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Relationship: 1509

Title

The title of the KER should clearly define the two KEs being considered and the sequential relationship between them (i.e., which is upstream and which is downstream). Consequently all KER titles take the form “upstream KE leads to downstream KE”.  More help

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

Upstream event
Upstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help
Downstream event
Downstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

This table is automatically generated upon addition of a KER to an AOP. All of the AOPs that are linked to this KER will automatically be listed in this subsection. Clicking on the name of the AOP in the table will bring you to the individual page for that AOP. More help
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 Do not cite EAGMST Under Review

Taxonomic Applicability

Select one or more structured terms that help to define the biological applicability domain of the KER. In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. Authors can indicate the relevant taxa for this KER in this subsection. The process is similar to what is described for KEs (see pages 30-31 and 37-38 of User Handbook) More help
Term Scientific Term Evidence Link
Homo sapiens Homo sapiens High NCBI

Sex Applicability

Authors can indicate the relevant sex for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of the User Handbook). More help
Sex Evidence
Unspecific High

Life Stage Applicability

Authors can indicate the relevant life stage for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of User Handbook). More help
Term Evidence
All life stages High

Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can provide details that aren’t inherent in the description of the KEs themselves (see page 39 of the User Handbook). This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below). Careful attention should be taken to avoid reference to other KEs that are not part of this KER, other KERs or other AOPs. This will ensure that the KER is modular and can be used by other AOPs. More help

Localized nuclear factor of activated T cells (NFAT) in the nucleus of T cells binds to form 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 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 calcineurin inhibitor (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

Assembly and description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for overall assessment of the AOP (see pages 49-56 of the User Handbook). To do this, biological plausibility, empirical support, and the current quantitative understanding of the KER are evaluated with regard to the predictive relationships/associations between defined pairs of KEs as a basis for considering WoE (page 55 of User Handbook). In addition, uncertainties and inconsistencies are considered. More help
Biological Plausibility
Define, in free text, the biological rationale for a connection between KEupstream and KEdownstream. What are the structural or functional relationships between the KEs? For example, there is a functional relationship between an enzyme’s activity and the product of a reaction it catalyses. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs is very well established, to the extent that it is widely accepted and consistently supported by so much literature that it is unnecessary and impractical to cite the relevant primary literature. Citation of review articles or other secondary sources, like text books, may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. The description of biological plausibility can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured (see page 40 of the User Handbook for further information).   More help

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 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/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.

Uncertainties and Inconsistencies
In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. Additionally, while there are expected patterns of concordance that support a causal linkage between the KEs in the pair, it is also helpful to identify experimental details that may explain apparent deviations from the expected patterns of concordance. Identification of uncertainties and inconsistencies contribute to evaluation of the overall WoE supporting the AOPs that contain a given KER and to the identification of research gaps that warrant investigation (seep pages 41-42 of the User Handbook).Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application don’t need to be described. In general, this section details evidence that may raise questions regarding the overall validity and predictive utility of the KER (including consideration of both biological plausibility and empirical support). It also contributes along with several other elements to the overall evaluation of the WoE for the KER (see Section 4 of the User Handbook).  More help

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.

Response-response Relationship
This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture.  More help

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
This sub-section should be used to provide information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). This can be useful information both in terms of modelling the KER, as well as for analyzing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours will generally be of higher priority than other potential AOs that take weeks or months to develop). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not considered. More help

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
This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors. More help

At present, no evidence is found.

Known Feedforward/Feedback loops influencing this KER
This subsection should define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits? In some cases where feedback processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response, time-course, behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these positive or negative feedback, it is recommended that a graphical annotation (page 44) indicating a positive or negative feedback loop is involved in a particular upstream to downstream KE transition (KER) be added to the graphical representation, and that details be provided in this subsection of the KER description (see pages 44-45 of the User Handbook).  More help

At present, no evidence is found.

Domain of Applicability

As for the KEs, there is also a free-text section of the KER description that the developer can use to explain his/her rationale for the structured terms selected with regard to taxonomic, life stage, or sex applicability, or provide a more generalizable or nuanced description of the applicability domain than may be feasible using standardized terms. More help

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

List of the literature that was cited for this KER description using the appropriate format. Ideally, the list of references should conform, to the extent possible, with the OECD Style Guide (OECD, 2015). More help
  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.
  14. 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
  15. 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
  16. 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,
  17. D.M. Lehmann, W.C. Williams.(2018) Development and Utilization of a Unique In Vitro Antigen Presentation Co-culture Model for Detection of Immunomodulating Substances. Toxicol In Vitro.53: 20–28.