Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1β) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.


Introduction
Several cellular enzymes including NADPH oxidase (Nox), nitric oxide synthase (NOS), cytochrome P450 reductase, myeloperoxidase (MPO), and xanthine oxidase are involved in generation of superoxide radicals in different cell types [1,2]. Earlier studies identified reactive oxygen species (ROS)-generating enzymes in several cell types of the human airway as a critical component of local host defense [1,3]. ROS produced by Nox enzymes can lead to increased phosphorylation of NF-κB [4] and can induce expression of pro-inflammatory mediators such as TNF-α, MCP-1 [5,6], and IL-6 [7]. Moreover, ROS, when produced in excess by leukocytes, can exert cytotoxicity and lead to increased tissue damage, especially in the lung tissue [8][9][10]. ) production by alveolar phagocytic cells obtained from influenza A virus (H2N2)-infected mice [11]. Imai et al. established an increase in ROS levels in human peripheral blood mononuclear cells (PBMC) treated with inactivated H1N1 and H5N1 influenza A virus subtypes [9]. In addition, other studies addressing the role of ROS generating enzymes on disease severity caused by influenza A virus found increased ROS levels in response to infection [8,12,13]. Furthermore, in mouse model of influenza infection, studies focused on suppression of ROS using ROS scavenger compounds showed improvement in influenza A virus-induced disease severity [8,11,12,[14][15][16]. In these studies, different types of ROS-inhibitors such as pyran-polymer conjugated super oxide dismutase (SOD), NG-monomethyl-l-arginine, ebselen, apocyanin, Glycyrrhizin, and recombinant human catalase were evaluated.
Quinazolin derivatives, a class of N-containing heterocyclic compounds, have shown an array of biopharmaceutical activities including anti-oxidant [17], anti-inflammatory [18], antitumor [19,20], and anti-bacterial [21,22] properties, in a variety of experimental models. In the present study, we anticipated that these anti-oxidant and anti-inflammatory characteristics would be demonstrated by TG6-44, a thioxo-dihydroquinazolin-one derivative [23], in a model of influenza infection. Notably, in our earlier studies, TG6-44 (Compound 1c) was shown to inhibit MPO-specific ROS generation (85% inhibition with an IC 50 of 0.8 ± 0.1 μM) in a neutrophil membrane cell-free system in a NADPH-dependent L-012 chemiluminescence assay [23].
In the current study, we evaluated TG6-44 for ROS inhibition in an in vitro model of influenza A virus infection. We observed increased ROS levels in lung monocytic cells (THP-1) infected with influenza A (A/X31) virus accompanied by an increase in inflammatory mediators and virus-induced cell death, all of which were suppressed by treatment with TG6-44. Many of these effects were also observed in freshly isolated PBMC. These results demonstrate that TG6-44 has anti-inflammatory activity in influenza-virus infected cells in vitro, suggesting that this compound, or similar quinazoline derivatives, may prove beneficial as adjunct therapeutics during influenza A virus infection.

Flow cytometry
Apoptosis was detected using Annexin V apoptosis detection kit FITC (e-Bioscience; catalog number 88-8005-72) per manufacturer's instructions. Briefly, after harvesting THP-1 and PBMC at the indicated times, cells were centrifuged and washed once with cold PBS followed by another wash with 1X cold Annexin V Binding Buffer. To the cells (10 6 cells/100μl), 5μL of fluorochrome-conjugated Annexin V was added and incubated in the dark for 15 min. Cells were washed once with 1X Annexin V Binding Buffer and re-suspended in 300μL of 1X Annexin V Binding Buffer containing 5μL Propidium iodide (eBioscience). Percent apoptosis in PBMC subsets (T cells, B cells, dendritic cells, and monocytes) was determined by staining with antibodies (BD Bioscience) specific for immune cell surface markers: HLA-DR-allophycocyanin-Cy7 (Catalog number 335796), CDllc-APC (Catalog number 340544), CD19-PerCP (Catalog number 340421), CD14-Alexa Fluor 700 (Catalog number 557923), CD3-PE-Cy7 (Catalog number 563423). Samples were analyzed using an LSRII flow cytometer (BD Bioscience).

PCR array
Total RNA was extracted from infected and control samples using TRIzol (Invitrogen) as described by the manufacturer. Total RNA (200ng) was reverse transcribed by the addition of 5X reverse transcriptase buffer (Roche), oligo (dT) primer (Invitrogen), dNTP (Invitrogen), random hexamers (Invitrogen), reverse transcriptase (Roche) and water to the RNA in a 20μL volume reaction. Thermocycler conditions for cDNA synthesis were 48˚C for one hour and 85˚C for 10 minutes. Real-time PCR was carried out using SYBR Green PCR Master Mix (Invitrogen) per manufacturer's instructions. Twenty-five μL of the SYBR Green PCR Master Mix/cDNA was added to each well of a 96-well PCR array plate (SABioscience, PAHS-012-A-12, RT 2 Profiler PCR Array Human Apoptosis). Thermocycler conditions were 1 cycle at 95˚C for 10 min, 40 cycles at 95˚C for 30 sec and 60˚C for 1 min and 1 cycle at 95˚C for 1 min, 55˚C for 30 sec and 95˚C for 30 sec using Stratagene MX3005P thermocycler (Agilent Technologies).

Confocal immunofluorescence
Following treatment, cells were pelleted, washed with cold PBS, fixed with 4% v/v paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS and blocked with 1% BSA and 5% normal goat serum in PBS. Cells were stained with mouse anti-Influenza A-virus-nucleoprotein (1:2500) overnight, followed by goat anti-mouse Alexa Fluor Flour 488 (1:5000, Molecular Probes) for 2h. Cells were mounted on slides with Prolong Antifade Mounting Media (Molecular Probes) containing DAPI to visualize nuclei. Images were captured using a LSM 710 inverted confocal microscope (Zeiss, Oberkochen, Germany) equipped with a x100 numerical aperture oil immersion objective. Images were processed using identical parameters and assembled into final figures using Abode Photoshop CS3 (Adobe Inc).
For calculating influenza nucleoprotein (NP)-positive cells, five independent 20x-image fields from each condition were evaluated. The total numbers of NP-positive cells was divided by the total numbers of DAPI-positive cells in each field to estimate the percent NP-positive cells (n = >500). For calculating cells expressing NP in the nucleus or cytoplasm, NP-positive cells (n = 50) were evaluated for nuclear or cytoplasmic expression of NP. Cells expressing NP in the cytoplasm or nucleus was divided by the total NP-positive cells counted in each field to estimate the percent expression in nucleus or in the cytoplasm.

Western blot
Cells washed with cold 1X PBS were lysed using 1X RIPA lysis buffer (Cell Signaling) containing a cocktail of protease inhibitors (Roche Diagnostics). Equal amounts of protein were loaded on to a 4-12% Bis-Tris Criterion XT precast gel (Biorad). Primary antibodies for Bcl-2, phosphorylated Bcl-2 (ser70) (Cell Signaling, Catalog number 2871), and Caspase3 (Cell Signaling, Catalog number 9661S), were all used at a dilution of 1:1000. Anti-rabbit secondary antibody (Cell Signaling, Catalog number 7074S) was used at 1:5000. Influenza A-virus-NP antibody was used at a dilution of 1:5000 and β-actin (Sigma, Catalog number A2228) antibody was used at 1:10000. Antibody dilution buffer and blocking buffer was 5% BSA in 1X PBS-0.1% Tween. Images were acquired with Kodak Image Station 400MM Pro (Molecular Imaging Systems) using Pico luminescence HRP detection kit (Thermo Scientific).

ROS assay
Cells washed in room temperature HBSS with calcium and magnesium (Gibco) were added to 96-well plates at 2x10 4 cells/150μL and allowed to equilibrate for 20 min at 37˚C. After the incubation period, PMA (50μM), diphenyleneidonium chloride (DPI) (10μM) (Sigma), SOD (5 Units) (MP Biomedicals), and/or TG6-44 (50 μM) were added to infected and control cells. L-012 (Wako) was added at a concentration of 50μM followed by 0.3 units of horseradish peroxidase (Sigma). Plates were immediately read using a BioTek plate reader set for continuous reading every minute for 80 min at 37˚C.

Bio-Plex
Following the manufacturer's protocol, a customized panel of 8 inflammatory cytokines and chemokines (IL-6, IL-8, INF-γ, IP-10, MCP-1, MIP-1β, RANTES and TNF-α) was measured using a Bio-Plex suspension array system (Bio-Rad). Briefly, cell culture supernatants and assay standards were added to a 96-well filter plate, followed by anti-cytokine-coupled beads, biotinylated bead detection antibodies, and PE-conjugated streptavidin. The plate was read using a Bio-Plex suspension array system and data were analyzed using Bio-Plex Manager 4.0 software (Bio-Rad) to calculate protein concentrations (pg/mL) based on standard curves.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software). A Student's t test was used to analyze differences in measures of ROS, inflammatory mediators, virus titer, and cell death. The data are presented as mean ± SEM. All differences were considered statistically significant when the p-value was �0.05.

TG6-44, a small molecule inhibitor of ROS, suppresses virus-induced ROS in A/X31-infected THP-1 cells and PBMC
Previous demonstration of the impact of ROS inhibition on influenza infection [8,11,12,14,15] prompted us to investigate the effects of TG6-44, a small molecule ROS inhibitor, on virusinduced ROS. TG6-44 is a quinazolin derivative developed in our laboratory by a HTS approach for small molecule inhibitors of ROS [23]. Previous studies demonstrated inhibition of MPOmediated ROS generation by TG6-44 (85% inhibition with an IC 50 of 0.8 ± 0.1 μM) in a neutrophil membrane cell-free system in a NADPH-dependent L-012 chemiluminescence assay [23]. In our current studies, in response to influenza A virus (A/X31) infection, THP-1 cells exhibited an increase in ROS production as early as 3 h p.i. (Fig 1, left panel). This increase in ROS production caused by influenza A virus infection has also been observed with a different subtype of influenza A virus and cell type [9]. As a positive control, ROS production by THP-1 cells was markedly stimulated by treatment with PMA, an activator of protein kinase C that phosphorylates and activates p47 phox of Nox2 in neutrophils and monocytes [27,28]. ROS level decreased significantly in A/X31-infected cells treated with TG6-44 (50 μM). As expected, ROS levels decreased significantly in infected cells treated with DPI, an inhibitor of flavoenzymes, or by SOD, an enzyme that catalyzes O 2 into oxygen and hydrogen peroxide [29,30]. Induction of ROS by virus infection, and its reduction by TG6-44, as well as DPI and SOD treatments, was also apparent at 6 h (Fig 1, mid i. in A/X31-infected human PBMC (Fig 1, right panel).

TG6-44 treatment decreases virus-induced inflammatory mediators in influenza A virus-infected cells
Since ROS is expressed in THP-1 cells in response to influenza virus infection (Fig 1) and, more importantly, is associated with enhanced inflammation and influenza A virus-induced disease severity [8,11,14],

TG6-44 treatment decreases X31-infectivity in THP-1 cells
Previous studies using ROS inhibitors to suppress influenza A virus-induced ROS in a mouse model showed suppression of lung inflammation as well as virus titer in the lung tissue [12,31]. Having found in this study that TG6-44 treatment suppressed influenza A virus-induced ROS and inflammatory mediators in THP-1 cells and PBMC (Figs 1, 2 and S2), we next examined its effects on virus infectivity in THP-1 cells. As a measure of virus infectivity, we  [32], NP was seen predominantly in the nucleus of A/X31-infected cells (Fig 3A, left panel). Addition of vehicle to the cells did not alter localization of NP in A/X31-infected cells. However, NP expression was restricted primarily to cytoplasm of A/X31-infected cells in the TG6-44-treated THP-1 cells (Fig 3A, lower panel and Fig  3C). At 18 h p.i, while majority of NP expression in A/X31 and vehicle-treated cells was relocalized to cytoplasm as expected [32], TG6-44 treated cells showed significantly higher percent cells with nuclear localization of NP (Fig 3A, right panel and Fig 3D). To determine the amount of NP in the cells, we performed the kinetic analysis of NP expression at 6, 12, and 24 h p.i. by western blot assay and found that NP expression in A/X31-infected cell cultures treated with TG6-44 (50 μM) at 6 and 12 h p.i. was relatively decreased when compared to A/ X31-infected cell cultures (Fig 4A). However, as shown in Fig 4B,

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were subjected to pre-incubation for 10 minutes prior to infecting THP-1 cells (Fig 4C). Likewise, reduction in NP expression was seen when TG6-44 treatment was delayed by 2 h after infection ( Fig 4C). However, the consequent virus titer by 24 h p.i did not change significantly with either pre-incubation of TG6-44 with A/X31 or with the delayed treatment regimen ( Fig  4D). Besides, analysis of IFN-β, an early anti-viral cytokine, in the virus infected cells failed to show any significant changes in IFN-β mRNA ( Fig 5A) as well as protein (Fig 5B) at 6 and 12 h time points. However, at 24 h p.i., both IFN-β mRNA and protein levels in the TG6-44 treated cells were significantly reduced when compared to vehicle control (Fig 5A and 5B).

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significant decrease in cell death both at 12 h and 24 h p.i. (Fig 6A and 6B). As shown in Figs  6B

TG6-44 treatment of A/X31-infected cells (THP-1 and PBMC) resulted in marked suppression of virus-induced inflammatory mediators and a significant decrease in virus-induced cell death (Figs 2, 6, S2 and S3). Previous studies of influenza A virus infection and virus-induced
cell death have demonstrated increase of pro-apoptotic as well as a reduction of anti-apoptotic molecules in several cell types [35][36][37][38]. To determine whether the decrease in virus-induced cell death in cultures treated with TG6-44 was associated with modifications in anti-apoptotic and pro-apoptotic molecules, we performed a gene profiling assay (PCR array) using naïve  [39][40][41] and pro-apoptotic (CASP10, CASP3, and CASP5) [42] molecules in A/X31-infected as well as TG6-44-treated conditions with levels detected in naïve THP-1 cells. Consistent with the Annexin V/PI data (Fig 6A and 6B), A/X31-infected cell cultures treated with TG6-44 (50 μM) contained higher mRNA levels of anti-apoptotic and lower mRNA levels of pro-apoptotic molecules (S1 Table). Western blot analysis for phopho-Bcl-2 and cleaved caspase-3, two well established indicators of cell survival and cell death [36,38], were used to confirm this finding. A/X31-infected cell cultures treated with TG6-44 had higher levels of phospho-Bcl-2 compared to A/X31-infected cell cultures ( Fig  6C, upper panel), particularly at 24 h p. i. Also, when compared to A/X31-infected cells, TG6-44-treated cells infected with A/X31 demonstrated lower levels of cleaved caspase-3 both at 12 h and 24 h p.i. (Fig 6C, middle panel).

Discussion
Expression of ROS and ROS-generating enzymes in different cell types is associated with boosting inflammation in tissues [5,6]. Studies addressing the role of ROS in influenza A virus infection have established a link between ROS-generating enzymes and severity of disease caused by the virus [9,12,43]. In these studies, investigators used either mice deficient in ROS-generating enzymes or treatment with ROS-inhibitors for suppressing virus-induced ROS. In our study, we demonstrated that ROS levels are enhanced by influenza A virus Previously, many studies have addressed the impact of ROS generation in different cell types and, more importantly, the consequence of its inhibition on health and disease (reviewed in [44,45]). In the context of influenza A virus infection and ROS, Oda et al. showed that influenza A virus (A/Kumamoto/Y5/67/H2N2)-infected mice treated with pyran polymer conjugated SOD (O 2 scavenger) demonstrated significantly higher survival and reduced pathology in the lung tissue [11]. Vlahos et al. used apocyanin, a pharmacological agent that scavenges ROS, to prevent Nox2 activity in influenza A virus-infected mice. In this study, apocynin treatment, by virtue of its ability to prevent the association of p47phox with Nox2 subunit, led to a reduction in airway cellularity, superoxide level, and lung viral titer [12]. Moreover, Yamatz et al. used ebselen, a glutathione peroxidase (family of enzymes that catalyze the reduction of H 2 O 2 into water and oxygen) mimetic, and found that ebselen treatment of influenza A virus (A/X-31/H3N2)-infected mice leads to reduction in lung mRNA levels of inflammatory mediators and proteases without any significant effects on either virus titer or virus-induced morbidity [8]. With reference to highly pathogenic influenza viruses and ROS, Perrone et al. used a NOS inhibitor (NG-monomethyl-l-arginine) and observed a delay in A/H1N1/1918 virusinduced morbidity and mortality [14]. Also, in an in vitro model of A/H5N1 influenza using infection, glycyrrhizin treated A549 cells showed a decrease in virus-induced ROS, inflammatory gene expression, as well as virus replication [15]. In the current experiments, we evaluated a quinazolin derivative (TG6-44) [23] for ROS inhibition in an in vitro model of influenza A virus infection. Based on our screening of TG6-44 for cytotoxicity on THP-1 cells (S1 Fig) and earlier investigations, by other groups, on different ROS-inhibitors [46,47], we used two different concentrations (10 and 50 μM) of TG6-44 for evaluating its effects on virus infectivity as well as virus-induced inflammatory mediators and virus-induced cell death. We used SOD and DPI treatment of cell cultures as positive controls for ROS inhibition and, as expected, ROS levels decreased significantly in infected cells treated with DPI, an inhibitor of flavoenzymes, or by SOD, an enzyme that catalyzes O 2 into oxygen and hydrogen peroxide [29,30].
Since SOD is a non-specific flavin-containing protein inhibitor, further studies using genetic approaches and specific NOX inhibitors are needed to define the specific sources of ROS modulated by MPO.
Since monocytes differentiate to phagocytes [48,49] and are permissive for productive infection by influenza A virus, we chose to address expression levels and consequences of ROS inhibition both in a monocytic cell line (THP-1) and PBMC. Reports from earlier work, including our own, suggest that influenza infection of PBMC largely impacts the monocyte subset leading to productive infection and secretion of inflammatory mediators [33,34]. Our current experiments demonstrate the anti-inflammatory effects of TG6-44 on host response to influenza A virus infection. Interestingly, addition of TG6-44 to THP-1 cell culture also resulted in decreased NP expression at early time points (Figs 3 and 4). To test whether the decrease in NP expression by TG6-44 treatment was the result of direct interference with virus infectivity by TG6-44, we pre-incubated TG6-44 with A/X31 prior to infection and then carried out infection of THP-1 cells. Also, to minimize the possibility of direct interference of TG6-44 on virus infection, we infected THP-1 cells with A/X31 and delayed TG6-44 treatment by 2 h. In both cases (Fig 4C), we found decreased levels of NP expression up to 12 h post infection. However, irrespective of the time point or sequence when TG6-44 was added to THP-1 cells, by 24 h p.i., there was no significant impact of TG6-44 treatment on virus titer. Also, analysis of cell cultures for IFN-β, a key anti-viral cytokine, showed no differences at 6 and 12 h p.i. However, by 24 h p.i., the levels of IFN-β were reduced in A/X31-infected cell cultures treated with TG6-44. Since NP expression was low irrespective of whether TG6-44 treatment was done along with or after virus infection but yet there was no major effect on key antiviral cytokine (IFN-β), it is possible that TG6-44 in some way interferes with virus entry and may not cause any alteration in anti-viral state in the cells. Moreover, the negative impact on IFN-β level by 24 h p.i. could be due to the suppressive effect of ROS inhibition on virusinduced cytokines (Fig 2). Notably, in vivo studies using Nox2-deficient mice [12,43] as well as studies using pharmacological agents known to suppress ROS [12,31] demonstrated reduction in lung virus titer. However, in the current study, TG6-44 mediated targeting of virusinduced ROS and inflammatory mediators does not seem to negatively impact the virus infectivity in in vitro model of influenza A virus infection.
Both influenza A virus infection as well as ROS production predisposes cells toward cell death [33,50]. We found that TG6-44 treatment led to a decrease in A/X31-induced cell death in THP-1 cells and PBMC. Interestingly, monocytes, a key cell type previously shown to be susceptible for influenza virus infection [34], were less susceptible to cell death in presence of TG6-44. In fact, in response to TG6-44 treatment, the decrease in virus-induced cell death in 12 and 24 h cultures may have contributed toward the observed increase in IFN-γ, MCP-1, and IP-10. Reduction in cell death may be attributed to decreased level of virus-induced ROS in the presence of TG6-44 since higher levels of ROS are known to enhance cell apoptosis [50]. It is also possible that TG6-44 interferes with virus infectivity as suggested by our experiments where NP expression was decreased irrespective of time and sequence when TG6-44 was added. Consequently, the reduction in virus infectivity could have contributed towards decrease in cell death. Yet another, although unlikely, possibility is that TG6-44 binds to virus thereby decreasing infectivity at early time points.
The link between up-regulation of ROS and influenza A virus induced pathology [9,43] raises the possibility and potential for evaluation of specific inhibitors that can suppress lung inflammation. However, experimental data in support of therapeutic role for ROS scavengers and antioxidants in the context of influenza virus infection and lung inflammation are necessary to support such a possibility. Further studies directly comparing the effects of TG6-44 treatment with ROS scavengers and antioxidants could support a therapeutic role for scavengers on suppression of influenza virus-induced overt cellular responses. Studies addressing the anti-tumor potency of quinazolin derivatives have established anti-proliferative and antiangiogenic properties by their ability to target epidermal growth factor receptor and platelet derived growth factor receptor [17]. Our earlier studies with TG6-44 showed ROS inhibition in MPO-specific manner [23] highlighting the possibility that modulation of MPO in cells could alter the course of host response to influenza infection. In fact, Sugamata et al. established a role for phagocyte-derived MPO in early phase of influenza infection in mice [51]. Notably, MPO is involved in multiple pathways in different cell types and its role is not limited only to regulation of ROS production. For example, MPO treatment is shown to activate ERK/ Akt pathway, p38 MAPK activation and enhanced nuclear translocation of NF-κB [52,53]. In our studies, although treatment with TG6-44 was effective in suppressing virus infectivity and virus-induced inflammation in our in vitro cell culture model, the specific signaling pathways leading to ROS inhibition and other observed effects are not clear. While both validation of TG6-44 treatment on MPO-mediated ROS inhibition as well its effect on the course of influenza infection in an in vivo model remain as the immediate goal of our investigations, it is however evident from our current findings that the strategy of MPO inhibition using quinazolin derivatives such as TG6-44 may be helpful in diminishing immune mediated injury seen during influenza A virus infections. This may be particularly applicable to situations, including that with highly pathogenic influenza virus infections such as avian H5N1, where antivirals are effective in limiting the virus loads but immune injury caused by overt host response necessitates adjunct therapeutic modalities. It remains to be seen whether the anti-infective and antiinflammatory properties of TG6-44 allow it to be used alone or in combination with currently available antivirals to effectively suppress the disease severity caused by influenza A virus infection of varying subtypes in in vitro and in in vivo models.

Disclaimer
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry.