Diethilde Theil

Exacerbation of Viral and Autoimmune Animal Models for Multiple Sclerosis by Bacterial DNA

Theilers murine encephalomyelitis virus (TMEV) infection and relapsing‐remitting experimental allergic encephalomyelitis (R‐EAE) have been used to investigate the viral and autoimmune etiology of multiple sclerosis (MS), a possibleTh1‐type mediated disease. DNA immunization is a novel vaccination strategy in which few harmful effects have been reported. Bacterial DNA and oligodeoxynucleotides, which contain CpG motifs, have been reported to enhance immunostimulation. Our objectives were two‐fold: first, to ascertain whether plasmid DNA, pCMV, which is widely used as a vector in DNA immunization studies, could exert immunostimulation in vitro; and second, to test if pCMV injection could modulate animal models for MS in vitro. We demonstrated that this bacterially derived DNA could induce interleukin (IL)‐12, interferon (IFN)γ (Th1‐promoting cytokines), and IL‐6 production as well as activate NK cells. Following pCMV injections, SJL/J mice were infected with TMEV or challenged with encephalitogenic myelin proteolipid protein (PLP) peptides. pCMV injection exacerbated TMEV‐induced demyelinating disease in a dose‐dependent manner. Exacerbation of the disease did not correlate with the number of TMEV‐antigen positive cells but did with an increase in anti‐TMEV antibody. pCMV injection also enhanced R‐EAE with increased IFNγ and IL‐6 responses. These results caution the use of DNA vaccination in MS patients and other possible Th1‐mediated diseases.

Prevalence of HSV-1 LAT in human trigeminal, geniculate, and vestibular ganglia and its implication for cranial nerve syndromes.

Herpes simplex virus type 1 (HSV‐1) enters sensory neurons and can remain latent there until reactivation. During latency restricted HSV‐1 gene expression takes place in the form of latency‐associated transcripts (LAT). LAT has been demonstrated to be important not only for latency but also for reactivation, which may cause cranial nerve disorders.

Antibody Association with a Novel Model for Primary Progressive Multiple Sclerosis: Induction of Relapsing-Remitting and Progressive Forms of EAE in H2S Mouse Strains

Multiple sclerosis (MS) can be divided into 4 clinical forms: relapsing‐remitting (RR), primary progressive (PP), secondary progressive (SP), and progressive relapsing (PR). Since PP‐MS is notably different from the other forms of MS, both clinically and pathologically, the question arises whether PP‐MS is immunologically similar to the other forms. The pathogenesis of the PP‐MS remains unclear, partly due to a lack of highly relevant animal models. Using an encephalitogenic peptide from myelin oligodendrocyte glycoprotein (MOG)92–106, we have established animal models that mimic different forms of MS in 2 strains of H‐2s mice, SJL/J and A.SW. We induced experimental allergic encephalomyelitis (EAE) using MOG92‐106 in the presence or absence of supplemental Bordetella pertussis (BP). Although, SJL/J mice developed RR‐EAE whether BP was given or not, A.SW mice developed PP‐EAE without BP and SP‐EAE with BP. Histologically, SJL/J mice developed mild demyelinating disease with T cell infiltration, while A.SW mice developed large areas of plaque‐like demyelination with immunoglobulin deposition and neutrophil infiltration, but with minimal T cell infiltration. In A.SW mice without BP, high titer serum anti‐MOG antibody was detected and the anti‐MOG IgG2a/IgG1 ratio correlated with survival times of mice. We hypothesized that, in A.SW mice, a Th2 response favors production of myelinotoxic antibodies, leading to progressive forms with early death. Our new models indicate that a single encephalitogen could induce either RR‐, PP‐, or SP‐ forms of demyelinating disease in hosts with immunologically different humoral immune responses.

Alterations in cytokine but not chemokine mRNA expression during three distinct Theiler's virus infections

DA, GDVII and H101 are neurovirulent strains of Theilers murine encephalomyelitis virus that cause very different neuropathology and CNS disease when inoculated into SJL/J mice. DA virus causes a chronic demyelinating disease, GDVII virus causes an acute fatal polioencephalomyelitis, and H101 virus causes an acute pachymeningitis with hydrocephalus. Performing RNase protection assays, we detected the same pattern of chemokine (RANTES, MCP-1, IP-10, MIP-1beta, MIP-1alpha and MIP-2) mRNA expression in brain and spinal cord during all three infections. In contrast, IFN-beta and IL-6 mRNA were highly expressed only in GDVII virus infection, whereas high levels of LT-alpha mRNA were only found during DA virus infection. Our study demonstrates that proinflammatory cytokines are involved in the neuropathogenesis of CNS disease and modulate the acute and chronic process underlying different pathologic features of disease.

HSV-1 not only in human vestibular ganglia but also in the vestibular labyrinth.

Reactivation of herpes simplex virus type 1 (HSV-1) in the vestibular ganglion (VG) is the suspected cause of vestibular neuritis (VN). Recent studies reported the presence of HSV-1 DNA not only in human VGs but also in vestibular nuclei, a finding that indicates the possibility of viral migration to the human vestibular labyrinth. Distribution of HSV-1 DNA was determined in geniculate ganglia, VGs, semicircular canals, and macula organs of 21 randomly obtained human temporal bones by nested PCR. Viral DNA was detected in 48% of the labyrinths, 62% of the VGs, and 57% of the geniculate ganglia. The potential significance of this finding is twofold: (1) Inflammation in VN could also involve the labyrinth and thereby cause acute unilateral vestibular deafferentation. (2) As benign paroxysmal positional vertigo often occurs in patients who have had VN, it could also be a sequel of viral labyrinthitis.

Viruses can silently prime for and trigger central nervous system autoimmune disease.

Although many viruses have been isolated from patients with multiple sclerosis (MS), as yet, no one agent has been demonstrated to cause MS. In contrast, epidemiological data indicate that viral infections are associated with exacerbations of MS. Here, we present data showing that virus infections can subclinically prime animals for central nervous system (CNS) autoimmune disease; long after the original infection has been eradicated, a nonspecific challenge/infection can trigger an exacerbation. The priming infectious agent must show molecular mimicry with self-CNS antigens such as glial fibrillary acidic protein (GFAP), myelin associated glycoprotein (MAG) or myelin proteolipid protein (PLP). The subsequent challenge, however, may be nonspecific; complete Freund’s adjuvant (CFA), or infection with a recombinant vaccinia virus encoding an irrelevant protein, could trigger CNS disease. In the CNS, we could detect a mononuclear cell infiltration, but no demyelination was found. However, if the pathogenesis of MS is similar to that of this novel animal model for CNS autoimmune disease, our findings could help explain why exacerbations of MS are often associated with a variety of different viral infections.

Latency of alpha-herpes viruses is accompanied by a chronic inflammation in human trigeminal ganglia but not in dorsal root ganglia

The immune response to latent herpesvirus infections was compared in human trigeminal ganglia (TG) and dorsal root ganglia (DRG) of 15 dead individuals. On the basis of our previous findings, we hypothesized that T-cells would be attracted to sensory neurons latently infected with herpes simplex virus type 1 (HSV-1), but not to those harboring latent varicella zoster virus (VZV). We showed that the TG contain a positive hybridization signal for HSV-1 latency-associated transcript (LAT), whereas the DRG from the same individuals lack detectable LAT. In contrast, immunohistochemistry revealed that latent VZV protein 62 stained positive in the vast majority of all tested TG and DRG. T-cell infiltrates prominently surrounded individual neurons in the TG but not in the DRG. TaqMan polymerase chain reaction also showed higher expression of CD8 and RANTES transcripts in the TG versus DRG. Only the infiltrates in the TG, but not in the DRG, produced RANTES at the protein level. Because it has been shown that RANTES protein is produced only after T-cell receptor stimulation, we assume that T-cell infiltration is associated with antigen recognition in the TG but not in the DRG.

HSV-1 genome subnuclear positioning and associations with host-cell PML-NBs and centromeres regulate LAT locus transcription during latency in neurons.

Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.). Latent HSV-1 genomes display two major patterns, called “Single” and “Multiple”, which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. “Single” and “Multiple” patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the “Multiple” pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction.

Latency of Herpes simplex virus type-1 in human geniculate and vestibular ganglia is associated with infiltration of CD8+ T-cells

Herpes simplex virus type‐1 latency and CD8+ T‐cell occurrence were investigated in the trigeminal, geniculate, and vestibular ganglia from seven deceased humans. The HSV‐1 “latency‐associated transcript” was assessed by in situ hybridization and quantitative RT‐PCR. Infiltration of CD8+ T cell was detected by immunohistochemistry and quantitative RT‐PCR. The data show that HSV‐1 latency and CD8+ T‐cell infiltration are not solely confined to the trigeminal ganglia but can also occur in other cranial ganglia along the neuroaxis. However, the HSV‐1 latency transcripts in the geniculate and vestibular ganglia were expressed at a very low level. The difference in CD8 transcript levels among HSV‐1 latently infected trigeminal ganglia, geniculate, and vestibular ganglia was less conspicuous. Colocalization of latent HSV‐1 and CD8+ T cells in geniculate and vestibular ganglia supports further the hypothesis that HSV‐1 reactivation is possible in these ganglia and is the cause of Bells palsy and vestibular neuritis. J. Med. Virol. 82:1917–1920, 2010.

Presence of HSV-1 Immediate Early Genes and Clonally Expanded T-cells with a Memory Effector Phenotype in Human Trigeminal Ganglia

The latent persistence of herpes simplex virus type 1 (HSV‐1) in human trigeminal ganglia (TG) is accompanied by a chronic CD8 T‐cell infiltrate. The focus of the current work was to look for HSV‐1 transcription activity as a potential trigger of the immune response and to characterize the immune cell infiltrates by this feature. We combined in situ hybridization, laser cutting microscopy, and single cell RT‐PCR to demonstrate the expression of the HSV‐1 immediate early (IE) genes ICP0 and ICP4 in human trigeminal neurons. Using CDR3 spectratyping, we showed that the infiltrating T‐cells are clonally expanded, indicating an antigen‐driven immune response. Moreover, the persisting CD8+ T‐cells had features of the memory effector phenotype. The voltage‐gated potassium channel Kv1.3, a marker of chronic activated memory effector cells, and the chemokines CCL5 and CXCL10 were expressed by a subpopulation of infiltrating cells. The corresponding chemokine receptors CCR5 and CXCR3 were co‐expressed on virtually all CD8 T‐cells. In addition, T‐cells expressed granzymes and perforin. In contrast to animal models of HSV‐1 latency, hardly any FoxP3‐positive regulatory T‐cells were detected in human TG. Thus, HSV‐1 IE genes are expressed in human TG and the infiltrating T‐cells bear several characteristics that suggest viral antigenic stimulation.