Anna Jurewicz

Caspase-8 Serves Both Apoptotic and Nonapoptotic Roles

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Notch3 Inhibition in Myelin-Reactive T Cells Down-Regulates Protein Kinase Cθ and Attenuates Experimental Autoimmune Encephalomyelitis

Among its varied functions, Notch signaling is involved in peripheral T cells responses. The activation and polarization of CD4+ T cells toward a Th1 lineage are essential steps in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Inhibition of all four Notch receptors with a γ-secretase inhibitor was shown to block Th1-type polarization and to attenuate the symptoms of experimental autoimmune encephalomyelitis. In this study, we have examined the role of individual Notch receptors in proliferation, cytokine production, and encephalitogenic potential of PLP-reactive T cells. Specific induction of Notch1 and Notch3 transcripts were noted in PLP-reactive T cells upon Ag stimulation. However, using γ-secretase inhibitor and Abs blocking distinct Notch receptors, we have found that selective inhibition of Notch3, but not Notch1, receptor abrogated proliferation, Th1- and Th17-type responses of PLP-reactive T cells. Moreover, Notch3 inhibition in T cells correlated with the down-regulated expression of protein kinase Cθ, a kinase with important regulatory function within mature T cells. Thus, selective inhibition of the Notch3 receptor may have important effects on peripheral T cell responses and may offer a new attractive target in treating autoimmune diseases, including multiple sclerosis.

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.

Overcoming failure to repair demyelination in EAE: γ-secretase inhibition of Notch signaling

In multiple sclerosis (MS), myelin destroyed by the immune attack is not effectively repaired by oligodendrocytes (OLs) and MS foci eventually undergo glial scarring. Although oligodendrocyte precursor cells (OPCs) are normally recruited to the lesion areas, they fail to mature and remyelinate the damaged fibers. Activation of the Notch pathway has been shown to inhibit OPC differentiation and to hamper their ability to produce myelin during CNS development. We have recently shown that inhibition of γ-secretase within the CNS of SJL/J mice with experimental autoimmune encephalomyelitis (EAE) blocks Notch pathway activation in OLs, promotes remyelination, reduces axonal damage and significantly enhances clinical recovery from the disease. Our results suggest that inhibiting the non-myelin permissive environment maintained by Notch pathways within the mature CNS offers a new strategy for treating autoimmune demyelination, including MS.

Brain-Derived Heat Shock Protein 70-Peptide Complexes Induce NK Cell-Dependent Tolerance to Experimental Autoimmune Encephalomyelitis

Heat shock proteins (Hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). In this study, we show that Hsp70-peptide complexes (pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139–151 (PLP139–151) immunization. In contrast, pure Hsp70 or Hsp70-pc derived from brains of healthy mice or other inflamed tissue did not modulate the expression of EAE. In animals in which EAE had been suppressed by Hsp70-pc, lymphocytes showed increased cell death in response to PLP139–151 that correlated with elevated IFN-γ and NO production. Coculture of spleen cells from Hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139–151. Transfer of NK cells from Hsp70-pc-immunized mice to recipients sensitized for EAE abolished disease development. Thus, we propose that Hsp70 demonstrate the ability to bind to peptides generated during brain inflammation and to induce a regulatory NK cell population that is capable of preventing subsequent autoimmunization for EAE.

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.

EAE Tolerance Induction with Hsp70-Peptide Complexes Depends on H60 and NKG2D Activity

Inflammation leads to induction of tissue stress conditions that might contribute to the generation of mechanisms limiting ongoing immune responses. We have shown previously that peptides derived from brain tissue of mice with experimental autoimmune encephalomyelitis (EAE) complexed with the chaperone heat shock protein 70 (Hsp70-pc) induce an NK-cell-dependent tolerance for subsequent EAE sensitization. We now present data that showed that the MHC class I-related glycoprotein H60 determines Hsp70-pc-induced EAE inhibition. Hsp70-pc led to significant and selective up-regulation of H60 expression in SJL/J mice, and Ab-blocking of H60 expression led to loss of EAE tolerance. Similarly, blocking of the NK cell receptor for H60, NKG2D, also reversed the Hsp70-pc-induced EAE inhibition. In contrast, in C57BL/6 mice H60 was not expressed, and Hsp70-pc-induced tolerance was not detected. The NK cell mediated Hsp70-pc-induced tolerance to EAE was dependent on modulation of dendritic cells function leading to diminished T cell reactivity to PLP. As, no increase of H60 expression on T cells from EAE mice immunized with PLP was detected, and no enhanced loss of CD3+H60+over CD3+H60− cells in Hsp70-pc-induced EAE tolerance was found direct killing of H60+ PLP-reactive cells seems not to be involved in the Hsp70-pc-induced tolerance induction. We have provided evidence that Hsp70-pc-induced tolerance for EAE, mediated by NK cells, involves induction of H60 ligand and its interaction with NKG2D receptor. NK cells tolerization of EAE depends on altered dendritic cells activity leading to enhanced death of Ag reactive cells.