Functional relevance of the multi-drug transporter abcg2 on teriflunomide therapy in an animal model of multiple sclerosis.

BACKGROUND: The multi-drug resistance transporter ABCG2, a member of the ATP-binding cassette (ABC) transporter family, mediates the efflux of different immunotherapeutics used in multiple sclerosis (MS), e.g., teriflunomide (teri), cladribine, and mitoxantrone, across cell membranes and organelles. Hence, the modulation of ABCG2 activity could have potential therapeutic implications in MS. In this study, we aimed at investigating the functional impact of abcg2 modulation on teri-induced effects in vitro and in vivo. METHODS: T cells from C57BL/6 J wild-type (wt) and abcg2-knockout (KO) mice were treated with teri at different concentrations with/without specific abcg2-inhibitors (Ko143; Fumitremorgin C) and analyzed for intracellular teri concentration (HPLC; LS-MS/MS), T cell apoptosis (annexin V/PI), and proliferation (CSFE). Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6J by active immunization with MOG35-55/CFA. Teri (10 mg/kg body weight) was given orally once daily after individual disease onset. abcg2-mRNA expression (spinal cord, splenic T cells) was analyzed using qRT-PCR. RESULTS: In vitro, intracellular teri concentration in T cells was 2.5-fold higher in abcg2-KO mice than in wt mice. Teri-induced inhibition of T cell proliferation was two fold increased in abcg2-KO cells compared to wt cells. T cell apoptosis demonstrated analogous results with 3.1-fold increased apoptosis after pharmacological abcg2-inhibition in wt cells. abcg2-mRNA was differentially regulated during different phases of EAE within the central nervous system and peripheral organs. In vivo, at a dosage not efficacious in wt animals, teri treatment ameliorated clinical EAE in abcg2-KO mice which was accompanied by higher spinal cord tissue concentrations of teri. CONCLUSION: Functional relevance of abcg2 modulation on teri effects in vitro and in vivo warrants further investigation as a potential determinant of interindividual treatment response in MS, with potential implications for other immunotherapies.

Vitamin D increases glucocorticoid efficacy via inhibition of mTORC1 in experimental models of multiple sclerosis.

The limited efficacy of glucocorticoids (GCs) during therapy of acute relapses in multiple sclerosis (MS) leads to long-term disability. We investigated the potential of vitamin D (VD) to enhance GC efficacy and the mechanisms underlying this VD/GC interaction. In vitro, GC receptor (GR) expression levels were quantified by ELISA and induction of T cell apoptosis served as a functional readout to assess synergistic 1,25(OH)2D3 (1,25D)/GC effects. Experimental autoimmune encephalomyelitis (MOG35-55 EAE) was induced in mice with T cell-specific GR or mTORc1 deficiency. 25(OH)D (25D) levels were determined in two independent cohorts of MS patients with stable disease or relapses either responsive or resistant to GC treatment (initial cohort: n = 110; validation cohort: n = 85). Gene expression of human CD8+ T cells was analyzed by microarray (n = 112) and correlated with 25D serum levels. In vitro, 1,25D upregulated GR protein levels, leading to increased GC-induced T cell apoptosis. 1,25D/GC combination therapy ameliorated clinical EAE course more efficiently than respective monotherapies, which was dependent on GR expression in T cells. In MS patients from two independent cohorts, 25D deficiency was associated with GC-resistant relapses. Mechanistic studies revealed that synergistic 1,25D/GC effects on apoptosis induction were mediated by the mTOR but not JNK pathway. In line, 1,25D inhibited mTORc1 activity in murine T cells, and low 25D levels in humans were associated with a reduced expression of mTORc1 inhibiting tuberous sclerosis complex 1 in CD8+ T cells. GR upregulation by 1,25D and 1,25D/GC synergism in vitro and therapeutic efficacy in vivo were abolished in animals with a T cell-specific mTORc1 deficiency. Specific inhibition of mTORc1 by everolimus increased the efficacy of GC in EAE. 1,25D augments GC-mediated effects in vitro and in vivo in a T cell-specific, GR-dependent manner via mTORc1 inhibition. These data may have implications for improvement of anti-inflammatory GC therapy.

1,25-OH2 vitamin D3 and AKT-inhibition increase glucocorticoid induced apoptosis in a model of T-cell acute lymphoblastic leukemia (ALL).

In acute lymphoblastic leukemia (ALL), steroid resistance and hypovitaminosis D are both associated with a poor prognosis. We show that methylprednisolone, calcitriol and the AKT-inhibitor MK-2206 have a synergistic effect on the apoptosis of steroid resistant T-ALL cells. Compared to methylprednisolone monotherapy, calcitriol increases methylprednisolone induced apoptosis dose-dependently (1.37-1.92-fold; p < 0.05). Pre-incubation with calcitriol increases the apoptotic effect of MK-2206 even further (3.6-fold; p < 0.05). It also potentiates synergism between MK-2206 and methylprednisolone (vehicle control 38% vs. calcitriol 58%, p < 0.01). The combination of calcitriol and AKT inhibition should be investigated further as treatment options for steroid resistance in T-ALL.

Fingolimod attenuates experimental autoimmune neuritis and contributes to Schwann cell-mediated axonal protection.

BACKGROUND: Fingolimod, a sphingosine-1-phosphate receptor modulator with well-described immunomodulatory properties involving peripheral immune cell trafficking, was the first oral agent approved for the treatment of relapsing remitting multiple sclerosis. Analogous immunomodulatory treatment options for chronic peripheral autoimmune neuropathies are lacking. METHODS: We tested fingolimod in the animal model of experimental autoimmune neuritis in Lewis rat. Six to eight-week-old female rats were immunized with P2 peptide and from this day on treated with fingolimod. Histology of the sciatic nerve was done to analyze T cell and macrophage cell count, intercellular adhesion molecule (ICAM) and amyloid precursor protein (APP) expression, as well as apoptotic Schwann cell counts. RESULTS: Preventive oral treatment with 0.1 mg/kg up to 3 mg/kg fingolimod once daily dissolved in rapeseed oil completely ameliorated clinical neuritis signs. It reduced circulating peripheral blood T cells and infiltrating T cells and macrophages in the sciatic nerve, whereas at the same time, it preserved blood-nerve barrier impermeability. Most importantly, fingolimod showed beneficial properties on the pathogenic process as indicated by fewer apoptotic Schwann cells and a lower amount of amyloid precursor protein indicative of axonal damage at the peak of disease course. CONCLUSIONS: Taken together, orally administered low-dose fingolimod showed an impressive immunomodulatory effect in the rat model of experimental autoimmune neuritis. Our current observations introduce fingolimod as an attractive treatment option for neuritis patients.

Dimethyl Fumarate Ameliorates Lewis Rat Experimental Autoimmune Neuritis and Mediates Axonal Protection.

BACKGROUND: Dimethyl fumarate is an immunomodulatory and neuroprotective drug, approved recently for the treatment of relapsing-remitting multiple sclerosis. In view of the limited therapeutic options for human acute and chronic polyneuritis, we used the animal model of experimental autoimmune neuritis in the Lewis rat to study the effects of dimethyl fumarate on autoimmune inflammation and neuroprotection in the peripheral nervous system. METHODS AND FINDINGS: Experimental autoimmune neuritis was induced by immunization with the neuritogenic peptide (amino acids 53-78) of P2 myelin protein. Preventive treatment with dimethyl fumarate given at 45 mg/kg twice daily by oral gavage significantly ameliorated clinical neuritis by reducing demyelination and axonal degeneration in the nerve conduction studies. Histology revealed a significantly lower degree of inflammatory infiltrates in the sciatic nerves. In addition, we detected a reduction of early signs of axonal degeneration through a reduction of amyloid precursor protein expressed in axons of the peripheral nerves. This reduction correlated with an increase of nuclear factor (erythroid derived 2)-related factor 2 positive axons, supporting the neuroprotective potential of dimethyl fumarate. Furthermore, nuclear factor (erythroid derived 2)-related factor 2 expression in Schwann cells was only rarely detected and there was no increase of Schwann cells death during EAN. CONCLUSIONS: We conclude that immunomodulatory and neuroprotective dimethyl fumarate may represent an innovative therapeutic option in human autoimmune neuropathies.

Laquinimod exerts strong clinical and immunomodulatory effects in Lewis rat experimental autoimmune neuritis.

Laquinimod is an immunomodulatory drug with neuroprotective potential. We used the animal model of experimental autoimmune neuritis (EAN) in the Lewis rat to study the effects of laquinimod treatment. After immunization with the neuritogenic peptide aa 53-78 of P2 myelin protein, preventive therapy with 12.5mg/kg laquinimod once daily inhibited neuritis in clinical and electrophysiological terms. Histology corroborated a lower degree of inflammatory lesions and demyelination in the sciatic nerve. The proportion of FoxP3-positive regulatory T cells in the peripheral lymph nodes of treated rats remained unchanged. We conclude that laquinimod may represent a therapeutic option in human autoimmune neuropathies.