Oligodendrocytes that survive acute coronavirus infection induce prolonged inflammatory responses in the CNS

Abstract

Neurotropic strains of mouse hepatitis virus (MHV), a coronavirus, cause acute and chronic demyelinating encephalomyelitis with similarities to the human disease multiple sclerosis. Here, using a lineage-tracking system, we show that some cells, primarily oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs), survive the acute MHV infection, are associated with regions of demyelination, and persist in the central nervous system (CNS) for at least 150 d. These surviving OLs express major histocompatibility complex (MHC) class I and other genes associated with an inflammatory response. Notably, the extent of inflammatory cell infiltration was variable, dependent on anatomic location within the CNS, and without obvious correlation with numbers of surviving cells. We detected more demyelination in regions with larger numbers of T cells and microglia/macrophages compared to those with fewer infiltrating cells. Conversely, in regions with less inflammation, these previously infected OLs more rapidly extended processes, consistent with normal myelinating function. Together, these results show that OLs are inducers as well as targets of the host immune response and demonstrate how a CNS infection, even after resolution, can induce prolonged inflammatory changes with CNS region-dependent impairment in remyelination.

Keywords: coronavirus; neuroinflammation; oligodendrocyte; virus-induced demyelination.

Fig. 1.

Development and characterization of Cre- and Venus-expressing MHV. (A) Genome of recombinant MHV expressing either enterobacteria phage P1 Cre recombinase (rMHV_Cre, shown in yellow) or Venus (rMHV_Venus, shown in green). (B) Schematic shows that expression of Cre resulted in excision of loxP-flanked stop sites leading to tdTomato protein expression in rMHV_Cre-infected cells. (C) Cre expression was confirmed by Western blot analysis using antibodies specific for Cre. (D) Venus expression was confirmed by fluorescent microscopy using 17Cl-1 cells infected with rMHV_Venus or mock infected. (E). Weights (Left) and clinical scores (Middle) of infected mice were monitored. rMHV, rMHV_Cre, or rMHV_Venus,-infected Ai14 mice develop indistinguishable clinical disease. (Right) Virus titers at day 5, the peak of infection. n.s., not significant. (F) Representative images of frozen midsagittal brain sections are shown at 30 d after intracranial injection of Rosa-tdTomato mice with 750 plaque-forming units (PFUs) of rMHV_Cre. tdTomato+ cells are red and DAPI-stained nuclei are blue. See also SI Appendix, Fig. S2. Data are representative of three independent experiments.

Fig. 2.

Time course of tdTomato and Venus expression in infected mice. Mice were infected intracranially with the indicated virus (750 PFUs/mouse). Infected mice were killed at the indicated times, and combined brains were harvested and analyzed by flow cytometry. (A) tdTomato⁺ and Venus⁺ populations in the brain are shown. Venus⁺ cells (blue, n = 5 mice/time point) were cleared more quickly than tdTomato⁺ cells (red, n = 5 mice/time point). (B) Venus⁺ and tdTomato⁺ populations were analyzed for CD11b and CD45 expression. Few Venus⁺ cells were detected by 20 dpi, and all were CD11b⁻/CD45⁻. Frequency of tdTomato⁺ cells did not vary significantly up to 40 dpi although the fraction of CD11b⁻/CD45⁻ increased with time. (C) CD11b⁻/CD45⁻ tdTomato⁺ cells in rMHV_Cre-infected mice were analyzed for O4 expression at the indicated time points (n = 8 mice/time point). *P ≤ 0.05, **P ≤ 0.01; two-tailed, unpaired Student’s t tests were used in all panels.

Fig. 3.

Regional differences in types of surviving cells in the brain. Confocal micrographs showing colocalization of tdTomato with Iba-1 (A and E), Olig2 (B), GFAP (C), and CSPG4 (D and F) in the olfactory bulb (OB, A), splenium (SPL, B–E), and SVZ (F) at 30 dpi. Boxed areas in A, B, and C are shown in a₁, b₁, c₁, and c₂, respectively. c₂ shows GFAP⁻tdTomato⁺ cells in the splenium, which are likely OLs (b₁). Images with colocalized cells are highlighted in white. Colocalization data were generated using ImageJ (A, F, and a₁–c₂). OLs and astrocytes but not OPCs colocalized with tdTomato in the splenium, while CSPG4⁺ cells (OPCs) in the SVZ were tdTomato⁺. Microglia/macrophages were the predominant tdTomato⁺ cells in the OB. (G) Summary of the day 30 p.i. data (n = 9 mice) from three individual experiments (Top) and list of antibodies used for detection of each cell type (Bottom). (H and h₁) Olig2⁺CSPG4⁺ OPCs were detected in the SVZ at day 30 p.i.; Olig2⁺CSPG4⁺tdTomato⁺ cells are pink and white. (I, i₁) CSPG4⁺ cells (OPCs) were detected in the SVZ at day 60 p.i. Boxed areas in H and I are shown in h₁ and i₁, respectively. Ventricles (V) are indicated in F, H, I, and i₁. (Scale bars: A–F, 100 μm; a₁–c₂, 10 μm; H and I, 200 μm; h₁ and i₁, 50 μm.)

Fig. 4.

Oligodendrocytes are the predominant surviving cells in the spinal cord. (A) Percentage of O4⁺ oligodendrocytes in the brain and spinal cord at day 30 p.i. (n = 5 mice). TdTomato-positive cells were identified as shown in Fig. 2A. **P ≤ 0.01. (B–D) Sections of spinal cord were stained with oligodendrocyte lineage-specific antibodies (Olig2: OLs and OPCs; CSPG4: OPCs only). Most tdTomato⁺ cells were OLs (B). (C) OPCs accumulated adjacent to demyelinating lesions in the spinal cord. tdTomato⁺ OPCs were found in the WML. Boxed area in C is shown in D. n = 5 mice. Representative images are shown. (E) Surviving cells localized in the gray matter that had a neuron-like morphology. (B–E) Nuclei are stained with DAPI (blue). (Scale bars: 50 μm.)

Fig. 5.

All regions of demyelination in the spinal cord contain surviving OLs and show site-specific immune responses at day 30 p.i. (A) Sagittal sections of cervical and upper thoracic spinal cords from mice infected with rMHV_Cre (750 PFUs) were prepared and stained with myelin gold (Left) or with anti–Iba-1 antibody (Right). Demyelinating lesions are indicated (yellow arrow). Extensive infiltration of myeloid cells is shown. (Scale bar: 1,000 μm.) (B) Sections of rostral pons, WMLs, and NAWM were immunolabeled for CD8 (blue) and Iba-1 (green) expression. More CD8 T cells and activated myeloid cells were detected in the WML than in the rostral pons even though numbers of surviving cells were similar. (C) Microglia/macrophages in B (Iba-1–stained) were converted to binary and skeleton images (The boxed areas in Fig. 5B are shown in SI Appendix, Fig. S5B, as an example of how we quantified branch length and endpoints.) Summary of microglia/macrophage endpoints/cell (Left) and process length/cell data (Right) in different regions that were measured as described in Materials and Methods. A cutoff value of three endpoint branches was used to minimize background effects. Microglia/macrophages in the spinal cord (SC) WML had increased numbers (C, Left) and increased length (C, Right) of branches, suggestive of increased activation. ***P < 0.001, ****P < 0.0001; one-way ANOVA; n = 3 mice and three sections/mouse.

Fig. 6.

Surviving OLs exhibit site-specific morphology and variable degrees of inflammation at 60 dpi. Sections were prepared from the rostral pons (A) and spinal cord (B, D, and F) and immunolabeled with Iba-1. Surviving cells in the rostral pons showed evidence of process extension consistent with remyelinating function and were associated with microglia/macrophages that had nearly normal morphology (A). tdTomato⁺ cells in the spinal cord were associated with activated myeloid cells and CD8 T cells (blue) (B, D, and E) and either remained rounded (E) or showed minimal process extension (D) compared to cells in the rostral pons. (C) Microglia/macrophages in A and B (Iba-1–stained) were converted to binary and skeleton images. Summary of microglia/macrophages process length/cell (Top) and endpoints/cell (Bottom) in different regions that were measured as described in Materials and Methods, n.s., not significant. (F) A tdTomato⁺ cell undergoing engulfment by a myeloid cell (from boxed area of E) is shown. Iso-surface rendition (f2, f4) obtained from the stacks (f1, f3) using Imaris is shown. (Scale bar: A and B, 50 μm; D and E, 20 μm; F, 16 μm.)

Fig. 7.

Long-term MHC class I up-regulation on tdTomato⁺ OLs. (A and B) OLs from uninfected and infected mice at day 30 p.i. were stained for MHC-I expression and analyzed by flow cytometry (mock-blue, tdTomato⁺-red, tdTomato⁻-gray) . Both tdTomato⁺ and tdTomato⁻ OLs up-regulated MHC-I, but up-regulation was more prolonged on tdTomato+ cells. Dashed line indicates MHC-I in mock-infected mice (normalized). Geometric mean fluorescence intensity (gMFI) data are summarized in B. Data are means ± SD of five mice/time point. *P < 0.05, **P < 0.01; Student’s t test for indicated pairwise comparison, coupled with one-way ANOVA for multigroup comparisons.

Fig. 8.

Next generation RNA-Seq analyses of gene expression profile in rMHV_Cre-infected and noninfected OLs at 30 d p.i. Gene expression analyses were performed using four independent biological replicates (tdTomato⁺ OLs [P], tdTomato⁻ OLs [N] from infected mice and OLs from mock-infected mice [M]). (A and B) Venn diagram (A) and heat map (B) summarizing the number and overlap of genes regulated in P versus M (blue) or N versus M (red). (C and D) Heat map (C) and circle plots (D) comparing targeted pathways, including antigen presentation, identified by pathway analysis. There is increased expression of antigen presentation and response to IFN-γ pathways in P compared to N. In D, the outer circle scatter plots represent differential gene expression within each term, with red points representing up-regulated genes and blue points representing down-regulated genes. The inner circle is colored based on Z score, which is a measure for predicting a bias in gene regulation, with blue predicting a decrease in the pathway and red predicting an increase. Additionally, the inner circle is sized based on P value, with the larger size correlating to a more significant P value. (E) MHC I antigen expression and related genes as depicted in the KEGG: Kyota Encyclopedia of Genes and Genomes database are shown. Red boxes denote up-regulated genes, when groups N and P were compared to M.