image_1_Distinct Gene Profiles of Bone Marrow-Derived Macrophages and Microglia During Neurotropic Coronavirus-Induced Demyelination.TIF

<p>Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and axonal loss. Demyelinating lesions are associated with infiltrating T lymphocytes, bone marrow-derived macrophages (BMDM), and activated resident microglia. Tissue damage is thought to be mediated by T cell produced cytokines and chemokines, which activate microglia and/or BMDM to both strip myelin and produce toxic factors, ultimately damaging axons and promoting disability. However, the relative contributions of BMDM and microglia to demyelinating pathology are unclear, as their identification in MS tissue is difficult due to similar morphology and indistinguishable surface markers when activated. The CD4 T cell-induced autoimmune murine model of MS, experimental autoimmune encephalitis (EAE), in which BMDM are essential for demyelination, has revealed pathogenic and repair-promoting phenotypes associated with BMDM and microglia, respectively. Using a murine model of demyelination induced by a gliatropic coronavirus, in which BMDM are redundant for demyelination, we herein characterize gene expression profiles of BMDM versus microglia associated with demyelination. While gene expression in CNS infiltrating BMDM was upregulated early following infection and subsequently sustained, microglia expressed a more dynamic gene profile with extensive mRNA upregulation coinciding with peak demyelination after viral control. This delayed microglia response comprised a highly pro-inflammatory and phagocytic profile. Furthermore, while BMDM exhibited a mixed phenotype of M1 and M2 markers, microglia repressed the vast majority of M2-markers. Overall, these data support a pro-inflammatory and pathogenic role of microglia temporally remote from viral control, whereas BMDM retained their gene expression profile independent of the changing environment. As demyelination is caused by multifactorial insults, our results highlight the plasticity of microglia in responding to distinct inflammatory settings, which may be relevant for MS pathogenesis.</p

RNase L mitigates spinal cord demyelination.

<p>Spinal cords of infected wt and RL^−/− mice at day 7 (top) and day 10 (bottom) p.i. stained with Luxol Fast Blue. Scale bar = 100 µm.</p

RNase L does not alter CNS inflammation.

<p>Numbers and composition of CD45^hi inflammatory cells derived from brains of infected wt and RL^−/− mice analyzed by flow cytometry at the indicated times p.i. Numbers of bone marrow derived CD45^hi cells/brain are shown in the top left panel. Composition of cell infiltrates is indicated by relative percentages of Ly-6G⁺ neutrophils (ND = not detected), F4/80⁺ macrophages, CD4 T cells, and CD8 T cells. Percentages of virus-specific CD8 T cells within the CD8 population is assessed by D^b/S510 tetramer staining (CD8+Tet+). Data are the mean of two independent experiments±SEM.</p

RNase L prevents spread of MHV-JHM to spinal cord grey matter.

<p>Viral antigen in spinal cords from infected wt and RL^−/− mice at day 7 p.i. Note spread of virus into the grey matter (GM) (arrows, bottom panel) is only observed in RL^−/− mice, whereas viral antigen is restricted to white matter (WM) in wt mice. Also note the absence of infected neurons. Scale bar = 50 µm.</p

Increased demyelination and axonal damage in brain stem.

<p>Brain stems of infected wt and RL^−/− mice at day 10 p.i. stained with Luxol Fast Blue (top panels). Scale bar = 100 µm. Immunohistochemical staining for axonal integrity in brain stem at day 10 p.i. (bottom panels) Scale bar = 100 µm. Note more abundant loss of myelin and axonal degeneration in infected RL^−/− relative to wt mice.</p

Levels of viral nucleocapsid protein encoding mRNA in CNS and liver.

a<p>relative to GAPDH.</p>b<p>SD from 3 individual mice.</p>c<p>below detection.</p

RNase L enhances microglia activation.

<p>Microglia activation in spinal cords from infected wt and RL^−/− mice at day 7 p.i. Microglia/monocytes detected by immunoperoxidase staining using anti Iba-1antibody.</p

RNase L ameliorates clinical symptoms and is required for recovery from MHV-JHM.

<p>Top Panel: Morbidity of wt and RL^−/− mice infected with gliotropic MHV-JHM. Data are the average of two independent experiments. Middle Panel: Survival rates of wt and RL^−/− (n = 16/group) following i.c. infection with MHV-JHM. Data are representative of two independent experiments. Bottom Panel: Virus replication in the brains of infected mice is not significantly altered by RNase L deficiency (n = 6/group).</p

RNase L protects microglia/macrophages from infection in grey matter.

<p>Virus infected cells detected using virus nucleocapsid protein specific mAb J.3.3 (Antigen - green) and macrophage/microglia cell marker Iba-1 (Iba-1 – red) in spinal cords from infected wt and RL^−/− mice at day 7 p.i. Co-localization of viral antigen and Iba-1 staining cells (Merge - yellow) indicates infected microglia/macrophages are present only in white matter (WM) of wt, but both white matter and grey matter (GM) in RL^−/− spinal cords. Scale bar = 100 µm.</p

RNase L does not alter IFN responses to MHV-JHM infection of the CNS.

<p>Expression of IFN-α4, IFN-β1, OAS2, IFIT-1, IFIT-2 and IRF-7 mRNA relative to the housekeeping gene GAPDH in brains of MHV-JHM infected wt and RL^−/− mice. Data are the average of individual mouse brains from two independent experiments (n = 6).</p