Mariola Matysiak

Immunoregulatory function of bone marrow mesenchymal stem cells in EAE depends on their differentiation state and secretion of PGE2

Bone marrow mesenchymal stem cells (BMSC)-induced amelioration of experimental autoimmune encephalomyelitis (EAE) was diminished with neuronal differentiantion of BMSC (nBMSC). BMSC secreted large amounts of PGE2, compared to nBMSC, which correlated with higher efficacy to EAE inhibition. EAE mice treated with PGE2 inhibitor, meloxicam showed decreased serum levels of PGE2 and in parallel decreased inhibitory effect on EAE course. In addition, high levels of PGE2 secretion correlated with high expression of indoleamine-2,3-dioxygenase (IDO). Meloxicam blocked IDO expression in BMSC transferred mice indicating functional relation between PGE2 and IDO induction. The current findings demonstrates PGE2 involvement in BMSC-induced inhibition of EAE and provides a mechanistic link between BMSC-derived PGE2 and IDO-dependent immunoregulation of this autoimmune condition.

Stem cells ameliorate EAE via an indoleamine 2,3-dioxygenase (IDO) mechanism

Syngeneic, pluripotent Lin(-)Sca1(+) bone marrow stem cells (SC), transferred to mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis, enhanced recovery, prevented relapses and promoted myelin repair. SC-treated mice showed elevated interferon-gamma production and induction of indoleamine 2,3-dioxygenase (IDO) in CD11c(+) dendritic cells (DC). IDO induction was specific since in the presence of IDO-producing CD11c(+) DC, PLP stimulated T cell proliferation was inhibited and the IDO-inhibitor, 1-MT, abrogated the SC effect. Relapse prevention during chronic disease correlated with decreased responsiveness to PLP(178-191) and MBP(85-99). Thus, pluripotent SC induce IDO in DC leading to inhibition of antigen reactivity and spreading in EAE.

Soluble Nogo-A, an inhibitor of axonal regeneration, as a biomarker for multiple sclerosis.

Background: CNS axons display a poor regenerative response to injury. In multiple sclerosis (MS), failure of damaged axons to regenerate may be a major factor underlying non-reversible neurologic dysfunction. Nogo is a development-related molecule inhibiting axonal regeneration and is a major component of CNS myelin. Methods: CSF from 114 patients with remitting relapsing MS (RR-MS) and secondary progressive MS (SP-MS) and 153 controls, as well as CNS tissue from 3 patients with MS and 2 controls, were used for this study. Results: We found soluble 20 kDa Nogo-A product in 96% (110/114) of CSF samples from patients with MS compared with 0/18 from meningo-encephalomyelitis, 0/125 from control subjects with other neurologic diseases, and 0/10 from CNS autoimmune diseases. Nogo-A products were present both in RR-MS and SP-MS, as well as in early cases of the disease, but not in neuromyelitis optica. The same Nogo A product was detected in CNS tissue from all patients with MS but not in control CNS tissue. Conclusion: Soluble Nogo-A may be specific for the CSF of patients with multiple sclerosis and its presence may predict failure of axonal regeneration within the CNS.

TRAIL-induced death of human adult oligodendrocytes is mediated by JNK pathway

Tumor necrosis‐related apoptosis‐inducing ligand (TRAIL) induces apoptosis of oligodendrocytes, target cells of immune attack in multiple sclerosis (MS). TRAIL‐induced human oligodendrocyte (hOL) death depends on TRAIL ligation with its receptor 1 (TRAIL‐R1). However, the intracellular signaling initiated with ligation of TRAIL‐R1 in hOLs is unknown. We defined that intracellular transduction signaling involved in TRAIL‐induced death of hOLs is associated with strong activation of c‐jun NH2‐terminal kinase (JNK) and a dominant negative mutant of MKK4/SEK1, MAP kinase upstream of JNK, inhibited TRAIL‐induced apoptosis of hOLs. The immunoprecipitation experiments showed that JNK3 isoform was predominantly activated upon hOLs exposure to TRAIL and JNK‐3 activation occurred before mitochondrial membrane dysfunction. The other mitogen‐activated protein kinase p38 and ERK, as well as calpains and serine proteases, were not activated during TRAIL‐induced hOL death. Accordingly, the calpain inhibitor, ZLLY.FMK, p38 kinase inhibitor, SB 203580, and serine proteases inhibitor, TPCK, did not protect hOLs from TRAIL‐induced apoptosis. These results demonstrate that JNK pathway is critically involved in hOL death induced by TRAIL and might have significant importance in designing new molecules to protect immune‐mediated hOLs demise.

MicroRNA-146a Negatively Regulates the Immunoregulatory Activity of Bone Marrow Stem Cells by Targeting Prostaglandin E2 Synthase-2

The molecular mechanisms that regulate the immune function of bone marrow–derived mesenchymal stem cells (BMSCs) are not known. We have shown previously that freshly isolated BMSCs when induced to express neuronal stem cell markers lose immunoregulatory function when transferred into mice sensitized to develop experimental autoimmune encephalomyelitis. Recently, microRNAs (miRs) have been shown to be involved in the regulation of several immune responses in both innate and acquired immunity. We now show that among several differentially expressed miRs, miR-146a was strongly upregulated in neuronally differentiated when compared with miR-146a expression in freshly isolated BMSCs or control BMSCs cultured in parallel but in nondifferentiating medium. Inhibition of miR-146a with a selective antagomir restored the immunoregulatory activity of nBMSCs. We mapped miR-146a to its multiple predicted target mRNA transcripts and found that miR-146a was predicted to block PGE2 synthase (ptges-2). We then showed that Ptges-2 was directly targeted by miR-146a using a luciferase reporter assay. Furthermore, increased expression of miR-146a in BMSCs correlated with inhibition of PGE synthase-2 and inhibition of PGE2 release. Accordingly, inhibition of miR-146a restored synthesis of PGE2. These data support the conclusion that miR-146a plays a critical role in the control of the immunoregulatory potential of BMSCs.

Aberrant stress‐induced Hsp70 expression in immune cells in multiple sclerosis

Heat shock protein 70 (Hsp70), a prominent member of the heat shock protein family, is a stress‐induced chaperone, contributing to the “protein triage” mechanism. However, we and others have previously shown that chaperonin activity of Hsp70 also promotes immune recognition of protein/peptide antigens, including myelin autoantigens. Hsp70 has been strikingly elevated in multiple sclerosis (MS) lesions. In a search for the mechanism of Hsp70 up‐regulation in MS, we analyzed Hsp70 expression in peripheral blood mononuclear cells (PBMCs) from MS patients (n = 49), healthy controls (n = 40), and patients with rheumatoid arthritis, (RA; n = 13). Hsp70 was detected by Western blot, and Hsp70 levels were quantified by ELISA. We found that Hsp70 was expressed at low levels in ex vivo PBMCs. However, after heat shock, Hsp70 was up‐regulated significantly more (up to sixfold) in MS patients compared with healthy controls. This significant overproduction of Hsp70 was also seen following another stress condition, LPS stimulation. Hsp70 is a product of several independent genes, and we found the HSPA1B gene product to be the major form responsible for Hsp70 protein overexpression in PBMCs. Hsp70 overexpression was preceded by increased nuclear presence of heat shock factor 1 (HSF1). HSF1 activation depends on phosphorylation, and we found that inhibition of the A group of protein kinase C isoenzymes significantly reduced inducible Hsp70 production. These results indicate that immune cells from MS patients are more prone to Hsp70 induction under stress conditions, suggesting a possible link between Hsp70 overexpression and development of autoimmunity.

Plasmocytoid dendritic cell deficit of early response to toll-like receptor 7 agonist stimulation in multiple sclerosis patients

Plasmacytoid dendritic cells (pDCs), an important immunoregulatory population, are characterized by vigorous secretion of type I interferons (IFNs) in response to toll-like receptor (TLR) 7 and 9 stimulation. We studied the function of pDCs in multiple sclerosis (MS) patients by analysis of TLR7 responses. We assessed a pDC secretion pattern of cytokines in the short term PBMC cultures stimulated with TLR7 agonist. pDCs sorted from PBMCs of both MS patients and controls were used to assess TLR7 expression profile. TLR7 induced signaling in pDCs has been analyzed with intracellular flow cytometry. We have identified a clinically correlated significant decrease of the TLR7-induced IFN-alfa (IFNa) secretion by pDCs from MS patients. This deficit has been accompanied by insufficient intracellular phosphorylation of protein kinase Akt and a decrease of the TLR7 gene expression in MS pDCs. Our results demonstrated a selective pDC deficit in MS supporting a relationship between pDCs and mechanisms of MS.

C5a-Preactivated Neutrophils Are Critical for Autoimmune-Induced Astrocyte Dysregulation in Neuromyelitis Optica Spectrum Disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune neuroinflammatory disease. In contrast to multiple sclerosis, autoantibodies against aquaporin-4 (AQP4) expressed on astrocytic end-feet have been exclusively detected in sera of NMOSD patients. Several lines of evidence suggested that anti-AQP4 autoantibodies are pathogenic, but the mechanism triggering inflammation, impairment of astrocyte function, and the role of neutrophils presented in NMOSD cerebrospinal fluid remains unknown. In this study, we tested how human neutrophils affect astrocytes in the presence of anti-AQP4 Ab-positive serum derived from NMOSD patients. An in vitro model of inflammation consisted of human astrocyte line, NMOSD serum, and allogenic peripheral blood neutrophils from healthy individuals. We showed evidence of pathogenicity of NMOSD serum, which by consecutive action of anti-AQP4 Abs, complement system, and neutrophils affected astrocyte function. Anti-AQP4 Ab binding astrocytes initiated two parallel complementary reactions. The first one was dependent on the complement cytotoxicity via C5b-9 complex formation, and the second one on the reverse of astrocyte glutamate pump into extracellular space by C5a-preactivated neutrophils. As a consequence, astrocytes were partially destroyed; however, a major population of astrocytes polarized into proinflammatory cells which were characterized by pathological glutamate removal from extracellular space.