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Relationship: 1509
Title
Reduction, NFAT/AP-1 complex formation leads to Suppression, IL-2 and IL-4 production
Upstream event
Downstream event
Key Event Relationship Overview
AOPs Referencing Relationship
AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding | Point of Contact | Author Status | OECD Status |
---|---|---|---|---|---|---|
Inhibition of Calcineurin Activity Leading to Impaired T-Cell Dependent Antibody Response | adjacent | High | High | Takumi Ohishi (send email) | Open for comment. Do not cite | WPHA/WNT Endorsed |
Taxonomic Applicability
Term | Scientific Term | Evidence | Link |
---|---|---|---|
Homo sapiens | Homo sapiens | High | NCBI |
Sex Applicability
Sex | Evidence |
---|---|
Unspecific | High |
Life Stage Applicability
Term | Evidence |
---|---|
All life stages | High |
Key Event Relationship Description
Localized nuclear factor of activated T cells (NFAT) in the nucleus of T cells forms complexes with activator protein-1 (AP-1) at the Interleukin (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 interaction 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).
Lowered nuclear localization of NFAT by calcineurin inhibitor (CNI) results in decreased formation of NFAT/AP-1 complex at the promoter region of IL-2 genes in the nucleus of T cells thereby reducing 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 Collection Strategy
Evidence Supporting this KER
Biological Plausibility
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. glucocorticoid-induced leucine zipper (GILZ) 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, calcineurin A (CnA)-knockout (KO) mice produce less Interferon (IFN)- γ, IL-2, and IL-4 than wild-type mice. However, primary antibody response in CnA-KO mice is normal in response to trinitrophenol-ovalbumin (Zhang et al. 1996).
The following phenotypes are observed in NFAT-KO 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 calcineurin (CN). Suppression of T-cell-derived cytokines is noted both in CnA-KO and NFAT-KO 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 inhibits. 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/phorbol 12-myristate-13-acetate (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
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 cyclosporin A (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).
- Treatment with CsA completely eliminated detectable IL-2 release from 3A9 T cells co-cultured with antigen-bearing Ch27 B cells with an IC25 and IC50 for IL-2 production of 1.19 nM and 1.99 nM. Treatment with other immunosuppressant compounds (dexamethasone, azathioprine, methotrexate, benzo(a)pyrene and urethane) also resulted in decreased IL-2 release from stimulated 3A9 T cells at non-cytotoxic concentrations. Urethane, a weakly immunosuppressive chemical, was least potent in the assay, with an IC25 and IC50 for IL-2 secretion of 4.24 mM and 13.26 mM (D.M. Lehmann. et al. 2018).
- In female B6C3F1 mice, 1,2:5,6-dibenzanthracene exposure reduced production of IL-2 in spleen cell culture supernatants after in vitro stimulation with Concanavalin A or lipopolysaccharide (Donna, C. et al. 2010).
- Treatment with CsA at 50 mg/kg BID via oral gavage or 2C1.1 (a fully human anti-ORAI1 monoclonal antibody) at 25 mg/kg single IV resulted in reduction of IL-2, IL-4, IL-5, and IL-17 cytokine production from PMA/ionomycin stimulation of whole blood in the cynomolgus monkey (Kevin, G. et al. 2014).
- In male CD-1 mice, chronic psychosocial stress (types of social outcome occurred: residents becoming subordinates) reduced IL-2 release in response to keyhole limpet hemocyanine (Alessandro, B. et al. 2003).
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
CNIs are reported to suppress IL-17 release from Th17 cells and development of Th17 cells from naïve T cells (Tsuda et al, 2012). On the other hand, Yadav reported that Th17 cells increased and Treg cells decreased in number and that the levels of RORC mRNA increased and those of FOXP3 decreased in renal transplanted patients with chronic calcineurin inhibitor toxicity (Yadav, 2015). From these findings, CNIs suppress the functions of Th17 and Treg cells,which enhance Th17 cells to develop chronic CNI toxicity.
FK506 suppresses expression of IL-2 receptor (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 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, early growth response 1, early growth response 4, interferon-regulatory factor 4, octamer-binding transcription factor, 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.
Known modulating factors
At present, no evidence is found.
Quantitative Understanding of the Linkage
Response-response Relationship
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-2R) 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 peripheral blood mononuclear cells (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
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 Feedforward/Feedback loops influencing this KER
At present, no evidence is found.
Domain of Applicability
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-2R) 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, FK506 is reported to inhibit expression of both IL-2 and mRNA in human anti-CD3/PMA-activated cells (Dumont et al. 1998).
References
- Schreiber, SL., and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42.
- 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.
- 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.
- Macián, F., López-Rodríguez, C. and Rao, A. (2001). Partners in transcription: NFAT and AP-1. Oncogene. 20(19): 2476-89.
- 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.
- Mittelstadt, PR. and Ashwell, JD. (2001). Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem. 276(31):29603-10.
- 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.
- 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.
- 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.
- Macian, F. (2005) NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol. 5(6): 472-84.
- 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.
- 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.
- 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.
- Alessandro B, Paola S, Alberto E. Paneraic, Tiziana P,Paola Palanzaa and Stefano P(2003). Chronic psychosocial stress-induced down-regulation of immunity depends upon individual factors Journal of Neuroimmunology 141: 58–64
- Donna C. S, Matthew J. S and Kimber L. W Jr. (2010) Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1 mice, Journal of Immunotoxicology, 7:3, 219-231
- Kevin G, Hossein S, Raju S, Valerie A, Anna K, Ming Z, Fen-Fen L, Hung Q. N, Lei Z, John K. S, Min W and Helen J. M(2015) Inhibition of CRAC with a human anti-ORAI1 monoclonal antibody inhibits T-cell-derived cytokine production but fails to inhibit a T-cell-dependent antibody response in the cynomolgus monkey, Journal of Immunotoxicology, 12:2, 164-173,
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