FAF1 downregulation by Toxoplasma gondii enables host IRF3 mobilization and promotes parasite growth

Abstract Fas‐associated factor 1 (FAF1) has gained a reputation as a member of the FAS death‐inducing signalling complex. However, the role of FAF1 in the immunity response is not fully understood. Here, we report that, in the human retinal pigment epithelial (RPE) cell line ARPE‐19 cells, FAF1 expression level was downregulated by Toxoplasma gondii infection, and PI3K/AKT inhibitors reversed T. gondii‐induced FAF1 downregulation. In silico analysis for the FAF1 promoter sequence showed the presence of a FOXO response element (FRE), which is a conserved binding site for FOXO1 transcription factor. In accordance with the finding, FOXO1 overexpression potentiated, whereas FOXO1 depletion inhibited intracellular FAF1 expression level. We also found that FAF1 downregulation by T. gondii is correlated with enhanced IRF3 transcription activity. Inhibition of PI3K/AKT pathway with specific inhibitors had no effect on the level of T. gondii‐induced IRF3 phosphorylation but blocked IRF3 nuclear import and ISGs transcription. These results suggest that T. gondii can downregulate host FAF1 in PI3K/AKT/FOXO1‐dependent manner, and the event is essential for IRF3 nuclear translocation to active the transcription of ISGs and thereby T. gondii proliferation.


| INTRODUC TI ON
Toxoplasma gondii is a ubiquitous, intracellular, apicomplexan parasite of warm-blooded animals and is one of the most common parasite of human and other animals, which affects approximately one third of the global human population. 1 Toxoplasma gondii plays a crucial role in retinochoroiditis in immunocompetent and immunosuppressed individuals, and ocular lesions may be present in up to 20% of infected patients. 2 The retinal pigment epithelium (RPE) cell is an integral part of the neuroretina in the posterior segment of the eye, whose normal functions are for light absorption, epithelial conveyance, spatial ion buffering, visual cycle, phagocytosis, secretion, elimination of oxidized or damaged materials and immune modulation. Various molecular responses of human RPE cells to infection with T. gondii has been presented by several research groups. [3][4][5][6][7] However, the host TBK1/IRF3 signalling for T. gondii infection and growth in ARPE-19 cells is elusive.
Interferon regulatory factor 3 (IRF3), key transcriptional mediator of type I interferon (IFN)-dependent immune responses, plays an essential role in the innate immune response against DNA and RNA viruses. 8,9 IRF3 is constitutively expressed in several tissues and localized in the cytoplasm in an unstimulated condition.
During the course of infection, single-or double-stranded infectious RNAs from virus accumulated inside cells are recognized by PRRs, which recruit the kinase TANK-binding kinase 1 (TBK1) and active IRF3 by phosphorylating its C-terminal region. 10 -12 The activation leads to IRF3 dimerization and translocation from cytosol to nucleus and binds to a specific promoter sequence, which conserved enhancer motifs named ISREs to induce transcription of ISGs, which would contribute to various immune responses.
Interestingly, it has also been reported that instead of detrimental effect to parasite, TBK1/IRF3 activity promotes T. gondii's efficient proliferation. 13,14 In addition to TBK1/IRF3 pathway, several reports have demonstrated that PI3K is also involved in the T. gondii infection event through activation of AKT. 15,16 In response to insulin or growth factors, AKT directly regulates the phosphorylation of FOXO transcription factors, which cause the export of FOXO from the nucleus to the cytoplasm, and thereby resulted in inhibition of FOXOs transcription activity and downstream gene expression. 17 Although this signalling pathway has been intensively studied in the view of metabolic syndromes, it also provided issues to other fields, especially in infection biology. There were challenges to reveal the role of FOXO transcription factors in the immunobiology [18][19][20] ; for example, FOXO3a was reported to suppress IRF7 transcription and negatively mediate innate immune response. 21 Given the important role of PI3K/AKT pathways in the regulation of metabolic processes and in view of emerging information regarding the T. gondii-related immunological and pathological activities of FOXO transcription factors, and our observation of PI3K/AKTdependent IRF3 activity by T. gondii, we established the hypothesis that FAF1 is an essential link between T. gondii-induced TBK1 and PI3K signalling pathways for T. gondii growth.
Fas-associated factor (FAF1), initially identified as a Fas-binding protein that potentiates Fas-induced apoptosis, participates in diverse mechanisms that promote cell death and thus is thought to act as a tumour suppressor. 22,23 In addition to its inhibitory action on tumorigenesis, FAF1 is also involved in diverse biological processes.
It has also been demonstrated that FAF1 plays critical roles in defence against pathogens infection, and as such, FAF1 can protect the host form Listeria infection by positively regulating NADPH oxidasemediated ROS production, 24 and FAF1 also plays a novel role in negatively regulating virus-induced IFNβ production and the antiviral response by inhibiting the translocation of active, phosphorylated IRF3 from the cytosol to the nucleus. 25 However, FAF1's role in protozoan infection is not fully understood yet.
In this study, we provide evidences that T. gondii requires activation of host TBK1/IRF3 and downstream genes (ISGs) expression for its efficient infection and growth in ARPE-19 cells. In addition, we also revealed that T. gondii controls the expression of host FAF1 in a PI3K/ AKT-dependent manner. A property of FAF1 as a "trapper" for IRF3 nuclear import negatively contributes to the required T. gondii-induced ISGs expression, and indeed, impairment of FAF1 expression resulted in enhanced transcription of host ISGs. Combined together, our results identified a regulatory link between the TBK1/IRF3 pathway and PI3K/AKT pathway, which is controlled by T. gondii infection and essential for its growth. The present study will provide the insights about how T. gondii modulates the host cell signalling pathway for its growth and about the immunopathological basis of ocular toxoplasmosis.

| Cell culture
The human retinal pigment epithelial cell line ARPE-19, which consists of highly polarized cells that from the outer blood-retina barrier between the photoreceptors of the neurosensory retina and vascularized choroid, was obtained from the American Type Culture Collection (ATCC). Cells were routinely cultured under 5% CO 2 at 37°C in DMEM/F12 medium (WelGENE) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco BRL) and 1% antibiotic-antimycotic reagents (Gibco BRL). The cells were passaged with 0.25% Trypsin-EDTA (Life Technologies) every 3-4 days. ARPE-19 cells between passages 4 and 8 were used in this study.

| Parasites
Toxoplasma gondii tachyzoites RH strain was maintained in ARPE-19 cells at 37°C, 5% CO 2 and passaged for every 2-3 days. RH expressing transgenic green fluorescent protein (GFP-RH) and red fluorescent protein (RFP-RH) was kindly provided by Dr. Yoshifumi Nishikawa (Obihiro University of Agriculture and Veterinary Medicine) and incubated same condition with RH.

| Preparation of T. gondii ESA
Excretory/secretory antigen (ESA) from T. gondii was produced as for 2 h at room temperature. After three times washing, the membranes were soaked with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore), and chemiluminescence was detected using a Fusion Solo System (Vilber Lourmat).

| Targeted gene silencing
One day before transfection, ARPE-19 cells were cultured in a 6well plate with 3 ml normal growth media. The medium was removed
We used the Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, ARPE-19 cells were cultured in a 6-well plate with 3 ml normal growth media to be 70%-80% confluent at transfection. We dilute Lipofectamine™ LTX Reagent in fresh DMEM/F12 medium, dilute DNA in DMEM/F12 medium and then add PLUS™ Reagent. We add diluted DNA to diluted Lipofectamine™ LTX Reagent (1:1 ratio) and incubate at room temperature for 5 min. Gene overexpression efficiency was assayed by Western blot or RT-PCR. The supernatant is the cytoplasmic fraction. We re-suspend the pellet in 50 μl complete lysis buffer by pipetting up and down and then incubate suspension for 30 min on ice on a rocking platform set at 150 rpm; vortex 30 s at the highest setting; and centrifuge for 10 min at 14,000 g in a microcentrifuge pre-cooled at 4°C. The supernatant is the nucleus fraction. We aliquot and store at −80°C, avoid freeze/thaw cycles.

| Translocation of IRF3
ARPE-19 cells seeded in 24-well plate with glass coverslips were infected with GFP-RH or RFP-RH at MOI5 for 24 h, and fixed with 4%

| RNA isolation and RT-PCR
Total RNA from stimulated cells was isolated using the Trizol reagent (Invitrogen), and cDNA was generated using M-MLV reverse transcriptase kit (Invitrogen Life Technologies) as described by the manufacturer. All PCR reactions were performed with a MyCycler (Bio-Rad) for 25 cycles. The primers were designed using Primer 3 software. mRNA sequence of the selected genes was obtained from NCBI website. The primer sequences are summarized in Table 1.
Amplified products were electrophoresed in a 1.5% agarose gel and visualized with ethidium bromide. Quantification of mRNA was performed using an imaging densitometer (Bio-Rad Laboratories, Inc).

| Real-time quantitative revere transcription polymerase chain reaction (qRT-PCR)
qRT-PCR was performed using Power SYBR ® Green PCR Master Mix (Applied Biosystems). The primers used in this study are summarized in Table 1. All reactions were performed with an ABI 7500 Fast Real-Time PCR system (Applied Biosystems) under the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative gene expression levels were quantified based on the cycle threshold (Ct) values and normalized to the reference gene GAPDH. Each sample was measured in triplicate, and the gene expression levels were calculated using the 2 -ΔΔCt method.

| Statistical analysis
All these experiments were repeated at least three times, independently, and the results were expressed as the mean ± standard deviation (SD). p values between groups were determined by a twotailed paired Student's t tests. p < 0.05 was considered as "statistically significant."

| T. gondii growth requires activation of host TBK1/IRF3 signalling pathway
To assess the capability of T. gondii to activate host TBK1/IRF3 sig-  an inhibitor of TBK1, clearly suppressed the T. gondii-induced IRF3 nuclear import, indicating that this event is under control of TBK1 ( Figure 1D).
To investigate the role of TBK1/IRF3 signalling pathway on T. gondii infection and growth, T. gondii infection and replication rate were measured under microscopy. As shown in Figure 1E, IRF3 deficiency had no effect on T. gondii infection rate, but clearly reduced the T. gondii replication that number of tachyzoites in each parasitophorous vacuole (PV) was mostly one or two, even after 24 h infection. Control group with normal intracellular IRF3 level showed commonly 8 tachyzoites in each PV ( Figure 1E). These results suggest that T. gondii infection stimulate host IRF3 activation and translocalization through TBK1 activation and this event is indispensable for T. gondii growth in the host.

| PI3K/AKT signalling pathway is involved in T. gondii infection-induced IRF3 translocation but not phosphorylation
To investigate whether PI3K/AKT signalling is involved in T. gondii-  (Figure 2A). Surprisingly, T. gondii-induced production of ISGs was dramatically decreased in accordance with the increasing concentrations of PI3K/AKT inhibitors ( Figure 2B).
This observation suggests that PI3K/AKT signalling is not involved in the phosphorylation of TBK1/IRF3, but plays a role in IRF3 transcriptional activity.
It is well known that IRF3 is constitutively expressed and lo- Immunofluorescence images also confirmed that T. gondii infection induced IRF3 nuclear import was dramatically inhibited with PI3K/ AKT inhibitors that most of IRF3 was accumulated in the host cytoplasm even with the parasite infection ( Figure 2D).
These data suggested that, PI3K/AKT signalling pathway is involved in the transcriptional activation of IRF3 by promoting IRF3 nuclear translocation in response to T. gondii infection in ARPE-19 cells.

| FAF1 expression is reduced in the host by T. gondii infection
Fas-associated factor 1 (FAF1) is a Fas-binding pro-apoptotic protein that is a component of the death-inducing signalling complex in Fas-mediated apoptosis. 27 It has also been demonstrated that FAF1 is involved in the immune response by physically interacts with NF-κB p65 to prevent its translocation into the nucleus and downregulate the target genes. 28 Thus, the change of host FAF1 protein and mRNA levels after T. gondii stimulation were measured to investigate whether FAF1 is associated with the process of parasite infection.
Western blot analysis showed that FAF1 expression decreased in a MOI-dependent manner upon T. gondii tachyzoite infection, and, at MOI 10, host intracellular FAF1 level reached its most minimal level ( Figure 3A). To confirm that endogenous FAF1 synthesis was downregulated by T. gondii, FAF1 mRNA expression level was measured using RT-PCR ( Figure 3B) and quantified by qPCR ( Figure 3C).
Indeed, FAF1 mRNA level was dramatically diminished in accordance with the increasing MOI of T. gondii ( Figure 3B,C). Coincidently, the measurement of intracellular FAF1 level by fluorescent microscopic also supported our hypothesis that T. gondii infection lead to a MOIdependent downregulation of FAF1 expression in the host cell ( Figure 3D).

| T. gondii infection-induced downregulation of FAF1 is dependent on PI3K/AKT/FOXO1 pathway
To identify the control mechanism for the reduction of Consistently, confocal microscopy images for endogenous FAF1 also illustrated that siRNA-mediated FOXO1 gene silencing caused a dramatic reduction of FAF1 compared to the control siRNA-treated group ( Figure 4C), whereas FOXO1 overexpression indeed enhanced the host FAF1 levels ( Figure 4D).

| FOXO1-mediated FAF1 expression modulates IRF3 nuclear translocation, downstream gene expression and infected T. gondii growth
Next, we examined whether FAF1 is involved in IRF3 nuclear translocation induced by T. gondii. FOXO1 overexpression-dependent FAF1 upregulation had no effect on the phosphorylation of TBK1/IRF3 by T. gondii infection ( Figure 5A), but the IRF3 nuclear import was dramatically suppressed by the condition ( Figure 5B). Accordingly, the production levels of IRF3 downstream ISGs were also obviously inhibited, even in the presence of T. gondii infection ( Figure 5C).
Silencing FAF1 gene revealed opposite results that IRF3 nuclear import ( Figure 5D left) and downstream ISGs expression were enhanced compare to control groups ( Figure 5E). This observation was also con-  29,30 The previous study shown that during the test of the antiviral effects of more than 350 individual human ISGs, 25 genes were revealed to enhance certain virus infectivity. 31 These genes expression is highly dependent on activity of TBK1/IRF3 signalling.
Interferon regulatory factor 3 (IRF3) has been known as a transcription regulator of many cellular responses in many cell types that is known to be critical for innate immunity against viral infection.
Activation of IRF3 requires phosphorylation on specific serine residues by the kinase TANK-binding kinase 1 (TBK1), which leads to the translocation of IRF3 into the nucleus. Although TBK1-IRF3 signal axis had its reputation for the host defence mechanism, it also has been reported that the activation of host IRF3 is also indispensable for the replication of the protozoan parasite, T. gondii. 13,14 This Parasite growth was also measured by RT-PCR using the SAG1 primer, and confocal microscope with T. gondii tachyzoites GFP-RH. Similar results were obtained in three independent experiments. **, p < 0.01; ***, p < 0.001, as compared to the control group requirement is also observed and confirmed in our T. gondii infection model that the TBK1 inactivation by specific inhibitor Amlexanox or IRF3 depletion by gene silencing dramatically restrained the parasite proliferation. These observations suggest that the activation of host TBK1/IRF3 signalling pathway is also essential for development of ocular toxoplasmosis.
Interestingly, T. gondii has been known for taking advantage of specific host signalling pathways, such as PI3K/AKT pathway, for changing host environment and for supporting its growth. More than a decade ago, there was evidence that T. gondii exploits host Gi-dependent PI3K/AKT signalling to maintain host cell survival and parasite persistence. 15 Toxoplasma gondii-mediated EGFR-dependent AKT activation also has been linked to inhibition of autophagy of T. gondii-infected cells and raised the possibility that AKT signalling as a pathway critical for the pathogen survival. 32 Toxoplasma gondii also has evolved a unique mechanism to suppress the host ROS generation by reducing NOX4, which expression is regulated by PI3K/ AKT signalling. 16 13 We also provide the supporting results that silencing of ISG56 in the host resulted in defect of T. gondii proliferation, significantly ( Figure S2), which means that it is also indispensable for T. gondii in ARPE-19 cells. These findings suggest that T. gondii has more complicated usage of host signal modifiers and further studies would be necessary to decode the antigen or host stimulating factors which would determine the fate of host and parasite.
Collectively, our results show that PI3K/AKT pathway is an indispensable component for controlling of host IRF3 nuclear import on response to T. gondii infection, and FAF1 is an essential element for connection and efficient activation the host TBK1-IRF3 signal axis, which contribute to the survival and growth of T. gondii ( Figure 6). These findings suggest that PI3K/AKT and FAF1 could be useful therapeutic targets to manipulate TBK1-IRF3 relied immune responses and to improve parasite control against T. gondii infection.

ACK N OWLED G EM ENTS
We would like to give our sincere appreciation to our colleague for their helpful suggestions on this article. We thank Dr. Young Joo Jeon (Chungnam National University, Korea) for her kindly provide p3x-FLAG-CMV-10-ISG15 plasmid DNA.

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest were disclosed.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.