation, but only a subset of genes was repeatedly differentially expressed at different time points during treatment. CONCLUSIONS: Overall, the observed gene regulatory effects of GA on monocytes were modest and not stable over time. However, our study revealed several genes that are worthy of investigation in future studies on the molecular mechanisms of GA therapy.

MicroRNA expression changes during interferon-beta treatment in the peripheral blood of multiple sclerosis patients.

MicroRNAs (miRNAs) are small non-coding RNA molecules acting as post-transcriptional regulators of gene expression. They are involved in many biological processes, and their dysregulation is implicated in various diseases, including multiple sclerosis (MS). Interferon-beta (IFN-beta) is widely used as a first-line immunomodulatory treatment of MS patients. Here, we present the first longitudinal study on the miRNA expression changes in response to IFN-beta therapy. Peripheral blood mononuclear cells (PBMC) were obtained before treatment initiation as well as after two days, four days, and one month, from patients with clinically isolated syndrome (CIS) and patients with relapsing-remitting MS (RRMS). We measured the expression of 651 mature miRNAs and about 19,000 mRNAs in parallel using real-time PCR arrays and Affymetrix microarrays. We observed that the up-regulation of IFN-beta-responsive genes is accompanied by a down-regulation of several miRNAs, including members of the mir-29 family. These differentially expressed miRNAs were found to be associated with apoptotic processes and IFN feedback loops. A network of miRNA-mRNA target interactions was constructed by integrating the information from different databases. Our results suggest that miRNA-mediated regulation plays an important role in the mechanisms of action of IFN-beta, not only in the treatment of MS but also in normal immune responses. miRNA expression levels in the blood may serve as a biomarker of the biological effects of IFN-beta therapy that may predict individual disease activity and progression.

Cilia proteins are biomarkers of altered flow in the vasculature.

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation-the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.

Mn porphyrins as a novel treatment targeting sickle cell NOXs to reverse and prevent acute vaso-occlusion in vivo.

In sickle cell disease (SCD), adhesion of sickle red blood cells (SSRBCs) and activated leukocytes in inflamed venules affects blood rheology, causing vaso-occlusive manifestations and vital reduction in microvascular blood flow. Recently, we found that NADPH oxidases (NOXs) create a vicious feedback loop within SSRBCs. This positive feedback loop mediates SSRBC adhesion to the endothelium. We show for the first time the therapeutic effectiveness of the redox-active manganese (Mn) porphyrins MnTnBuOE-2-PyP5+ (MnBuOE; BMX-001) and MnTE-2-PyP5+ (MnE; BMX-010, AEOL10113) to treat established vaso-occlusion in a humanized sickle mouse model of an acute vaso-occlusive crisis using intravital microscopy. These Mn porphyrins can suppress SSRBC NOX activity. Subcutaneous administration of only 1 dose of MnBuOE or MnE at 0.1 to 2 mg/kg after the inflammatory trigger of vaso-occlusion, or simultaneously, reversed and reduced leukocyte and SSRBC adhesion, diminished leukocyte rolling, restored blood flow, and increased survival rate. Furthermore, MnBuOE and MnE administered to sickle mice subcutaneously at 0.1 to 1 mg/kg for 28 days (except on weekends) did not exacerbate anemia, which seemed to be due to downregulation of both SSRBC reactive oxygen species production and exposure of the eryptotic marker phosphatidylserine. In addition, Mn porphyrins ameliorated leukocytosis, venous blood gases, endothelial activation, and organ oxidative damage. Our data suggest that Mn porphyrins, likely by repressing NOX-mediated adhesive function of SSRBCs and activated leukocytes, could represent a novel, safe therapeutic intervention to treat or prevent the establishment of acute pain crises. These NOX-targeted antioxidants merit further assessment in SCD clinical trials.

Multiple sclerosis: modulation of toll-like receptor (TLR) expression by interferon-ß includes upregulation of TLR7 in plasmacytoid dendritic cells.

Interferon-ß is an established treatment for patients with multiple sclerosis (MS) but its mechanisms of action are not well understood. Viral infections are a known trigger of MS relapses. Toll-like receptors (TLRs) are key components of the innate immune system, which sense conserved structures of viruses and other pathogens. Effects of interferon-ß on mRNA levels of all known human TLRs (TLR1-10) and the TLR adaptor molecule MyD88 were analyzed in peripheral blood mononuclear cells (PBMCs) of healthy donors by quantitative real-time PCR and by transcriptome analysis in PBMCs of 25 interferon-ß-treated patients with relapsing-remitting MS. Regulation of TLR protein expression by interferon-ß was investigated by flow cytometry of leukocyte subsets of healthy subjects and of untreated, interferon-ß-, or glatiramer acetate-treated patients with MS. Interferon-ß specifically upregulated mRNA expression of TLR3, TLR7, and MyD88 and downregulated TLR9 mRNA in PBMCs of healthy donors as well as in PBMCs of patients with MS. Plasmacytoid dendritic cells (pDCs) were identified as the major cell type responding to interferon-ß with increased expression of TLR7 and MyD88 protein. In line with this, expression of TLR7 protein was increased in pDCs of interferon-ß-treated, but not untreated or glatiramer acetate-treated patients with MS. Interferon-ß-induced upregulation of TLR7 in pDCs is of functional relevance since pre-treatment of PBMCs with interferon-ß resulted in a strongly increased production of interferon-α upon stimulation with the TLR7 agonist loxoribine. Flow cytometry confirmed pDCs as the cellular source of interferon-α production induced by activation of TLR7. Thus, upregulation of TLR7 in pDCs and a consequently increased activation of pDCs by TLR7 ligands represents a novel immunoregulatory mechanism of interferon-ß. We hypothesize that this mechanism could contribute to a reduction of virus-triggered relapses in patients with MS.

MicroRNAs in multiple sclerosis and experimental autoimmune encephalomyelitis.

MicroRNA (miRNA) are small non-coding RNA molecules about 21-25 nucleotides long. They control gene regulation by translational repression and cleavage. Several studies have shown that many miRNA are associated with the etiology of different diseases. Recent developments in diverse miRNA profiling platforms like microarray and quantitative real-time PCR may enable the identification of specific miRNA as novel diagnostic and predictive markers for various diseases. MiRNAs could even be used as therapeutic drug targets. Multiple sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system. Dysregulated immune system processes result in demyelination of neurons and consequently, electrical impulses that travel along the nerves are disrupted resulting in the impairment of organs. In the past three years, there has been an increased interest in establishing miRNA-based biomarkers for MS. So far, there are six studies on miRNA expression in MS patients in which first miRNAs were discovered as potential disease markers. For instance, one study showed that blood levels of miR-145 can discriminate MS patients from healthy controls, and another showed that active lesions in the brain are characterized by a strong up-regulation of miR-155. Studies on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, further support the significance of miRNA as e.g. mice with miR-155 deletion are highly resistant to EAE. Such investigations help to understand the molecular processes involved in the disease. The identification of miRNA markers that are associated with type of MS, individual disease activity or clinical progression under treatment may open new avenues for early diagnosis and optimized therapy of MS.

Integration of MicroRNA databases to study MicroRNAs associated with multiple sclerosis.

MicroRNAs (miRNAs) are small non-coding RNAs which regulate many genes post-transcriptionally. In various contexts of medical science, miRNAs gained increasing attention over the last few years. Analyzing the functions, interactions and cellular effects of miRNAs is a very complex and challenging task. Many miRNA databases with diverse data contents have been developed. Here, we demonstrate how to integrate their information in a reasonable way on a set of miRNAs that were found to be dysregulated in the blood of patients with multiple sclerosis (MS). Using the miR2Disease database, we retrieved 16 miRNAs associated with MS according to four different studies. We studied the predicted and experimentally validated target genes of these miRNAs, their expression profiles in different blood cell types and brain tissues, the pathways and biological processes affected by these miRNAs as well as their regulation by transcription factors. Only miRNA-mRNA interactions that were predicted by at least seven different prediction algorithms were considered. This resulted in a network of 1,498 target genes. In this network, the MS-associated miRNAs hsa-miR-20a-5p and hsa-miR-20b-5p occurred as central hubs regulating about 500 genes each. Strikingly, many of the putative target genes play a role in T cell activation and signaling, and many have transcription factor activity. The latter suggests that miRNAs often act as regulators of regulators with many secondary effects on gene expression. Our present work provides a guideline on how information of different databases can be integrated in the analysis of miRNAs. Future investigations of miRNAs shall help to better understand the mechanisms underlying different diseases and their treatments.