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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 13, 2005 as doi:10.1096/fj.05-4650fje. |
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,1
Departments of
* Neurology and
Immunology, Mayo Clinic, Rochester, Minnesota, USA
1 Correspondence: Department of Neurology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA. E-mail: rodriguez.moses{at}mayo.edu
SPECIFIC AIMS
While investigating the CD4+ T cell Th1 (STAT4) and Th2 (STAT6) response to Theiler’s virus infection, we found mice with a demyelinating phenotype that satisfies the requirements for a long incubation period prior to inflammatory demyelination proposed for the human disease multiple sclerosis (MS). Infection of STAT4–/– mice resulted in a phenotype where prominent inflammatory demyelination similar to MS did not occur until after 180 days after intracerebral infection.
PRINCIPAL FINDINGS
1. STAT4–/– but not STAT6–/– mice developed spinal cord demyelinating lesions very late after virus infection
At 45 and 90 days after infection demyelination in STAT4–/– mice was limited (Fig. 1
), and we would have characterized these mice as resistant to TMEV-induced demyelination. For comparison, prototypic susceptible SJL/J mice injected with the same lot of virus resulted in a severe demyelination at the same time points after infection. However, by 180 days after infection, STAT4–/– mice presented with severe demyelination and meningeal inflammation. From day 270 to 390 after infection, 27 to 29% of spinal cord quadrants contained demyelination (Fig. 1)
. Lesions were characterized by primary demyelination with relative preservation of axons, intense inflammation consisting of mononuclear cells, absence of oligodendrocyte-mediated, or Schwann cell-mediated remyelination (Fig. 2
). In contrast STAT6–/– mice or BALB/cJ mice showed minimal or no demyelination throughout the course of the experiment.
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To assess neurologic function in chronically infected STAT4–/–and STAT6–/– mice (>270 days after infection), spontaneous activity was monitored continuously for 3 days. Vertical activity, which measures rearing, is related to hind limb paralysis and is the more sensitive indicator of disability. A rank sum comparison of the combined hourly nocturnal vertical activity (6 AM to 5 PM) between groups demonstrated a significant difference in rearing (P<0.001). STAT4–/––deficient mice broke the vertical beam 181 ± 44-fold/h compared with 398 ± 30 vertical beam breaks/h for STAT6–/– mice (median values±SD) during their normally active nighttime period.
2. (STAT4–/– X STAT6–/–) F1 mice did not develop significant spinal cord demyelination
We crossed STAT4–/–mice to STAT6–/– mice to determine whether STAT mutations resulted in spinal cord demyelination. F1 mice were generally resistant to chronic demyelinating disease.
3. STAT4–/– mice had normal humoral immune responses to TMEV infection
Deficiency of transcription factors from birth could have affected protective humoral responses directed against virus and predisposition to the late demyelinating disease. We assessed antibody responses in serum directed against purified virus antigens by ELISA and by virus-specific neutralization. There were no major differences in the humoral immune response between the strains.
4. Lesions in STAT4–/– mice showed CD4+ T cells, CD8+ T cells, macrophages, and expression of MHC Class I and Class II antigens
Immunostaining was performed on spinal cord sections from STAT4–/–, STAT6–/–, and F1 mice at 270 days after infection. CD4+ and CD8+ T cells were observed in the spinal cords of STAT4–/– mice, but not in STAT6–/–or F1 mice. There was greater staining for F4/80, which marks microglia and a subset of macrophages, in STAT4–/–mice than STAT6–/– mice or F1 mice. In STAT4–/– mice, Class I was distributed within the lesion in inflammatory cells, blood vessels and in cells with glial morphology, but only minimally in STAT6–/– mice or F1 mice. In STAT4–/– mice, Class II was expressed in macrophages and microglia, but less so in STAT6–/– mice or F1 mice. The late demyelinating lesions in the STAT4–/– mice were densely inflammatory suggesting an immune-mediated mechanism of pathology such as seen in MS.
5. Deletion of STAT4 or STAT6 did not alter brain neuronal damage after virus infection
We asked whether deficiency in STAT4 or STAT6 would predispose specific populations of brain neurons to late virus-induced injury. All 3 strains demonstrated a similar extent of pathology on day 7 in cortex, hippocampus, and striatum, with minimal disease in the cerebellum. By day 90, most neuronal brain disease had subsided in all strains with only occasional mice showing persistent neuronal pathology. On day 270, when STAT4–/– mice showed late demyelination in the spinal cord, there was minimal neuronal disease in the brain. Thus, STAT4 deficiency exclusively altered the late demyelinating disease.
6. STAT4–/– mice propagated viral RNA in the spinal cord during all stages of infection
High expression of virus RNA was observed during acute infection (7, 21, and 45 days) in the brain and spinal cord of all 3 strains. At 7 days there was an increased level of virus RNA expression in the brain (P=0.001) and in the spinal cord (P<0.001) in STAT4–/– mice compared with STAT6–/– or F1 mice. No difference in virus RNA in the brain was measured at 21 and 45 days. In the spinal cord of STAT4–/– mice, more viral RNA was measured as compared with F1 mice (21 days, P=0.04) and STAT6–/– mice (45 days, P=0.002). There was more viral RNA in the brain and spinal cord of STAT4–/– mice at 90, 180, and 360 days compared with STAT6–/– or to F1 mice (P<0.001). While all strains replicated TMEV during the early stages of infection, STAT4–/– and STAT6–/– mice maintained a high level of viral RNA throughout the course of infection, whereas F1 mice controlled viral RNA.
F1 mice that remained resistant to demyelination appeared to control virus RNA after 45 days of infection. To test whether virus RNA was truly "cleared" in F1 mice or was under "control" by the immune system, we treated F1 mice that were infected for 270 days with cyclophosphamide (50mg/Kg) which is immunosuppressive. All mice treated with cyclophosphamide had detectable virus RNA (3 to 4 log10 of virus RNA), whereas none of the PBS-treated mice did (P<0.001, Chi square test). In mice that had controlled virus infection, persistent virus RNA could be detected after immune suppression.
7. STAT4–/– mice expressed virus antigen in spinal cord primarily during the late demyelinating stages of disease
Virus antigen was expressed to a limited extent in all strains at 45 days despite the absence of demyelination in any strain. At both 90 and 270 days after infection there was a significant (P=0.006) increase in the percent of white matter quadrants with virus in STAT4–/– compared with F1 and STAT6–/– mice.
8. Epitope spreading to myelin antigens did not account for the late demyelination in STAT4–/– mice
Epitope spreading has been proposed to explain the chronic demyelination observed in the prototypic SJL/J strain. We determined the proliferative responses of T cells obtained from spleens of chronically TMEV-infected STAT4–/–, STAT6–/–, or F1 mice in response to various CNS specific myelin antigens. Unexpectedly, T cells from nondemyelinating F1 mice showed the greatest proliferative response to several antigens, including bovine MBP. T cells from F1 mice and STAT4–/– mice responded to MOG and PLP139-154 and PLP179-198. Cells from F1 mice responded strongly to PLP41-60 and PLP91-100, whereas a minimal response was measured in STAT4–/– mice. F1 mice responded to PLP209-228 only at high concentrations whereas STAT4–/– did not respond.
9. The mechanism of the late demyelinating model of the late demyelinating phenotype is dependent on immune cells of the STAT4–/– strain
We explored the mechanism of why STAT4–/– mice developed demyelination using bone marrow chimeric adoptive transfer. Mature T cells were deleted from donor bone marrow and transplanted into lethally irradiated recipient mice. Recipient mice developed an immune system comprised of donor cells, but maintained radiation-resistant cells of the host CNS. F1 mice were reconstituted with STAT4–/– bone marrow and allowed 8 wk to recover and develop a new immune repertoire. Mice were then infected with TMEV and killed after chronic infection. All 17 F1 mice reconstituted with STAT4–/– bone marrow developed severe demyelination. Reciprocal STAT4–/– mice reconstituted with F1 bone marrow had significantly less demyelination than F1 mice reconstituted with STAT4–/– bone marrow (P=0.001, Student’s t test). The mechanism of the late demyelinating phenotype of STAT4–/– mice was due to immune dysregulation rather than an intrinsic abnormality of the CNS.
CONCLUSIONS AND SIGNIFICANCE
We established that the mechanism by which deletion of STAT4 predisposes mice to late demyelination depends on immune dysregulation in STAT4–/– mice rather than on an intrinsic characteristic of the CNS of BALB/cJ mice. We propose that virus is partially controlled such that virus antigen is not expressed in the spinal cord of mice that do not demyelinate. However, as a consequence of the STAT4 deletion and a function of age, virus antigen becomes expressed in the spinal cord white matter triggering demyelination. By understanding how the virus was controlled in STAT4–/– mice during the first 180 days, then why virus antigen emerged, may provide insight into the immune dysregulation that has been proposed for MS.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4650fje;
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