Nuclear Factor of Activated T Cells and Cytokines Gene Expression of the T Cells in AIDS Patients with Immune Reconstitution Inflammatory Syndrome during Highly Active Antiretroviral Therapy

Background. The etiology of immune reconstitution inflammatory syndrome (IRIS) in AIDS patients after the initiation of HAART remains unknown. Several researches indicated that the development of IRIS is associated with the production and variation of cytokines, whose gene expression are closely related to the Ca2+/CN-nuclear factor of activated T cells (NFAT) pathway. Methods. We studied the expression of NFAT isoforms and their major target cytokines genes in peripheral blood CD3+ T cells of subjects through fluorescence quantitative PCR and explored the expression changes of these genes before and after HAART. Results. After the initiation of HARRT, NFAT1, IL-6, and IL-8 gene expression showed a reversal trend in the CD3+ T cells of the IRIS group and changed from low expression before HARRT to high expression after HARRT. In particular, the relative gene expression of NFAT1 was markedly higher compared with the other three isoforms. The IRIS group also showed higher NFAT4, NFAT2, NFAT1, IL-1β, IL-10, IL-2, IL-18, and TNF-α gene expression than the non-IRIS group. Conclusion. This study suggested that high expression levels of IL-2, IL-6, IL-8, TNF-α, IL-1β, IL-10, IL-12, and IL-18 can predict the risk of IRIS. The increased expression of NFAT1 and NFAT4 may promote the expression of cytokines, such as IL-6, IL-8, and TNF-α, which may promote the occurrence of IRIS.


Introduction
Highly active antiretroviral therapy (HAART) has substantially reduced the incidence and mortality of HIV-related diseases in AIDS patients. However, 8-40% of patients still have excessive inflammatory responses to various infectious or noninfectious pathogens, mostly within six months of HAART, leading to increased clinical symptoms. These patients present with aggregated opportunistic infections that have been effectively treated or with emerging infections, which is called immune reconstitution inflammatory syndrome (IRIS) [1][2][3][4][5]. IRIS is associated with various infectious diseases, and tuberculosis-related IRIS constitutes the highest incidence of IRIS at 8-43%; the incidence is even higher in developed countries (17-43%) [6]. Other infections associated with IRIS include Mycobacterium avium infection, cryptococcosis, pneumocystis pneumonia, and cytomegalovirus retinitis; noninfectious diseases include various autoimmune diseases such as Graves' disease and Kaposi's sarcoma [7].
To further elucidate the pathogenesis of IRIS, we analyzed gene expression changes of NFAT1-NFAT4 and the major target genes IL-2, IL-6, IL-8, TNF-, IL-1 , IL-10, and IL-12 in peripheral blood CD3 + T cells before and during the course of IRIS. subjects had IRIS and 33 did not. Above all, there were 30 patients with IRIS (IRIS group: 28 males and 2 females) and 33 patients without IRIS (non-IRIS group: 30 males and 3 females). The IRIS group included 13 cases with TB-IRIS, five cases with CMV-IRIS, three cases with C. neoformans-IRIS, five cases with varicella zoster virus-associated IRIS, two cases with pneumocystis pneumonia-associated IRIS, one case with cytomegalovirus/tuberculosis coinfected IRIS, and one case with Penicillium marneffei-associated IRIS. IRIS was diagnosed according to the criteria of the International Network for the Study of HIV-Associated IRIS. The baseline conditions of the subjects are detailed in Table 1. This study was approved by the hospital ethics committee, and each patient who was enrolled had signed an informed consent form.

Specimen Collection and Processing.
Whole blood specimens (5-8 mL each) were collected from the all subjects at the initiation and 3 months after starting HAART. Moreover, for the IRIS group, blood specimens were collected at the diagnosis time point of IRIS and 1 month after HAART for the non-IRIS group. The specimens were anticoagulated with EDTA. Plasma and PBMCs were isolated within 10 hours of collection. The PBMCs were stored in Zenoaq's CELL-BANKER cryopreservation medium at −80 ∘ C. We examined gene expression changes in the IRIS and non-IRIS groups before and 1 month after HAART.

Detection of NFAT and the Expression of Its Target
Genes in T Cells

Magnetic Bead Separation of CD3 + T Cells and RNA
Extraction of Samples. The PBMCs were thawed in a warm bath at 37 ∘ C and centrifuged at 300 g/min for 5 min. The supernatant was aspirated, and the CD3 + T cells were isolated using CD3 human MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) by strictly following the manufacturer's instructions. The isolated CD3 + T cells were added to 1 mL of TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA), and total RNA extraction was performed according to the manufacturer's instructions. The extracted RNA was dissolved with 20 L of DEPC water before the RNA concentration measurement. The synthesized cDNA was stored at −20 ∘ C.

Fluorescence Quantitative RT-PCR.
We performed qPCR reactions using iQ 6 SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The reaction system contained 12.5 L of Bio-Rad iQ SYBR Green Supermix, 1 L of sense primer (50 pg), 1 L of antisense primer (5 pg), 1 L of cDNA, and 9.5 L of DEPC H 2 O, resulting in a total volume of 25 L. The reaction conditions were as follows: 95 ∘ C for 3 min, followed by 45 cycles of 95 ∘ C for 15 sec, 60 ∘ C for 30 sec, and 72 ∘ C for 30 sec. A melting curve analysis was performed using the following conditions: 65 ∘ C to 95 ∘ C for 5 s. The primer sequences are shown in Table 2. The primers were synthesized by Sangon (Shanghai, China). The PCR reactions were performed on a quantitative PCR machine (Bio-Rad).

Statistical Methods
All of the data were analyzed using IBM SPSS Statistics 19 (IBM SPSS, Somers, NY, USA) unless otherwise stated. We first performed a one-sample K-S test to examine the normal distribution of the data. Then, a -test was used to analyze the normally distributed data, and the Mann-Whitney test was used to analyze the nonnormally distributed data. Differences were considered statistically significant at < 0.05. All of the graphs were generated using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). We compared previous gene expression changes to 1 month after HAART in the IRIS and non-IRIS groups, respectively. Meanwhile, we compare IRIS group to non-IRIS group at the time before HAART and 1 month after HAART separately by using the fold difference (2 −ΔΔCT ) method, with human GAPDH as an internal reference. The Delta-Delta-Threshold cycle (ΔΔCT) equation was used to compare the expressions of NFAT and cytokine genes in IRIS and non-IRIS patients before and after

NFAT and Cytokine Gene Relative Expression Levels Compared before and after HAART in the IRIS and Non-IRIS Groups of AIDS Patients.
A comparative analysis of the relative gene expression by fold difference before and after HAART revealed that the IRIS group had higher NFAT1, IL-6, IL-8, and TNF-expression in CD3 + T cells after receiving HAART for 1 month. The gene expression increase was the most obvious for NFAT1, whose expression after HAART was 3.058-fold greater than that before HAART. However, gene expression of IL-2, IL-10, and IL-18 was lower after HAART. The differences of gene expressions of NFAT4, NFAT2, and IL-1 were not obvious after initiation of HAART (fold difference: 0.969, 0.981, and 1.03, resp.). The non-IRIS group showed lower NFAT4, NFAT2, NFAT1, IL-1 , IL-6, IL-8, IL-10, IL-2, and IL-18 gene expressions after HAART than before HAART. The gene expression of TNF-after HAART changed little, and it was 1.03-fold higher than that before HAART ( Figure 1 and Table 3).

NFAT and Cytokine Gene Relative Expression Levels
Comparing the IRIS to Non-IRIS Groups of AIDS Patients before and after HAART. We compared the relative gene expression levels of NFAT and cytokines between the IRIS and non-IRIS groups before HAART. NFAT1 was expressed at significantly lower levels than the other three NFAT isoforms in the CD3 + T cells of the IRIS group. The IRIS group showed lower IL-6 and IL-8 expression levels than the non-IRIS group, whereas other genes including NFAT4, NFAT3, NFAT2, IL-1 , IL-10, IL-2, IL-18, and TNF-were expressed at higher levels in the IRIS group than in the non-IRIS group. After 1 month of HAART, NFAT1, IL-6, and IL-8 expressions showed a reversal trend and changed from low expression levels before HAART to high expression levels after HAART. The relative expressions of NFAT1, IL-6, and IL-8 genes in the IRIS group were 1.61-, 3.406-, and 3.368-fold greater than those in the non-IRIS group, respectively; other genes like NFAT4, NFAT2, IL-1 , IL-2, IL-10, TNF-, and IL-18 were also expressed at higher levels compared with those in the non-IRIS group. However, the relative expressions of IL-10, IL-2, and IL-18 showed a larger decreasing amount than those in the non-IRIS group ( Figure 2 and Table 3).

Discussion
IRIS easily occurs in patients who have a short interval between the treatment of opportunistic infections and the start of HAART [5]. As a result, the occurrence of IRIS was related with residual antigen burden. Taken the active tuberculosis as an example, before antituberculosis therapy, there existed high level of TB antigen, so the first step for TB AIDS patients was anti-TB therapy and then HAART, which was similar in the therapy of CMV AIDS. Generally speaking, the development of IRIS associated with pathogen infection; therefore the major principle of IRIS treatment is the antipathogen therapy for a period of time before the initiation of HAART, through which it can not only effectively decrease the antigen burden but also cut down the scale of residual antigen. Meanwhile, to reduce the occurrence of IRIS, methods like T-SPOT, CMV-DNA, and VZV-DNA are widely used to measure the antigen burden and residual antigen in clinic.  Figure 1: NFAT and cytokine gene relative expression levels compared before and after HAART in the immune reconstitution inflammatory syndrome (IRIS) and non-IRIS groups of AIDS patients. (a) NFAT expression in IRIS groups before and after HAART; NFAT1 expression from low to high after HAART; the relative expression of mRNA for NFAT1 gene to GAPDH after HAART was 3.002-fold higher as compared to that before HAART. (b) Cytokine gene expression in IRIS groups before and after HAART; IL-1 , IL-6, IL-8, and TNF-expression from low to high; TNF-gene expression also increased in non-IRIS group after HAART; ‰‰ the gene expressions after HAART were higher than those before treatment.

Mediators of Inflammation
Before HAART After HAART NFAT1 NFAT2 NFAT4 The pathogenesis of IRIS has not been clarified yet. Tadokera et al. found that IL-1 , IL-5, IL-6, IL-10, IL-13, IL-17A, IFN-, GM-CSF, and TNF levels are elevated in the cell culture of PBMCs from TB-IRIS patients after in vitro stimulation with inactivated Mycobacterium tuberculosis. This finding indicates that the release of cytokines promotes the occurrence of TB-IRIS [17]. The cytokine release syndrome (also known as the cytokine storm) can be observed in infectious or noninfectious diseases, such as graft-versus-host disease and acute respiratory distress syndrome [17]. Currently, calcineurin inhibitors including cyclosporine (CSA) and tacrolimus (FK506) are available to block the Ca 2+ -CN/NFAT pathway and suppress the function and spread of effector T cells. These agents have been extensively used in transplantation patients. Moreover, calcineurin inhibitors can inhibit the formation of IL-2, IL-4, and TNF- [16]. 6 Mediators of Inflammation Therefore, we speculate that the Ca 2+ -CN/NFAT pathway may play a role in the excessive immune response of AIDS patients receiving HAART.
We performed fluorescence quantitative PCR analysis to detect NFAT1-NFAT4, IL-2, IL-6, IL-8, TNF-, IL-1 , IL-10, and IL-12 genes in the CD3 + T cells of IRIS and non-IRIS patients. The results showed that the relative gene expression of NFAT1, IL-6, IL-8, and TNF-was obviously higher after 1 month of HAART than before HAART in the CD3 + T cells of IRIS patients and that the most significant gene expression increase was obtained for NFAT1. When the IRIS group was compared to the non-IRIS group, we found that, before HAART, IL-1 , IL-10, and IL-2 expressions of CD3 + T cells were higher in the former than in the latter group; after 1 month of HAART, NFAT2, NFAT4, IL-6, IL-8, TNF-, IL-1 , IL-10, and IL-2 expression levels were all higher in the former than in the latter group. IL-1 , IL-10, and IL-2 expressions were reduced in both the IRIS and non-IRIS groups after HAART; the reduction was larger in the non-IRIS group than in the IRIS group. Maintaining relatively high expression levels of IL-1 , IL-10, and IL-2 may play an important role in the development and progression of IRIS. The increase in NFAT1 expression in IRIS patients after 1 month of HAART likely plays an essential role in the occurrence of IRIS by contributing to IL-6, IL-8, and TNF-expression, NFAT can bind to the promoter/enhancer of various cytokine genes and can contribute to the expression of IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IFN-, and TNF-. During HAART for AIDS, we found NFAT expression changes in the T lymphocytes of the IRIS patients. Among four NFAT1 isoforms, NFAT1 and NFAT4 showed an increase in expression; the expression increase was more significant for NFAT1. These results provided a new idea for the treatment of IRIS: we can use selective inhibitors of iRNA or NFAT1 to block the CN/NFAT pathway and reduce IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IFN-, and TNFgeneration for the treatment of IRIS to reduce the occurrence of IRIS and improve the prognosis of IRIS patients in the clinic.
Taken powerful immunosuppressive action of calcineurin inhibitors, such as CSA and FK506, into consideration, we replace thalidomide, which is an anti-inflammatory, immunomodulatory, and antiangiogenic agent [18] and has already been effectively used in our recent case report of Mycobacterium avium complex-(MAC-) associated IRIS, whose cutaneous MAC lesions continue exacerbation despite antimycobacterial treatment. But rapid clinical remission occurred after adding thalidomide to the antituberculosis treatment. Furthermore, immunomodulatory effect of thalidomide on cytokine levels such as decreasing of TNFand increasing of IL-4 and IL-6 was observed in the patient [19]. The limitation for this approach of blocking the CN/NFT pathway is that this inhibition could negatively affect immune responses activation when these patients have real/new infections, as the immune system is still under severely immune suppression. Anyway, the development of IRIS also indicated that these patients have an excellent effect in response to HAART. As it is a condition of immunoimbalance, under the written informed consents from all subjects, we are conducting a case-control study by using CN/NFT drugs from small doses in study group and observe the effects intensively.

Consent
Written consent was obtained from the patients.