C. Jane Welsh

Drosophila Perlecan modulates FGF and Hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.

Chronic restraint stress during early Theiler's virus infection exacerbates the subsequent demyelinating disease in SJL mice.

Chronic restraint stress, administered during early infection with Theilers virus, was found to exacerbate the acute central nervous system (CNS) viral infection and the subsequent demyelinating phase of disease (an animal model of Multiple Sclerosis (MS)) in SJL male and female mice. During early infection, stressed mice displayed decreased body weights and spontaneous activity; while increased behavioral signs of illness and plasma corticosterone (CORT) levels. During the subsequent chronic demyelinating phase of disease, previously stressed mice had greater behavioral signs of the chronic phase, worsened rotarod performance, and increased inflammatory lesions of the spinal cord. In addition, mice developed autoantibodies to myelin basic protein (MBP), proteolipid protein peptide (PLP139-151), and myelin oligodendrocyte glycoprotein peptide (MOG33-55).

CHARACTERISTICS OF CLONED CEREBROVASCULAR ENDOTHELIAL CELLS FOLLOWING INFECTION WITH THEILER'S VIRUS. I: ACUTE INFECTION

The present study describes the replication of Theilers virus in cloned cerebrovascular endothelial cells (CVE) isolated from strains of mice that are either susceptible or resistant to Theilers virus-induced demyelination (TVID). CVE isolated from all strains of mice were equally permissive to Theilers virus infection. Interferon-gamma and tumor necrosis factor-alpha were found to inhibit the replication of Theilers virus in CVE. A correlation between susceptibility to demyelination and the ability of Theilers virus to induce MHC Class I on CVE was demonstrated.

Interleukin-6 as a mechanism for the adverse effects of social stress on acute Theiler's virus infection

Prior exposure to social disruption stress (SDR) exacerbates both the acute and chronic phase of Theilers murine encephalomyelitis virus infection (TMEV; [Johnson, R.R., Storts, R., Welsh, T.H., Jr., Welsh, C.J., Meagher, M.W., 2004. Social stress alters the severity of acute Theilers virus infection. J. Neuroimmunol. 148, 74--85; Johnson, R.R., Prentice, T.W., Bridegam, P., Young, C.R., Steelman, A.J., Welsh, T.H., Welsh, C.J.R., Meagher, M.W., 2006. Social stress alters the severity and onset of the chronic phase of Theilers virus infection. J. Neuroimmunol. 175, 39--51]). However, the neuroimmune mechanism(s) mediating this effect have not been determined. The present study examined whether stress-induced increases in the proinflammatory cytokine interleukin-6 (IL-6) contributes to the adverse effects of SDR on acute TMEV infection. Experiment 1 demonstrated that SDR increases central and peripheral levels of IL-6 and that this effect is reversed by intracerebral ventricular infusion of neutralizing antibody to IL-6 prior to each of six SDR sessions. Although SDR reduced the sensitivity of spleen cells to the anti-inflammatory effects of corticosterone, the neutralizing antibody to IL-6 did not alter this effect. To investigate whether stress-induced increases in IL-6 contribute to the exacerbation of acute TMEV infection, Experiment 2 examined whether intracerebral administration of neutralizing antibody to IL-6 during SDR would prevent the subsequent exacerbation of acute TMEV infection. Experiment 3 then replaced the social stress with intracerebral infusion of IL-6 to assess sufficiency. As expected, prior exposure to SDR subsequently increased infection-related sickness behaviors, motor impairment, CNS viral titers, and CNS inflammation. These deleterious effects of SDR were either prevented or significantly attenuated by intracerebral infusion of neutralizing antibody to IL-6 during the stress exposure period. However, infusion of IL-6 alone did not mimic the adverse effects of SDR. We conclude that IL-6 is necessary but not sufficient to exacerbate acute TMEV infection.

Galectin-9 Protein Is Up-regulated in Astrocytes by Tumor Necrosis Factor and Promotes Encephalitogenic T-cell Apoptosis

Background: Galectins are increased in astrocytes of patients with multiple sclerosis. Results: TNF up-regulates galectin-9 in primary astrocytes via the TNFR1/JNK/c-Jun pathway and can induce apoptosis of encephalitogenic T-cells. Conclusion: Astrocytes up-regulate galectin-9 in response to the proinflammatory cytokine TNF. Significance: Astrocyte-derived galectin-9 may function to restrict encephalitogenic T-cell-mediated inflammation in the CNS. Demyelination and axonal damage in multiple sclerosis (MS) are thought to be a consequence of inflammatory processes that are perpetuated by activated glia and infiltrating leukocytes. Galectin-9 is a β-galactoside binding lectin capable of modulating immune responses and appears to be up-regulated in MS. However, its role in the pathogenesis of MS has yet to be determined. Here, we report that proinflammatory cytokines induce galectin-9 (Gal-9) expression in primary astrocytes and the mechanism by which TNF up-regulates Gal-9. Astrocytes did not express Gal-9 under basal conditions nor did IL-6, IL-10, or IL-13 trigger Gal-9 expression. In contrast, IL-1β, IFN-γ, and particularly TNF up-regulated Gal-9 in astrocytes. TNF-induced Gal-9 expression was dependent on TNF receptor 1 (TNFR1) as TNF failed to induce Gal-9 in TNFR1−/− astrocytes. Blockade of the JNK MAP kinase pathway with the JNK inhibitor SP600125 abrogated TNF-induced Gal-9, whereas p38 and MEK inhibitors had minimal effects. Furthermore, specific knockdown of c-Jun via siRNA in astrocytes before TNF treatment greatly suppressed Gal-9 transcription, suggesting that TNF induces astroglial Gal-9 through the TNF/TNFR1/JNK/cJun signaling pathway. Finally, utilizing astrocytes from Lgals9 mutant (Gal-9−/−) mice as well as a myelin basic protein-specific Tim-3+ encephalitogenic T-cell clone (LCN-8), we found that conditioned medium from TNF-stimulated Gal-9+/+ but not Gal-9−/− astrocytes increased the percentage of apoptotic encephalitogenic T-cells. Together, our results suggest that Gal-9 is induced in astrocytes by TNF via the JNK/c-Jun pathway and that astrocyte-derived Gal-9 may function as an immunoregulatory protein in response to ongoing neuroinflammation.

Restraint stress modulates virus specific adaptive immunity during acute Theiler’s virus infection

Multiple sclerosis (MS) is a devastating CNS disease of unknown origin. Multiple factors including genetic background, infection, and psychological stress affect the onset or progression of MS. Theilers murine encephalomyelitis virus (TMEV) infection is an animal model of MS in which aberrant immunity leads to viral persistence and subsequently results in demyelination that resembles MS. Here, we examined how stress during acute TMEV infection altered virus-specific cell mediated responses. Using immunodominant viral peptides specific for either CD4(+) or CD8(+) T cells, we found that stress reduced IFN-gamma producing virus-specific CD4(+) and CD8(+) T cells in the spleen and CD8(+) T cells CNS. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus or viral peptides, indicated that stress decreased both type 1 and type 2 responses. Glucocorticoids were implicated in the decreased T cell function as the effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone. As T cells mediate viral clearance in this model, our data support the hypothesis that stress-induced immunosuppression may provide a mechanism for enhanced viral persistence within the CNS.

Neuroimmune Interactions in a Model of Multiple Sclerosis

Psychological stress has been implicated in both the onset and exacerbation of multiple sclerosis (MS). Our research has focused on the role of stress at the onset of MS, using the mouse model Theilers murine encephalomyelitis virus‐induced demyelination. Theilers virus is a natural pathogen of mice that causes a persistent infection of the central nervous system (CNS) and inflammatory immune‐mediated demyelination that is very similar to MS. Our research has shown that restraint stress sufficiently increases corticosterone secretion to cause immunosuppression. Stressed mice develop decreased innate and adaptive immune responses, including decreased chemokine and cytokine responses, to virus, which leads to increased viral replication within the CNS. Higher levels of virus then cause increased later demyelinating disease. These findings may have important implications in our understanding of the interactions between stress and the development of autoimmune diseases induced by infectious agents.

Glucocorticoid exposure alters the pathogenesis of Theiler's murine encephalomyelitis virus during acute infection

Previous research has shown that chronic restraint stress exacerbates Theilers virus infection, a murine model for CNS inflammation and multiple sclerosis. The current set of experiments was designed to evaluate the potential role of glucocorticoids in the deleterious effects of restraint stress on acute CNS inflammatory disease. Exposure to chronic restraint stress resulted in elevated levels of corticosterone as well as increased clinical scores and weight loss (Experiment 1). In addition, corticosterone administration alone exacerbated behavioral signs of TMEV-induced sickness (i.e. decreased body weight, increased symptoms of encephalitis, and increased mortality) and reduced inflammation in the CNS (Experiment 2). Infected subjects receiving exogenous corticosterone showed exacerbation of acute phase measures of sickness and severe mortality as well as decreased viral clearance from CNS (Experiment 3). These findings indicate that corticosterone exposure alone is sufficient to exacerbate acute CNS inflammatory disease.

Castration of male C57L/J mice increases susceptibility and estrogen treatment restores resistance to Theiler's virus-induced demyelinating disease.

Intracerebral inoculation of Theilers murine encephalomyelitis virus (TMEV) results in immune‐mediated demyelination in selective mouse strains. We have previously demonstrated that the males of C57L mice are significantly more susceptible to TMEV‐induced demyelinating disease. To assess further the hormonal influence for this gender‐associated differential susceptibility, estrogen‐treated, castrated C57L mice were infected with TMEV and compared with sham‐operated and/or placebo‐treated mice. Interestingly, castration further elevated the susceptibility to virally induced demyelinating disease compared with sham‐castrated control mice, and prolonged treatment of castrated mice with estrogen restored the resistance to the level of control mice. These results strongly suggest that sex hormone levels contribute to the gender‐biased susceptibility to TMEV‐induced demyelinating disease. Mice treated with estrogen showed a significantly decreased level of virus‐specific Th1 responses both in the periphery and in the CNS. In addition, in vitro estrogen treatment was able to inhibit viral replication directly in macrophages, consistent with the lower level of viral RNA in microglia/macrophages in the CNS from castrated estrogen‐treated mice compared with controls. Also, estrogen treatment inhibited VCAM‐1 expression induced by tumor necrosis factor‐α in cerebral vascular endothelial (CVE) cells via inhibition of nuclear factor‐κB (NFκB), which is produced in various glial cells upon TMEV infection. Overall, estrogen treatment appears to exert its effects on viral replication, induction of immune responses, as well as infiltration of activated immune cells into the CNS via inhibition of NFκB function.

Effects of Stress on the Immune Response to Theiler's Virus - Implications for Virus-Induced Autoimmunity

Psychological stress is an important factor in susceptibility to many diseases. Our laboratory has been investigating the impact of stress on the susceptibility to Theiler’s virus-induced demyelination (TVID), a mouse model of multiple sclerosis. Using immunodominant viral peptides specific for identification of either CD4+ or CD8+ T cells, stress reduced IFN-γ-producing virus-specific CD4+ and CD8+ T cells in the spleen and CD8+ T cells in the CNS. Expression of mRNA for the Th1 transcription factor T-bet and the Th2 transcription factor GATA-3 were decreased in spleen cells isolated from stressed mice. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus indicated that stress decreased both type 1 and type 2 responses. The adverse effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone, indicating a major role for glucocorticoids. Global stress-induced immunosuppression resulted in higher levels of virus replication and dissemination. The higher viral load subsequently led to an earlier disease onset and more severe clinical and histological signs of demyelinating disease. Our results have important implications for understanding the pathogenesis of MS, and suggest that stressful events during early infection with an agent capable of inducing demyelination may result in immunosuppression and failure to eliminate the pathogen, which in turn may lead to the development of MS.