Treatment of autoimmune encephalomyelitis with a histone deacetylase inhibitor

Abstract

We have previously shown that treatment of female NOD mice with a potent nonselective histone deacetylase inhibitor attenuated experimental autoimmune encephalomyelitis, a model for progressive multiple sclerosis. Herein we show that immunization with the MOG_35-55 peptide induced prolonged upregulation of genes encoding interleukin 17A (IL-17A), aryl hydrocarbon receptor, and histone deacetylase 11 in the spinal cord whereas the subunits of IL-27, IL-27p28 and IL-27ebi3 were significantly increased in secondary lymphoid organs after a lag period. Interestingly, the nitric oxide synthase gene was prominently expressed in both of these anatomic compartments following immunization. Treatment with the histone modifier repressed the transcription of all of these genes induced by immunization. Moreover, the drug suppressed the steady-state levels of the migration inhibitory factor and CD274 genes in both the spinal cord and peripheral lymphoid tissues. At the same time, the CD39 gene was downregulated only in secondary lymphoid organs. Paradoxically, the epigenetic drug enhanced the expression of Declin-1 in the spinal cord, suggesting a protective role in neuronal disease. Immunization profoundly enhanced transcription of the chemokine CCL2 in the secondary lymphoid tissues without a corresponding increase in the translation of CCL2 protein. Histone hyperacetylation neither altered the transcription of CCL2 nor its cognate receptor CCR2 in the central nervous system and peripheral lymphoid tissues. Surprisingly, the drug did not exert modulatory influence on most other immune response-related genes previously implicated in encephalomyelitis. Nevertheless, our data uncover several potential molecular targets for the intervention of experimental autoimmune encephalomyelitis that have implications for the treatment of progressive multiple sclerosis.

Keywords: Autoreactive T cells; Epigenetic regulation; Experimental autoimmune encephalomyelitis; Gene expression; Multiple sclerosis; Trichostatin A.

Figure 1. TSA treatment improved clinical disease.

Randomly chosen littermates of female NOD mice were immunized with MOG_35-55 and treated with TSA or DMSO three times a week for the indicated time interval. Clinical scores of five mice/group were determined on indicated time intervals and depicted as mean ± SEM. The statistical significance between DMSO (blank bars) and TSA-treated mice (hatched bars) was determined using the area under the ROC curve test and indicated. Individual data for DMSO (empty circles) and TSA-treated (empty squares) groups are also indicated. Mice were killed on day 14 (A), 21 (B), 28 (C), 54 (D), and 115 (E), and their spinal cord and peripheral lymphoid tissues were harvested and used for gene expression analysis.

Figure 2. Differential impact of the histone modifier on gene expression in the CNS.

Total RNA was extracted individually from five mice per group from the spinal cord (SC) at various time points. At each time point, RNA pooled from five mice/group was analyzed in triplicate. The expression of indicated genes was normalized to the housekeeping gene, Gapdh, and depicted as mean ± SEM of triplicate samples. Statistical significance (P<0.05) between DMSO (blank bars) and drug-treated mice (hatched bars) was determined using an unpaired two-tailed t-test and indicated by asterisks. Individual replicates for DMSO (empty circles) and TSA-treated samples (empty squares) are also shown. Unimmunized mice (10/group) were treated with DMSO or TSA and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization.

Figure 3. Different patterns of gene regulation in the SLO by TSA treatment.

Expression levels of indicated genes were determined in control DMSO (blank bars) and TSA-treated mice (hatched bars) using the total RNA derived from the spleen and draining lymph nodes (SP) from individual mice at the indicated time points. At each time point, RNA pooled from five mice/group was analyzed in triplicate. Individual data points for DMSO (empty circles) and TSA-treated (empty squares) are shown. Gene expression was normalized to the housekeeping gene, Gapdh, and depicted as mean ± SEM of triplicate samples. Statistical significance (P<0.05) between control and drug-treated mice was determined using an unpaired two-tailed t-test and indicated by asterisks. Unimmunized mice (10/group) were treated with DMSO or TSA and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization.

Figure 4. Epigenetic regulation failed to influence the prominent chemokine system.

Expression levels of indicated genes were determined in DMSO (blank bars) and TSA-treated mice (hatched bars) using the total RNA derived from the spinal cord (SC) (A, C) and peripheral lymphoid tissues (SP) (B, D) at various time points. At each time point, RNA was extracted from five individual mice/group, pooled, and analyzed in triplicate. Gene expression was normalized to the housekeeping gene, Gapdh, and depicted as mean ± SEM of triplicate samples. Individual data points are shown for DMSO (blank circles) and TSA-treated (empty squares) groups. CCL2 protein was estimated in duplicate from pooled sera of five mice per group using ELISA (E). Unimmunized mice (10/group) were treated with DMSO or TSA and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. Statistical significance (P<0.05) between control and drug-treated mice (n=five/group) was determined using an unpaired two-tailed t-test and indicated by asterisks.

Figure 5. TSA treatment repressed the transcription of Hdac11 selectively in the CNS.

Expression levels of indicated Hdac genes were determined in DMSO (blank bars) and TSA-treated mice (hatched bars) in the spinal cord as shown. At each time point, RNA was isolated from five individual mice/group, pooled and analyzed in triplicate. Gene expression was normalized to the housekeeping gene Gapdh and depicted as mean ± SEM of triplicate samples. Individual data points are shown for DMSO (blank circles) and TSA-treated (empty squares) groups. Statistical significance (P<0.05) between control and the drug-treated group was determined using an unpaired two-tailed t-test and indicated by asterisks. ns, not significant. Unimmunized mice (10/group) were treated with DMSO or TSA and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization.