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, 214 (2), 168-80

Combined Medication of Lovastatin With Rolipram Suppresses Severity of Experimental Autoimmune Encephalomyelitis

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Combined Medication of Lovastatin With Rolipram Suppresses Severity of Experimental Autoimmune Encephalomyelitis

Ajaib S Paintlia et al. Exp Neurol.

Abstract

Combinations of new medications or existing therapies are gaining momentum over monotherapy to treat central nervous system (CNS) demyelinating diseases including multiple sclerosis (MS). Recent studies established that statins (HMG-CoA reductase inhibitors) are effective in experimental autoimmune encephalomyelitis (EAE), an MS model and are promising candidates for future MS medication. Another drug, rolipram (phosphodiesterase-4 inhibitor) ameliorates the clinical severity of EAE via induction of various anti-inflammatory and neuroprotective activities. In this study, we tested whether combining the suboptimal doses of these drugs can suppress the severity of EAE. Prophylactic studies revealed that combined treatment with suboptimal doses of statins perform better than their individually administered optimal doses in EAE as evidenced by delayed clinical scores, reduced disease severity, and rapid recovery. Importantly, combination therapy suppressed the progression of disease in an established EAE case via attenuation of inflammation, axonal loss and demyelination. Combination treatment attenuated inflammatory T(H)1 and T(H)17 immune responses and induced T(H)2-biased immunity in the peripheral and CNS as revealed by serological, quantitative, and immunosorbant assay-based analyses. Moreover, the expansion of T regulatory (CD25(+)/Foxp3(+)) cells and self-immune tolerance was apparent in the CNS. These effects of combined drugs were reduced or minimal with either drug alone in this setting. In conclusion, our findings demonstrate that the combination of these drugs suppresses EAE severity and provides neuroprotection thereby suggesting that this pharmacological approach could be a better future therapeutic strategy to treat MS patients.

Figures

Figure 1
Figure 1. Combination therapy with suboptimal doses of lovastatin and rolipram are complementary in EAE
EAE was induced in female Lewis rats using guinea pig MBP antigen emulsified in CFA and treatment with lovastatin (LOV) and rolipram (RLP) in combination or individually was performed as described under Materials and Methods. Drug treatments were started from the day of immunization of rats with MBP and continued until the end of the study. (A) Plot depicts clinical score associated with EAE in treated/untreated rats with different doses of LOV. (B) Plot depicts clinical score associated with EAE in treated/untreated rats with different doses of RLP. (C) Plot depicts clinical score associated with EAE in treated/untreated rats with suboptimal doses of LOV and RLP in combination and individually including PDEI-4 (another inhibitor of phosphodiesterase-4). Vehicle (VEH)- and LOV plus RLP-treated rats immunized with CFA emulsion without MBP were referred to as CON (VEH) and CON (LOV + RLP), respectively. Data in plots are presented as Mean ± SD of 9 rats treated similarly in a group. Statistical significance indicated as ** p<0.01 and *** p<0.001 versus EAE (VEH).
Figure 2
Figure 2. Combined treatment of lovastatin and rolipram attenuates cellular infiltration and demyelination in the SC of EAE rats
Treatment with lovastatin (LOV; 1 mg/kg) and rolipram (RLP; 1 mg/kg) in combination or individually was commenced in rats after the establishment of EAE (clinical score ≥2.0) as described under methods. Plot depicts clinical score in EAE animals treated with LOV and RLP in combination or individually (A). Plot depicts cellular infiltration and demyelination in the SC of EAE rats treated with LOV and RLP in combination or individually (B). Representative photograph of the lateral funiculus of SC stained with H&E (C) and both H&E and LFB (D) from each group of rats i.e., a) CON (VEH), b) CON (LOV + RLP), c) EAE (VEH), d) EAE (RLP), e) EAE (LOV) and f) EAE (LOV + RLP) demonstrates existing cellular infiltration and demyelination. Yellow arrowhead depicts cellular infiltration in the white mater and V indicates ‘vessel’ (C). Data in plots are expressed as Mean ± SD from three independent experiments (A) and histological images of SC (n = 9)/group (B). Statistical significance * p<0.05, ** p<0.01 and *** p<0.001 versus EAE (VEH).
Figure 3
Figure 3. Attenuation of axonal loss and demyelination in the SC of EAE rats by combination therapy of lovastatin and rolipram
Treatment with lovastatin (LOV; 1 mg/kg)) and rolipram (RLP; 1 mg/kg) in combination or individually was commenced in rats after the establishment of EAE (clinical score ≥2.0) as described under Methods. Autoradiograph of immunoblotting depicts level of MBP isoforms in the SC of treated/untreated control and EAE animals (A). Plot depicts histological score of axonal loss in the white matter of lateral funiculi of SC of EAE rats treated with drugs in combination or individually or with vehicle (VEH) only (B). Representative photograph of the lateral funiculus of SC demonstrate impregnation of axons by Beilschowsky’s silver staining (C). Labeling of micrographs indicates a) CON (VEH), b) CON (LOV + RLP), c) EAE (VEH), d) EAE (RLP), e) EAE (LOV) and f) EAE (LOV + RLP) in both B and D. Data in plot is presented as Mean ± SD from three independent experiments with 9 rats/group and histological images of SC (n = 9)/group. Statistical significance * p<0.05, ** p<0.01 and *** p<0.001 versus EAE (VEH).
Figure 4
Figure 4. Expression of transcripts for inflammatory infiltrates, adhesion molecules and chemokines including receptors in the SC of EAE rats treated with lovastatin and rolipram in combination or individually
Plots depict the level of transcripts A; CD4 and CD8 T cells, B; CD11b monocytes, C; ICAM-1 and VCAM-1, and D; MCP-1 and CCR-2 in the SC of EAE (n = 9) and control (n = 6) rats treated with lovastatin (LOV) and rolipram (RLP) in combination or individually as determined by QRT-PCR as described under methods. Data in plots are expressed as Mean ± SD from three independent experiments. Statistical significance * p<0.05, ** p<0.01, *** p<0.001 and ‘NS’ (not-significant) versus EAE (VEH).
Figure 5
Figure 5. Level of transcripts for pro-inflammatory and anti-inflammatory mediators in the SC and anti-MBP immunoglobulin isotypes in the serum of EAE rats treated with lovastatin and rolipram in combination or individually
Plots depict the level of transcripts A; IFN-γ, TNF-α, IL-1β and iNOS, B; IL-23, IL-17, RORγt and CD25, C; IL-4, IL-10 and TGF-β1, and D; Foxp3 and IDO in the SC of EAE (n = 9) and control (n = 6) rats treated with lovastatin (LOV) and rolipram (RLP) in combination or individually as determined by QRT-PCR as described under methods. Plot depicts the level of ant-MBP immunoglobulin isotypes in the serum of EAE (n = 9) and control (n = 6) rats treated with LOV and RLP in combination or individually as determined by ELISA-based assay described under methods (E). Data in plots are expressed as Mean ± SD from three independent experiments. Statistical significance * p<0.05, ** p<0.01, *** p<0.001 and ‘NS’ (not-significant) versus EAE (VEH).
Figure 6
Figure 6. Protein level of pro-inflammatory and anti-inflammatory mediators in the SC of EAE rats following treatment with lovastatin and rolipram in combination or individually
Plots depict the level of IFN-γ (A), IL-17 (B), IL-4 (E) and IL-10 (F) in the SC homogenate of EAE (n = 9) and control (n = 6) rats treated with lovastatin (LOV) and rolipram (RLP) in combination or individually as determined by ELISA-based assay described under methods. Autoradiograph (C) and plot (D) depict immunoblotting of iNOS protein in the SC of EAE rats in each group. Data in plots are expressed as Mean ± SD from three independent experiments. Statistical significance * p<0.05, ** p<0.01 and ‘NS’ (not-significant) versus EAE (VEH).
Figure 7
Figure 7. Level of proteins associated with Treg cells and immunotolerance in the SC of EAE rats
Immunoblot depicts CD25, IDO, and β-actin in the SC of EAE animals treated/untreated with LOV and RLP in combination or individually (A). Plot depicts TGF-β1 level in the SC homogenate of EAE (n = 9) and control (n = 6) animals treated with lovastatin (LOV) and rolipram (RLP) in combination or individually as determined by ELISA-based assay described under methods (B). Representative photograph of the lateral funiculus of SC of EAE animals treated with LOV and RLP i.e., a) CON (VEH), b) CON (LOV + RLP), c) EAE (VEH), d) EAE (RLP), e) EAE (LOV) and f) EAE (LOV + RLP) demonstrating the expression of IDO in the SC (C). Data in plot is expressed as Mean ± SD from three independent experiments. Statistical significance * p<0.05, ** p<0.01 and ‘NS’ (not-significant) versus EAE (VEH).

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