Evaluating the effects of immunosuppressants on human immunity using cytokine profiles of whole blood

Abstract

It is critical to determine the status of the immune system in transplant recipients, but there are currently no clinical methods available to do so. To address this, we have developed an innovative method to evaluate the effects of immunosuppressants that involves measuring phytohemagglutinin (PHA)-stimulated cytokine secretions in vitro in response to treatments with dexamethasone (DEX), FK506 or mycophenolic acid (MPA). The results revealed that DEX nonspecifically and dose-dependently inhibited the production of 12 cytokines (IL-2, IFN-γ, TNF-α, IL-8, IL-1β, IL-17, IL-4, IL-5, IL-6, IL-10, IL-13, and G-CSF). In contrast, FK506 and MPA selectively inhibited the secretion of IL-2 and IL-13, and MPA unexpectedly increased the production of IL-1β. Therefore, different immunosuppressants have distinct cytokine signatures that can be used to determine whether there is under- or over-immunosuppression in transplant recipients. Immunosuppressants could be adjusted according to the cytokine profiles to maximize immunosuppressive effects while minimizing the risk of infection.

Introduction

The development of sensitive and accurate methods to evaluate the immune status of transplant recipients is of paramount importance. Such assays would be powerful tools for clinicians, allowing them to adjust the type or dose of immunosuppressants to reduce the occurrence or severity of rejection or infection, thereby prolonging the survival of grafts and patients. Since rejection or infection is mainly due to under- or over-immunosuppression, methods assessing the function of immune cells would be ideal for predicting the risks of forthcoming rejection or infection before clinical manifestations become evident [1], [2], [3], [4], [5]. Unfortunately, the lack of applicable immunological methods to assess patient immune status forces clinicians to depend completely on clinical symptoms, graft functional tests, and other examinations for the diagnosis of rejection or infection [3], [6]. These methods are only able to detect the ultimate consequences of inappropriate immunosuppression, which are usually only visible several days or weeks after profound changes in the immune system have occurred [6].

Researchers have long been trying to establish immunological methods for evaluating the immune status of transplant recipients. Most methods have included detecting mRNAs or cytokines (secreted or intracellular) in peripheral blood mononuclear cells (PBMCs) or diluted whole blood [6], [7], [8], [9]. A major problem with the detection of mRNAs is that the transcription level is not always consistent with the amount of protein expression, since modification often occurs after mRNA transcription [9], [10]. For the detection of cytokines in PBMCs or diluted whole blood, the isolation and culture procedures may wash out important immune molecules and immunosuppressive drugs, and disrupt molecular networks between immune cells. Thus, these methods may be unable to mimic the in vivo immunosuppressive environment [11], [12]. Furthermore, sample preparation procedures are also complicated and time consuming, which prevents an immediate readout of the immune status and slows the implementation of effective medication strategies [3], [12]. Consequently, the inaccurate and time consuming nature of those methods are major obstacles to their clinical application, as rejection or infection often occur unexpectedly and proceed very rapidly [12]. Thus, simple, fast, and accurate methods for evaluating immune status in transplant recipients are urgently required.

Actually, cytokine levels in stimulated whole blood have been used to evaluate the immune status in chronic heart failure patients [13]. In this study, we detected multiple cytokines simultaneously with the multi-plex technique to determine the effects of immunosuppressants. To mimic the in vivo environment, we used undiluted whole blood with a short-term in vitro stimulation. We found that a 12 h stimulation with PHA could maximize the secretion of most cytokines, and that blood treated with different immunosuppressants displayed distinct cytokine profiles. Therefore, the detection of blood cytokine profiles not only helps to evaluate whether there is under- or over-immunuosuppression, but it can also provide specific information on the effect of individual immunosuppressants [2], [14], [15]. This information will provide transplant clinicians with the opportunity to optimize the types and doses of immunosuppressants to allow better control of rejection and to minimize the risks of toxicity and infection. The method also has a potential application in patients with cancer, autoimmune disease and hypersensitive disease [16], [17], [18], [19], [20].