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Clofazimine inhibits human Kv1.3 potassium channel by perturbing calcium oscillation in T lymphocytes.

Ren YR, Pan F, Parvez S, Fleig A, Chong CR, Xu J, Dang Y, Zhang J, Jiang H, Penner R, Liu JO - PLoS ONE (2008)

Bottom Line: A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases.These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells.Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
The Kv1.3 potassium channel plays an essential role in effector memory T cells and has been implicated in several important autoimmune diseases including multiple sclerosis, psoriasis and type 1 diabetes. A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases. We report herein the identification of clofazimine, a known anti-mycobacterial drug, as a novel inhibitor of human Kv1.3. Clofazimine was initially identified as an inhibitor of intracellular T cell receptor-mediated signaling leading to the transcriptional activation of human interleukin-2 gene in T cells from a screen of the Johns Hopkins Drug Library. A systematic mechanistic deconvolution revealed that clofazimine selectively blocked the Kv1.3 channel activity, perturbing the oscillation frequency of the calcium-release activated calcium channel, which in turn led to the inhibition of the calcineurin-NFAT signaling pathway. These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells. Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo. Together, these results suggest that clofazimine is a promising immunomodulatory drug candidate for treating a variety of autoimmune disorders.

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Clofazimine inhibits human T cell-mediated skin graft rejection in immunodeficient mice.(A) Representative skin grafts at different days post-transplantation. Human foreskin was transplanted onto Pfp/Rag2−/− mice. 1.0×108 human peripheral blood lymphocytes (PBL) were adoptively transferred into each animal at Day 7 post-transplantation. Administration of clofazimine or carrier control (olive oil) was also initiated at Day 7. * Days after skin transplantation/cell transfer. (B) Effect of clofazimine on the mean survival time of transplanted human (n = 5) and mouse (n = 4) skin grafts. The mouse skin transplantation was performed using Balb/c mice as skin donors, B6 Rag1−/− mice as recipients and PBL from B6 for adoptive transfer.
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pone-0004009-g006: Clofazimine inhibits human T cell-mediated skin graft rejection in immunodeficient mice.(A) Representative skin grafts at different days post-transplantation. Human foreskin was transplanted onto Pfp/Rag2−/− mice. 1.0×108 human peripheral blood lymphocytes (PBL) were adoptively transferred into each animal at Day 7 post-transplantation. Administration of clofazimine or carrier control (olive oil) was also initiated at Day 7. * Days after skin transplantation/cell transfer. (B) Effect of clofazimine on the mean survival time of transplanted human (n = 5) and mouse (n = 4) skin grafts. The mouse skin transplantation was performed using Balb/c mice as skin donors, B6 Rag1−/− mice as recipients and PBL from B6 for adoptive transfer.

Mentions: Kv1.3 has been implicated in T cell activation and has served as a molecular target for developing novel immunosuppressive agents [22], [23]. Given that clofazimine is already used in the clinic, albeit for a completely different indication, we wondered whether it is efficacious in animal models of organ transplantation. Initial experiments using mouse skin or heart transplant models revealed no beneficial effects of clofazimine in those models. These negative results are not surprising given that Kv1.3 plays distinct roles in humans and rodents, as it has been shown that Kv1.3 is dispensable in mice due to the up-regulation of other chloride channels [9]. Consistent with this notion, we also failed to observe a dose-dependent inhibition of IL-2 production in primary mouse T cells (Fig. S7A) and murine mixed lymphocyte reaction (Fig. S7B). Moreover, similar results were obtained for mixed lymphocyte reaction using cells derived from rats, making it difficult to evaluate the in vivo effects of clofazimine using well-established animal models. To overcome this problem, we turned to a model of reconstituted human T cell-mediated human skin rejection in immunodeficient mice [37]. We thus transplanted human foreskin into Pfb-Rag2−/− mice that lack T, B and NK cells. Upon healing of the skin graft for about 7 days, a total of 100 million human peripheral blood lymphocytes from an unrelated donor were adoptively transferred into the same animals. The animals were administered orally either olive oil (control) or clofazimine at 50 mg/kg/day for a total of 10 days (Fig. 6A). For the control group, the transplanted foreskin was rejected with a median survival time of 11 days (Fig. 6B). For the group treated with clofazimine, the skin survived even beyond the cessation of the drug treatment with a mean survival time of 35 days (Fig. 6B), which is comparable to the efficacy for FK506 treatment (data not shown). It is noteworthy that in a parallel experiment using murine skin and total murine T cells, clofazimine had no effect on the survival of murine skin transplant (Fig. 6B). Together, these results demonstrated that clofazimine is uniquely effective in inhibiting human T cell-mediated graft rejection with no significant effect on murine T cells.


Clofazimine inhibits human Kv1.3 potassium channel by perturbing calcium oscillation in T lymphocytes.

Ren YR, Pan F, Parvez S, Fleig A, Chong CR, Xu J, Dang Y, Zhang J, Jiang H, Penner R, Liu JO - PLoS ONE (2008)

Clofazimine inhibits human T cell-mediated skin graft rejection in immunodeficient mice.(A) Representative skin grafts at different days post-transplantation. Human foreskin was transplanted onto Pfp/Rag2−/− mice. 1.0×108 human peripheral blood lymphocytes (PBL) were adoptively transferred into each animal at Day 7 post-transplantation. Administration of clofazimine or carrier control (olive oil) was also initiated at Day 7. * Days after skin transplantation/cell transfer. (B) Effect of clofazimine on the mean survival time of transplanted human (n = 5) and mouse (n = 4) skin grafts. The mouse skin transplantation was performed using Balb/c mice as skin donors, B6 Rag1−/− mice as recipients and PBL from B6 for adoptive transfer.
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Related In: Results  -  Collection

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pone-0004009-g006: Clofazimine inhibits human T cell-mediated skin graft rejection in immunodeficient mice.(A) Representative skin grafts at different days post-transplantation. Human foreskin was transplanted onto Pfp/Rag2−/− mice. 1.0×108 human peripheral blood lymphocytes (PBL) were adoptively transferred into each animal at Day 7 post-transplantation. Administration of clofazimine or carrier control (olive oil) was also initiated at Day 7. * Days after skin transplantation/cell transfer. (B) Effect of clofazimine on the mean survival time of transplanted human (n = 5) and mouse (n = 4) skin grafts. The mouse skin transplantation was performed using Balb/c mice as skin donors, B6 Rag1−/− mice as recipients and PBL from B6 for adoptive transfer.
Mentions: Kv1.3 has been implicated in T cell activation and has served as a molecular target for developing novel immunosuppressive agents [22], [23]. Given that clofazimine is already used in the clinic, albeit for a completely different indication, we wondered whether it is efficacious in animal models of organ transplantation. Initial experiments using mouse skin or heart transplant models revealed no beneficial effects of clofazimine in those models. These negative results are not surprising given that Kv1.3 plays distinct roles in humans and rodents, as it has been shown that Kv1.3 is dispensable in mice due to the up-regulation of other chloride channels [9]. Consistent with this notion, we also failed to observe a dose-dependent inhibition of IL-2 production in primary mouse T cells (Fig. S7A) and murine mixed lymphocyte reaction (Fig. S7B). Moreover, similar results were obtained for mixed lymphocyte reaction using cells derived from rats, making it difficult to evaluate the in vivo effects of clofazimine using well-established animal models. To overcome this problem, we turned to a model of reconstituted human T cell-mediated human skin rejection in immunodeficient mice [37]. We thus transplanted human foreskin into Pfb-Rag2−/− mice that lack T, B and NK cells. Upon healing of the skin graft for about 7 days, a total of 100 million human peripheral blood lymphocytes from an unrelated donor were adoptively transferred into the same animals. The animals were administered orally either olive oil (control) or clofazimine at 50 mg/kg/day for a total of 10 days (Fig. 6A). For the control group, the transplanted foreskin was rejected with a median survival time of 11 days (Fig. 6B). For the group treated with clofazimine, the skin survived even beyond the cessation of the drug treatment with a mean survival time of 35 days (Fig. 6B), which is comparable to the efficacy for FK506 treatment (data not shown). It is noteworthy that in a parallel experiment using murine skin and total murine T cells, clofazimine had no effect on the survival of murine skin transplant (Fig. 6B). Together, these results demonstrated that clofazimine is uniquely effective in inhibiting human T cell-mediated graft rejection with no significant effect on murine T cells.

Bottom Line: A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases.These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells.Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
The Kv1.3 potassium channel plays an essential role in effector memory T cells and has been implicated in several important autoimmune diseases including multiple sclerosis, psoriasis and type 1 diabetes. A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases. We report herein the identification of clofazimine, a known anti-mycobacterial drug, as a novel inhibitor of human Kv1.3. Clofazimine was initially identified as an inhibitor of intracellular T cell receptor-mediated signaling leading to the transcriptional activation of human interleukin-2 gene in T cells from a screen of the Johns Hopkins Drug Library. A systematic mechanistic deconvolution revealed that clofazimine selectively blocked the Kv1.3 channel activity, perturbing the oscillation frequency of the calcium-release activated calcium channel, which in turn led to the inhibition of the calcineurin-NFAT signaling pathway. These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells. Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo. Together, these results suggest that clofazimine is a promising immunomodulatory drug candidate for treating a variety of autoimmune disorders.

Show MeSH
Related in: MedlinePlus