Hiroyuki Komatsu (1) Junichiro Sugimoto (1) Ken Goto (1) Kiyoshi Kushima (1) Naohisa Tsutsui (1) Shigeru Hisada (1) Shiho Ito (1) Tadashi Kosaka (1) Takumi Ohishi (1) Yasuharu Otsubo (1) Yoshihiro Takahashi (1)
(1) AOP Working Group, Testing Methodology Committee, The Japanese Society of Immunotoxicology
Corresponding author: Kiyoshi Kushima (email@example.com)
Point of Contact
- Kiyoshi Kushima
|Author status||OECD status||OECD project||SAAOP status|
|Open for comment. Do not cite||EAGMST Under Review||1.38||Included in OECD Work Plan|
This AOP was last modified on May 21, 2017 19:32
|Inhibition, Calcineurin Activity||May 18, 2017 08:28|
|Interference, nuclear localization of NFAT||May 19, 2017 02:30|
|Reduction, NFAT complex formation||September 16, 2017 10:16|
|Impairment, T-cell dependent antibody response||May 21, 2017 19:43|
|Binding, Immunophilins||May 18, 2017 08:17|
|Suppression, IL-2 and IL-4 production||May 19, 2017 02:39|
|Binding, Immunophilins leads to Inhibition, Calcineurin Activity||May 19, 2017 10:18|
|Inhibition, Calcineurin Activity leads to Interference, nuclear localization of NFAT||May 19, 2017 02:57|
|Interference, nuclear localization of NFAT leads to Reduction, NFAT complex formation||May 21, 2017 19:46|
|Reduction, NFAT complex formation leads to Suppression, IL-2 and IL-4 production||May 21, 2017 19:40|
|Suppression, IL-2 and IL-4 production leads to Impairment, T-cell dependent antibody response||May 21, 2017 19:54|
|Tacrolimus||November 29, 2016 18:42|
|Cyclosporin||May 18, 2017 08:31|
Calcineurin is a type of protein phosphatase that is known to impair immune function when phosphatase activation is inhibited. The relationship between calcineurin and immune functions are well understood, and immunosuppressants that work by inhibiting calcineurin have been developed.
This AOP describes the linkage between the impairment of T-cell dependent response and immunosuppression that occurs due to inhibition of calcineurin.
Calcineurin activity is inhibited when stressors bond with immunophilins, which interferes with the nuclear localization of nuclear factor of activated T cells (NFAT), a substrate of calcineurin. As a result, the formation of functional NFAT complexes that bind at the site of IL-2 and other cytokine promoters is reduced, thereby suppressing production of these cytokines. Thus, T-cell dependent antibody response (TDAR) is impaired by the suppression of production of IL-2, IL-4, and other types of cytokines, which affects the proliferation and differentiation of B-cells.
We have identified a number of key events from within this pathway, and based on these key event relationships, created an AOP for inhibition of calcineurin activity leading to impaired T-cell dependent antibody response.
Calcineurin expresses in cells among vast variety species, because of which, this AOP is applicable to many mammal species, including humans and rodents.
Although there are numerous stressors that inhibit calcineurin activity, this AOP is based on an understanding of immunosuppression caused by FK506 and FKBP12 complexes, on which a significant body of scientific literature has been published.
We look forward to future amendments to this AOP with up-to-date information on other stressors, which will clarify the linkage between inhibition of calcineurin activity and T-cell dependent antibody response.
Summary of the AOP
Molecular Initiating Event
|Binding, Immunophilins||Binding, Immunophilins|
|Inhibition, Calcineurin Activity||Inhibition, Calcineurin Activity|
|Interference, nuclear localization of NFAT||Interference, nuclear localization of NFAT|
|Reduction, NFAT complex formation||Reduction, NFAT complex formation|
|Suppression, IL-2 and IL-4 production||Suppression, IL-2 and IL-4 production|
|Impairment, T-cell dependent antibody response||Impairment, T-cell dependent antibody response|
Relationships Between Two Key Events (Including MIEs and AOs)
|Binding, Immunophilins leads to Inhibition, Calcineurin Activity||Directly leads to||Strong||Strong|
|Inhibition, Calcineurin Activity leads to Interference, nuclear localization of NFAT||Directly leads to||Strong||Strong|
|Interference, nuclear localization of NFAT leads to Reduction, NFAT complex formation||Directly leads to||Strong||Strong|
|Reduction, NFAT complex formation leads to Suppression, IL-2 and IL-4 production||Directly leads to||Strong||Strong|
|Suppression, IL-2 and IL-4 production leads to Impairment, T-cell dependent antibody response||Directly leads to||Strong||Strong|
Life Stage Applicability
|All life stages||Moderate|
|Homo sapiens||Homo sapiens||Strong||NCBI|
|Mus musculus||Mus musculus||Strong||NCBI|
|Macaca fascicularis||Macaca fascicularis||Strong||NCBI|
|Rattus norvegicus||Rattus norvegicus||NCBI|
Graphical RepresentationClick to download graphical representation template
Overall Assessment of the AOP
Calcineurin activity is inhibited when stressors bond with immunophilins, which interferes with the nuclear localization of nuclear factor of activated T cells (NFAT), a substrate of calcineurin. As a result, the formation of functional NFAT complexes that bind at the site of IL-2 and other cytokine promoters is reduced, thereby suppressing production of these cytokines. Thus T cell dependent antibody response (TDRA) is impaired by the suppression of production of IL-2, IL-4, and other types of cytokines, which affect the proliferation and differentiation of B-cells. We have identified a number of key events from within this pathway, and based on these key event relationships, created an AOP for inhibition of calcineurin activity leading to impaired T cell dependent antibody response. There are many varieties of calcineurins, because of which, this AOP is applicable to many mammal species, including humans and rodents.
Domain of Applicability
The proposed AOP of inhibited immunophilin activation leading to immunosuppression is not associated with life stage-, sex-, or age-dependency. The relevant life stages for the AOP are from child to adult, and since tacrolimus ointment (Protopic) is approved for pediatric atopic dermatitis, the MOA for immunosuppression appears to be applicable to all of life stages. Since FK506-induced outcomes in humans are mimicked by similar responses in a variety of animal models, immunosuppression induced by immunophilin-calcineurin inhibitor complexes are considered to be preserved across a variety of mammalian species.
Essentiality of the Key Events
In calcineurin subunit A knockout (CnA-/-) mice, T cell proliferation in response to ovalbumin stimulation is lower than for wild-type mice and is not complemented by normal antibody producing cells.1 In addition, when stimulated with ovalbumin, CnA-/- mice produce less IFN-g, IL‑2, and IL‑4 than wild-type mice.1 However, primary antibody response in CnA-/- mice is normal in response to TNP-ovalbumin.1
There is no evidence of a relationship between FKBP12 KO and the immune system in the FKBP12 knockout mouse model.
The following phenotypes are observed in NFAT knockout mice2: 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; mild hyperactivation of peripheral T cells.
The study of NFAT-/- mice shows that NFAT is involved in a wide range of immune-cell phenomenon, and some of these phenomenon are known to be regulated by calcineurin. This indicates that the production of T-cell derived cytokine is regulated by CN-NFAT.
Weight of Evidence Summary
The inhibition of CN phosphatase activity due to the formation of immunophilin-CN ihibitor complexes, which are CN stressors, is well known as an effect of CsA-Cuclophilin complexes orFK506-FKBP12 complexes. The information in this AOP concerns FKBP12. Immunophilins are a general class of proteins that exhibit peptidyl-propyl isomerase (PPIase) activity, but there is no commonality between their two amino acid sequences. The three-dimensional structure of immunophilin complexes is essential to inhibition of CN phosphatase activity, but it is known that these enzyme activities are not related to inhibition of CN.
CN is expressed in immune cells and other tissue cells throughout the body, and experimentation with T cells indicates that TCR stimulation brings about intracellular increases in concentrations of Ca2+, which triggers CN activity, thereby inducing nuclear localization per dephosphorylation of its substrate NFAT, which forms complexes with AP-1 at the site of T‑cell cytokine promoters and induces production of these cytokines.
It is also well known that one of the effects on immune function when FK506 forms complexes with immunophilins and inhibits CN activity is the suppression of IL-2 and other T-cell derived cytokine production. It is further well known that inhibition of CN leads to suppression of T-cell dependent antibody production because IL‑2, IL‑4, and other cytokines promote the proliferation, class switching, differentiation, and maturation of B-cells.
Furthermore, CN-NFAT also exists in B‑cells, and although it has been reported that CN inhibitors do suppress production of certain cytokines, at the time of our review of the literature, we did not find any reports of a direct effect on B‑cells that affected the proliferations, class switching, differentiation, or maturation of B‑cells.
Also, although CN-NFAT is known to exist in dendritic, NKT, and other types of cells, where it regulates the expression of IL-2 receptors, there are no reports of effects on the production of antibodies.
The binding of CsA with cyclophilin can be detected quantitatively using ELISA kits.
Inhibition, calcineurin activity
Phosphatase activity of CN can be measured quantitatively using a phosphatase assay.
Interference, nuclear localization of NFAT
Interference with the translocation of NFAT to the nucleus can be detected quantitatively using a gel mobility shift assay.
Reduction, NFAT complex formation
Reduction in generation of NFAT/AP-1 complexes can be detected using a gel shift assay. Cytokine mRNA levels after NFAT complex formation can be measured using RNase protection assay in vitro and ex vivo.
In so far as the formation of NFAT/AP-1 complexes during T‑cell cytokine production is dependent on the quantity of NFAT that undergoes nuclear localization, however, there would appear to be no meaning to measuring false formations of NFAT/AP-1 complexes.
Suppression, IL-2 and IL-4 production
Quantification of cytokine content can be measured using a Sandwich ELISA kit, and cytokine mRNA levels can be determined using a RiboQuant MutiProbe RPA system (PharMingen, San Diego, CA).
Impairment, T-cell dependent antibody response
Total IgM and IgG levels as well as antigen-specific antibodies can be determined in vitro and in vivo. The effects on immunoglobulin class switching can also be evaluated in vitro. These all can be determined quantitatively.
Considerations for Potential Applications of the AOP (optional)
At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale. Detailing such considerations can aid the process of transforming narrative descriptions of AOPs into practical tools. In this context, it is necessarily beneficial to involve members of the regulatory risk assessment community on the development and assessment team. The Network view which is generated based on assessment of weight of evidence/degree of confidence in the hypothesized AOP taking into account the elements described in Section 7 provides a useful summary of relevant information as a basis to consider appropriate application in a regulatory context. Consideration of application needs then, to take into consideration the following rank ordered qualitative elements: Confidence in biological plausibility for each of the KERs Confidence in essentiality of the KEs Empirical support for each of the KERs and overall AOP The extent of weight of evidence/confidence in both these qualitative elements and that of the quantitative understanding for each of the KERs (e.g., is the MIE known, is quantitative understanding restricted to early or late key events) is also critical in determining appropriate application. For example, if the confidence and quantitative understanding of each KER in a hypothesised AOP are low and or low/moderate and the evidence for essentiality of KEs weak (Section 7), it might be considered as appropriate only for applications with less potential for impact (e.g., prioritisation, category formation for testing) versus those that have immediate implications potentially for risk management (e.g., in depth assessment). If confidence in quantitative understanding of late key events is high, this might be sufficient for an in depth assessment. The analysis supporting the Network view is also essential in identifying critical data gaps based on envisaged regulatory application. Instructions To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page. The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page.
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