API

Relationship: 1509

Title

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Reduction, NFAT/AP-1 complex formation leads to Suppression, IL-2 and IL-4 production

Upstream event

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Reduction, NFAT/AP-1 complex formation

Downstream event

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Suppression, IL-2 and IL-4 production

Key Event Relationship Overview

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AOPs Referencing Relationship

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AOP Name Adjacency Weight of Evidence Quantitative Understanding
Inhibition of Calcineurin Activity Leading to Impaired T-Cell Dependent Antibody Response adjacent High High

Taxonomic Applicability

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Term Scientific Term Evidence Link
Homo sapiens Homo sapiens High NCBI

Sex Applicability

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Sex Evidence
Mixed High

Life Stage Applicability

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Term Evidence
All life stages High

Key Event Relationship Description

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Localized NFAT in the nucleus of T cells binds to form complexes with AP-1 at the IL-2 promoter region (Schreiber and Crabtree 1992; Jain et al. 1992), which induces transcription of IL-2 (Jain et al. 1993). In addition to IL-2, NFAT localized in the nucleus of T cells also binds to the promoter region of the other classes of cytokines including IL-4 and IL-13.

For IL-2, NFAT proteins are necessary for IL-2 gene expression and cooperation of NFAT with AP-1 is required for IL-2 gene transcription. For IL-4, At least five different NFAT sites have been described in the IL-4 promoter with at least three of them being composite sites binding NFAT and AP-1 (Macián et al. 2001).

Decreased formation of NFAT/AP-1 complex at the promoter region of IL-2 genes in the nucleus of T cells following lowed nuclear localization of NFAT by CNI treatment reduces the transcription of IL-2 (Dumont et al. 1998). Production in T cells of IL-4 and other classes of cytokines is also suppressed in the same manner as IL-2 (Dumont et al. 1998).

Evidence Supporting this KER

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Biological Plausibility

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T-5224, a selective c-Fos/AP-1 inhibitor, inhibits the DNA-binding activity of AP-1 in primary murine T cells. T-5224 also inhibits CD25 (one of IL-2 receptors) up-regulation, IL-2 production, and c-Fos DNA-binding activity in mice (Yoshida et al. 2015).

Dexamethasone represses the IL-2 mRNA induction. GILZ (glucocorticoid-induced leucine zipper) is one of the most prominent glucocorticoid-induced genes, and inhibited the induction of the NFAT reporter and interferes with the AP-1 component of the NFAT/AP-1 complex. GILZ also inhibits the IL-2 promoter (Mittelstadt et al. 2001).

Ursolic acid suppressed activation of three immunoregulatory transcription factors NF-kB, NFAT and AP-1. Treatment of lymphocytes and CD4+ T cells with ursolic acid inhibited secretion of IL-2 and IL-4 cytokines. Treatment of CD4+ T cells with ursolic acid suppressed mRNA level of IL-2. Treatment of lymphocytes with ursolic acid inhibited the upregulation of CD25 expression on T cells (Checker et al. 2012).

NFATp- and NFAT4-deficient mice indicate decreased production of Th1 cytokine including IL-2 (Ranger et al. 1998).

It is generally accepted that NFAT, translocated to the nucleus after T-cell stimulation, binds with AP-1 to the promoter regions of the cytokine genes to mount transcription, which follows production of these T-cell–derived cytokines. Of these cytokines, IL-2 and IL-4 promote proliferation, maturation, and class-switching of B cells to enhance TDAR.

There is also sufficient evidence to support the hypothesis that CNI-induced decreases in T-cell–derived cytokine production is mediated through suppressed nuclear localization of NFAT, with a resultant decrease in the amount of NFAT/AP-1 complex binding to the promoter regions of T‑cell–derived cytokines.

When stimulated with ovalbumin, CnA-KO mice produce less IFN-g, IL-2, and IL-4 than wild-type mice. However, primary antibody response in CnA-KO mice is normal in response to TNP‑ovalbumin (Zhang et al. 1996).

The following phenotypes are observed in NFAT knockout mice: moderate hyperproliferation with splenomegaly; moderately enhanced B- and T-cell responses, with bias towards Th2- cell responses; decreased IFN-γ production in response to TCR ligation; reduced proliferative responses by T cells; impaired repopulation of the thymus and lymphoid organs; impaired Th2-cell responses and IL-4 production; grossly impaired T-cell effector functions, with profound defects in cytokine production and cytolytic activity; B-cell hyperactivity; impaired development of CD4 and CD8 single-positive cells, with increased apoptosis of double-positive thymocytes; and mild hyperactivation of peripheral T cells (Macian, 2005).

Therefore, the study of NFAT-KO mice shows that NFAT is involved in a wide range of immune responses, and some of these phenomenon are known to be regulated by CN. Suppression of T-cell-derived cytokines is noted both in CnA-knockout and NFAT-knockout mice, which indicates that the production of T-cell derived cytokines such as IL-2 and IL-4 is regulated by the CN-NFAT system.

FK506-FKBP12 complex decreased CN phosphatase activity, which leads to inhibit translocation of NFAT to the nucleus. Because NF-ATp is an essential transcription factor regulating the IL-2 gene, FK506 ultimately blocks the T-cell response by inhibiting IL-2 transcription (Panhans-Gross A et al. 2001). FK506 inhibited IL-2 mRNA expression in anti-CD3/PMA-activated cells (Dumont et al. 1998).

These facts indicate that although NFAT is widely involved in the function of T cells, the effect of CNIs is to suppress production of some classes of T‑cell–derived cytokines through reducing the formation of NFAT/AP-1 complexes induced by inhibition of CN phosphatase activity.

Empirical Evidence

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Empirical support of Reduction, NFAT/AP-1 complex formation leading to Suppression, IL-2 and IL-4 production is strong.

Rationale:

  • In purified T cell from male C57BL/6J mice, T-5224 (a selective c-Fos/AP-1 inhibitor) inhibits the DNA-binding activity of AP-1 and CD25 (one of IL-2 receptors) up-regulation at 80 μg/mL, and IL-2 production in a dose-dependent manner from 40 to 80 μg/mL (Yoshida et al. 2015).
  • In splenic lymphocytes stimulated with concanavalin A for 24 h in C57BL/6 mice, ursolic acid suppressed products of NF-kB, NFAT and AP-1 at 5 μM for 4 h. Secretion of IL-2 and IL-4 was inhibited in lymphocytes stimulated with concanavalin A for 24 h at concentrations of 0.5, 1 and 5 μM of ursolic acid, and lymphocytes and CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h at concentration of 5 μM of ursolic acid. In CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h, ursolic acid suppressed mRNA level of IL-2 at 5 μM for 4 h. In lymphocytes stimulated with concanavalin A for 24 h, ursolic acid inhibited CD25 expression at 5 μM for 4 h (Checker et al. 2012).
  • In NFATp- and NFAT4-deficient mice, cultured splenocytes bound anti-CD3 for 48 h indicates decreased production of Th1 cytokine including IL-2 (Ranger et al. 1998).
  • It is well established that inhibition of NFAT/AP-1 complex formation at the promoter sites reduces the production of T-cell–derived cytokines including IL-2 and IL-4, which are mainly involved in T-cell–dependent antibody response.
  • NFAT/AP-1 complex formation is inhibited by CNI shown by gel shit mobility assay using human T cell line or CD4+ T cells from heathy donners after 2 hours treatment with CsA at 1µM.. Preceding NFAT nuclear localization after T cell activation is suppressed with FK506 at the dose range of 0.01nM (Jarkat T cells) or 10nM (CD4+ T cells) to 1µM (Maguire et al. 2013), and NFAT nuclear localization and NFAT/AP-1 complex formation is shown to be strongly related (Jain et al. 1992, Jain et al. 1993).
  • In CD3/PMA-activated human T cells, FK506 suppressed production of IL-2, IL-4, and IFN-γ at the concentrations of 1.2 to 12.5 nM after 22 to 24 hours culture as well as inhibited expression of IL-2, IL-4, and IFN-γ mRNA in a dose-dependent (10 nM) manner after 3 day culture (Dumont et al. 1998).

Reduced nuclear translocation of NFAT followed by NFAT/AP-1 complex formation and suppression of IL-2/IL-4 productions are shown to occur under similar dose ranges and treatment duration.

Uncertainties and Inconsistencies

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FK506 suppresses expression of IL-2R (CD25) and costimulatory molecules CD80 (B7.1)/CD40 in Langerhans cells (Panhans-Gross A et al. 2001).

In human NK cells, FK506 suppresses IL-2 responsive proliferation and cytokine production as well as lowers cytotoxicity directed toward K562 tumor cells (Kim et al. 2010). FK506 suppresses IL-2 production of NKT cell line DN32.D3 induced by stimulus from phorbol 12- myristate 13-acetate (PMA)/calcium -ionophore (van Dieren et al. 2010).

The relationship between these FK506-induced mechanisms and NFAT and contribution of those to TDAR are unclear.

In addition to NFAT/AP-1 complexes, NFAT forms complexes at the site of IL-3 and IL-4 enhancers with avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), early growth response 1 (EGR1), early growth response 4 (EGR4), interferon-regulatory factor 4 (IRF4), octamer-binding transcription factor (OCT), and other transcriptional partners to induce transcription of a variety of cytokines (Macian 2005). The production of cytokine induced by these transcriptional partners also suppressed by CNI; however, contribution of these additional transcription factors to TDAR is also unclear.

Quantitative Understanding of the Linkage

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Response-response Relationship

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In purified T cells from male C57BL/6J mice, T-5224 (a selective c-Fos/AP-1 inhibitor) inhibits the DNA-binding activity of AP-1 at 80 μg/mL. On the other hand, T-5224 inhibits IL-2 production in a dose-dependent manner from 40, 60 and 80 μg/mL after 48 hours culture. T-5224 also inhibits CD25 (IL-2 receptor) up-regulation at 80 μg/mL (Yoshida et al. 2015).

In splenic lymphocytes stimulated with concanavalin A for 24 h in C57BL/6 mice, ursolic acid suppressed products of NF-kB, NFAT and AP-1 at 5 μM. In lymphocytes stimulated with concanavalin A for 24 h, ursolic acid inhibits secretion of IL-2 and IL-4 at 0.5, 1 and 5 μM. In lymphocytes and CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h, ursolic acid also inhibits secretion of IL-2 and IL-4 at 5 μM. In CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h, ursolic acid suppressed mRNA level of IL-2 at 5 μM. In lymphocytes stimulated with concanavalin A for 24 h, ursolic acid inhibited CD25 expression at 5 μM (Checker et al. 2012).

These findings showed that T-5244 and ursolic acid treated for 24 hours inhibit NFAT/AP-1 complex formation at a single concentration each and that these compounds suppress IL-2 and IL-4 production with dose dependent manner including the doses for inhibition of NFAT/AP-1 complex formation.

FK506 suppressed proliferation in human T cells induced by anti-CD3 mAb in the presence of adherent autologous PBMC (mean IC50 = 0.06 nM). FK506 suppressed, in a dose-dependent (1.2 to 12.5 nM) manner after 22-24 hours culture, production of IL-2, IL-4, and IFN-γ by human T cells stimulated with anti-CD3 mAb in the presence of PMA, as well as inhibited, also in a dose-dependent (10 nM) manner, expression of IL-2, IL-4, and IFN-γ mRNA in anti-CD3/PMA- activated cells (Dumont et al. 1998). On the other hand, the quantitative data for the decreased formation of NFAT/AP-1 complexes by CNI is insufficient, although the formation was suppressed by FK506 at the concentration within the range needed for suppressed production of IL2/IL-4 by FK506 after 2 hours culture.

Time-scale

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Inhibition of NFAT/AP-1 complex is detected by gel mobility shit assay after 2 hours culture with CNI; however, suppression of IL2/IL-4 could be measured after 22-48 hours in vitro culture.

Known modulating factors

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(To be described)

Known Feedforward/Feedback loops influencing this KER

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(To be described)

Domain of Applicability

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In purified T cell from male C57BL/6J mice, T-5224 (a selective c-Fos/AP-1 inhibitor) inhibits the DNA-binding activity of AP-1, IL-2 production and CD25 (IL-2 receptor) up-regulation (Yoshida et al. 2015).

In splenic lymphocytes and/or CD4+ T cells, ursolic acid suppressed products of NF-kB, NFAT and AP-1, and inhibits secretion of IL-2 and IL-4, mRNA level of IL-2 and CD25 expression (Checker et al. 2012).

NFATp- and NFAT4-deficient mice indicate decreased production of IL-2 (Ranger et al. 1998).

NFAT/AP-1 complex formation in the nucleus was shown using murine and human T cells lines (Jain J et al. 1992). In addition to data on suppression of cytokine production by CNI in rodents, the following is known from the use of human cells: FK506 inhibited expression of both IL-2 and mRNA in human anti-CD3/PMA-activated cells (Dumont et al. 1998).

References

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  1. Schreiber, SL., and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42.
  2. Jain J., McCaffrey P.G., Valge-Archer V.E., Roa A.(1992). Nuclear factor of activated T cells contains Fos and Jun. Nature 356(6372):801-804.
  3. Jain J., Miner Z., Rao A. (1993). Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. Journal of Immunology 151(2): 837-848.
  4. Macián, F., López-Rodríguez, C. and Rao, A. (2001). Partners in transcription: NFAT and AP-1. Oncogene. 20(19): 2476-89.
  5. Yoshida, T., Yamashita, K., Watanabe, M., Koshizuka, Y., Kuraya, D., Ogura, M., Asahi, Y., Ono, H., Emoto, S., Mizukami, T., Kobayashi, N., Shibasaki, S., Tomaru, U., Kamachi, H., Matsushita, M., Shiozawa, S., Hirono, S. and Todo, S. (2015). The Impact of c-Fos/Activator Protein-1 Inhibition on Allogeneic Pancreatic Islet Transplantation. Am J Transplant. 15(10): 2565-75.
  6. Mittelstadt, PR. and Ashwell, JD. (2001). Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem. 276(31):29603-10.
  7. Checker, R., Sandur, SK., Sharma, D., Patwardhan, RS., Jayakumar, S., Kohli, V., Sethi, G., Aggarwal, BB. and Sainis, KB. (2012). Potent Anti-Inflammatory Activity of Ursolic Acid, a Triterpenoid Antioxidant, Is Mediated through Suppression of NF-κB, AP-1 and NF-AT PLoS One. 7(2): e31318.
  8. Ranger, AM., Oukka, M., Rengarajan, J. and Glimcher, LH. (1998). Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity. 9(5):627-35.
  9. Dumont, F.J., Staruch, M.J., Fischer, P., DaSilva, C. and Camacho, R. (1998). Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK MAP kinase or calcineurin signaling pathways results in differential modulation of cytokine production. Journal of immunology 160 (6): 2579-89.
  10. Macian, F. (2005) NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol. 5(6): 472-84.
  11. Panhans-Gross, A., Novak, N., Kraft, S., and Bieber, T. (2001). Human epidermal Langerhans’ cells are targets for the immunosuppressive macrolide tacrolimus (FK506). Journal of Allergy and Clinical Immunology 107(2): 345-52.
  12. van Dieren, J.M., Lambers, M.E.H., Kuipers, E.J., Samsom, J.N., van der Woude, C.J. and Nieuwenhuis, E.E.S. (2010). Local immune regulation of mucosal inflammation by tacrolimus. Digestive diseases and sciences 55(9): 2514-19.
  13. Zhang, BW., Zimmer, G., Chen, J., Ladd, D., Li, E., Alt, FW., Wiederrecht, G., Cryan, J., O'Neill, EA., Seidman, CE., Abbas, AK., Seidman, JG. (1996). T cell responses in calcineurin A alpha-deficient mice. J Exp Med. 183(2): 413-20.