Overexpression of glia maturation factor reinstates susceptibility to myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in glia maturation factor deficient mice

Abstract

Glia maturation factor (GMF), a primarily CNS localized protein was discovered and characterized in our laboratory. We previously demonstrated that GMF is the upstream regulator for excessive production and release of proinflammatory cytokines/chemokines in brain cells leading to the destruction of oligodendrocytes, the myelin forming cells, and neurons. We also reported that mice lacking endogenous GMF (GMF-deficient, GMF-KO) were resistant to myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)) induced EAE, since immunization induced only delayed EAE with diminished severity. In the present study we show that a replication-defective adenovirus-GMF construct caused expression of GMF in CNS of GMF-KO mice and reinstated MOG(35-55) induced early and severe EAE. Our results show that MOG(35-55) immunization caused only a muted EAE and inflammation/demyelination in mice lacking endogenous GMF. The diminished incidence of EAE in GMF-KO mice was consistent with the significantly reduced expressions of cytokines/chemokines. The muted severity of EAE in GMF-KO mice was restored to full blown levels upon reintroduction of GMF using an adeno-GMF-virus (Adv-GMF) vector. Consistent with the clinical findings, histological examination of the CNS of mice with EAE revealed profound differences between wild type (Wt), GMF-KO, and GMF-KO mice with re-introduced GMF (GMF-KO+Adv-GMF). Spinal cord sections from mice with EAE were analyzed for the infiltration of mononuclear cells (inflammation) and myelin loss (demyelination). In Wt mice, 40% of spinal cord quadrants were positive for demyelination and 45% of spinal cord quadrants were positive for inflammation at the peak of EAE. Drastically reduced infiltrates (15%) and demyelination (10%) were found in GMF-KO mice that developed reduced severity of EAE. Upon GMF reintroduction in GMF-KO mice, MOG(35-55) immunization caused extensive monocytes infiltration (48%) and demyelination (46%), similar to that observed in the immunized Wt mice. The levels of cytokine/chemokine in the spinal cords of mice at three time points, corresponding to the onset, peak severity and recovery period of EAE, show a distinct pattern of very large increases in IFN-γ, TNF-α, GM-CSF and MCP-1 in Wt and GMF-KO+Adv-GMF mice compared to GMF-KO and GMF-KO+Adv-LacZ mice.

Figure 1. Expression of GMF in brain and spinal cord of EAE mice

Mice were injected with a replication-defective adenovirus vector containing a full length GMF cDNA (Adv-GMF) or cytoplasmic lacZ cDNA (Adv-LacZ) and two mice in each group were analyzed by Western blot analysis at the peak of EAE. Brain or spinal cord tissue homogenates (20 μg protein per lane) were subjected to SDS-polyacrylamide gel electrophoresis followed by electroblotting. The blots were probed with a monoclonal anti-GMF (G2-09) antibody and actin antibody. β-Actin served as an internal marker showing equal sample loading. The data shown are representative of at least three experiments. Br, brain; SpC, spinal cord

Figure 2. Overexpression of GMF exacerbates EAE induced by MOG_35–55 peptide

Mice were immunized with MOG_35–55 peptide and monitored for the development of disease. Adv-GMF or Adv-LacZ was injected intravenously every day, starting at day 2 to day 8 after immunization with MOG_35–55 peptide. Mean clinical score of MOG-induced EAE in Wt and GMF-KO (KO) along with Adv-GMF or Adv-LacZ injected mice (n = 10 for each group). Note this is a representative of three independent experiments.

Figure 3. Overexpression of GMF significantly increases the inflammation in MOG_35–55 induced EAE

Animals were sacrificed at day 12 -post immunization with MOG_35-55 and spinal cord inflammation was assessed by hematoxylin and eosin (H&E) staining. Representative microscopic photographs of the spinal cord showing significantly increased infiltration of inflammatory mononuclear cells in Wt and GMF-KO mice injected with Adv-GMF as compared to control-Adv-LacZ-injected mice at the peak of EAE. (Original magnification: X10; n =3 for each group)

Figure 4. Histopathological scores for inflammation and demyelination in MOG_35-55 induced EAE

Mice were injected with Adv-GMF or Adv-LacZ as described in Figure 2. The spinal cord sections are assessed for inflammation and demyelination in EAE mice. At the peak of the disease, five-micrometer thick transverse sections (five sections) from cervical, upper thoracic, lower thoracic and lumbar regions of spinal cord were stained. Each section was further subdivided into anterior, posterior and two lateral columns (4 quadrants). Each quadrant showing the infiltration of mononuclear cells was assigned a score of one inflammation and the quadrant that showed perivascular lesion and loss of myelin staining a score of one demyelination. Thus, each animal had a potential maximum score of 80. The average number of quadrants examined per mouse was 16. The pathologic score (inflammation or demyelination) for each group was expressed as the percentage positive over the total number of quadrants examined. Differences between Wt, GMF-KO, and GMF-KO+ Adv-GMF mice were highly significant (P < 0.001) at the peak of EAE.

Figure 5. Expression of pro inflammatory cytokines/chemokines in the spinal cord of MOG_35-55 induced EAE mice

The lumbar and lower thoracic spinal cord tissue (100 mg wet weight) was homogenized and TNF-α, IFN-γ, GM-CSF and MCP-1 levels in spinal cord homogenates were determined by ELISA in EAE mice at the onset, at the peak and recovery period of the disease. The results are obtained from three pools of material, each consisting of three mice.