extfieldsofstudy: Biology Medicine

text:
Bax Inhibitor-1 Acts as an Anti-Influenza Factor by Inhibiting ROS Mediated Cell Death and Augmenting Heme-Oxygenase 1 Expression in Influenza Virus Infected Cells

Influenza virus remains a major health concern worldwide, and there have been continuous efforts to develop effective antivirals despite the use of annual vaccination programs. The purpose of this study was to determine the anti-influenza activity of Bax inhibitor-1 (BI-1). Madin-Darby Canine Kidney (MDCK) cells expressing wild type BI-1 and a non-functional BI-1 mutant, BI-1 ∆C (with the C-terminal 14 amino acids deleted) were prepared and infected with A/PR/8/34 influenza virus. BI-1 overexpression led to the suppression of virus-induced cell death and virus production compared to control Mock or BI-1 ∆C overexpression. In contrast to BI-1 ∆C-overexpressing cells, BI-1-overexpressing cells exhibited markedly reduced virus-induced expression of several viral genes, accompanied by a substantial decrease in ROS production. We found that treatment with a ROS scavenging agent, N-acetyl cysteine (NAC), led to a dramatic decrease in virus production and viral gene expression in control MDCK and BI-1 ∆C-overexpressing cells. In contrast, NAC treatment resulted in the slight additional suppression of virus production and viral gene expression in BI-1-overexpressing cells but was statistically significant. Moreover, the expression of heme oxygenase-1 (HO-1) was also significantly increased following virus infection in BI-1-overexpressing cells compared to control cells. Taken together, our data suggest that BI-1 may act as an anti-influenza protein through the suppression of ROS mediated cell death and upregulation of HO-1 expression in influenza virus infected MDCK cells.

Introduction
Influenza viruses, which are enveloped RNA viruses belonging to the family Orthomyxoviridae, are a significant cause of respiratory infections, causing millions of deaths annually and imposing significant economic losses [1]. Influenza virus infection is a serious health problem worldwide, resulting in morbidity and mortality in humans in the form of epidemics or pandemics [2]. Until now, annual immunization and antiviral drugs have represented the main approaches for dealing with influenza. Antiviral drugs have been proven to be effective as a preventive and therapeutic regime over the last several decades [3]. To date, three classes of anti-influenza virus drugs are available for influenza management with different modes of action: the M2 channel blockers such as amantadine and rimantadine, neuraminidase (NA) inhibitors (e.g., oseltamivir) [4], and RNA polymerase inhibitors (e.g., ribavirin) [5]. All of these drugs are effective against virus infection if administered quickly, but concerns have been raised because of their adverse effects and the emergence of drug-resistant influenza A/H1N1 viruses [6,7]. The development of new therapeutic agents is urgently needed to treat influenza infections.
Several novel agents that may be effective against influenza virus are currently under development. Naturally available antiviral nutrients that are usually accessible are now being used as part of the diet to combat diseases, including influenza infection [8,9]. Reactive oxygen species (ROS), which are by-products of natural metabolic pathways, can dramatically increase during periods of cellular stress, inhibiting cellular protein function and promoting cell death. Influenza virus replication in infected cells is influenced by the cellular redox status [10,11]. The inhibition of virus-induced ROS formation using different strategies, including the use of the antioxidant N-acetyl-L-cysteine (NAC), has been shown to inhibit influenza A virus replication [12][13][14]. Identifying cellular molecules that have activities against apoptosis and ROS will be useful to counteract influenza virus infections in cells.
Characterization of the molecular mechanisms of novel antiviral proteins is also needed to combat influenza infections, as it will aid in developing new therapeutic strategies based on the identified mechanisms. Several identified novel small molecules that have inhibited Bax [15,16] and both Bax and Bak to protect cells from apoptotic cell death [17,18]. Bax inhibitor-1 (BI-1) is a membrane protein containing several transmembrane domains, and it has been reported to be an anti-apoptotic protein that has a protective function against ER stress [14,19,20]. BI-1 is widely conserved in both animal and plant species. BI-1 has been reported to regulate ROS production in the ER by modifying heme oxygenase-1 (HO-1) expression [14,21]. Heme oxygenase-1 is an oxygenase enzyme that blocks ROS activity and is expressed to counteract ROS accumulation, thereby promoting cell survival [22][23][24][25]. HO-1 has also been reported to act as an antiviral factor against porcine reproductive and respiratory syndrome virus infection [26] and to be induced by influenza virus infection [27]. Previous studies reported that a C-terminal mutant of BI-1 (BI-1∆C) relieved the effect of BI-1 overexpression regarding cell growth in transgenic mice and acted as BI-1 dominant negative mutant [19,28] and enhanced the survival and neural differentiation of embryonic stem cells by differential regulation of ROS production [29]. Thus, in this study, we determined whether BI-1 overexpression would promote cell survival against viral infection by increasing the production of antioxidant enzymes and by destabilizing the complex responsible for ROS production, which will be helpful for the further development of novel antiviral therapeutic strategies.

Overexpression of Bax Inhibitor-1 (BI-1) Relieves Virus Induced Cell Death in Madin-Darby Canine Kidney (MDCK) Cells
After preparing Madin-Darby Canine Kidney (MDCK) cells expressing the lentiviral construct containing wild type Bax inhibitor-1 (BI-1) or the non-functional deletion mutant form (BI-1 ∆C; deletion of 9 amino acids (EKDKKKEKK) from the C-terminal region of BI-1) ( Figure 1A) [19,29], we confirmed the endogenous or exogenous expression of BI-1 from the control, wild type BI-1, and non-functional BI-1 ∆C overexpressing MDCK cells by RT-PCR analysis using endogenous or exogenous specific primer set and overexpression was also confirmed by western blot analysis ( Figure 1B,C). Then, the BI-1 or BI-1 ∆C-overexpressing MDCK cells were used in an antiviral experiment against influenza virus A/PR/8/34, as illustrated in Figure 1D. First, the anti-influenza activity of BI-1 overexpressing cells was determined using cell viability assays after influenza virus administration, revealing that the overexpression of BI-1 in MDCK cells led to the significant suppression of virus-induced cell death compared to that in mock or BI-1 ∆C-overexpressing cells (Figure 2A). Flow cytometric analysis of the apoptotic sub G0/G1 populations also confirmed the significant anti-influenza virus activity of BI-1 ( Figure 2B), which supported our previous report that C-terminal amino acid deletion of BI-1 acted in a dominant negative fashion [19,29]. significant anti-influenza virus activity of BI-1 ( Figure 2B), which supported our previous report that C-terminal amino acid deletion of BI-1 acted in a dominant negative fashion [19,29]. BI-1 or BI-1 ΔC was inserted into the lentiviral pEF vector. The pEF lentivirus containing wild type BI-1 or the non-functional mutant BI-1 ΔC was produced and used to infect MDCK cells. (B) Illustration of specific primer sets which was used to analyze endogenous or exogenous expression of BI-1. (C) The endogenous or exogenous expression of BI-1 was confirmed by RT-PCR analysis in indicated cells; and the expression of HA was confirmed by western blot analysis in indicated cells. The expression of GAPDH and Actin was used as loading control. (D) The illustration of whole experimental protocol used in this study. (Abbreviations: chimeric Rous sarcoma virus (RSV) sequence, Rev response elements (RRE), central polypurine tract (cPPT), elongation factor 1 alpha (EF-1α) promoter region, a copGFP (copepod GFP) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), cells growth media (CGM), virus growth media (VGM), and hemagglutinin assay (HA assay)).

Overexpression of BI-1 Inhibits Viral Replication and Viral Gene Expression in MDCK Cells
To further analyze the antiviral activity of BI-1 on influenza virus production, we conducted a hemagglutination assay [30] using red blood cells (RBCs). We determined whether overexpression of BI-1 would interfere with viral adsorption to RBCs ( Figure 3A). A virus yield reduction HA assay with the media produced in the culture of the virus-infected BI-1or BI-1 ∆C-overexpressing MDCK cells showed that BI-1 overexpression, but not BI-1 ∆C overexpression, led to a substantial reduction in virus yield compared to the control. More than a four-fold reduction in virus production was observed in BI-1-overexpressing cells compared to that in the mock control or BI-1 ∆C-overexpressing cells.

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RT-PCR analysis of hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) gene expression revealed that the expression of these viral genes was significantly suppressed with BI-1 overexpression but not mock control and BI-1 ∆C overexpression ( Figure 3B,C). significantly suppressed with BI-1 overexpression but not mock control and BI-1 ∆C overexpression ( Figure 3B,C).

BI-1 Overexpression Suppresses ROS-Mediated Influenza Virus Infection in MDCK Cells
As influenza virus infection modulates ROS production in infected cells [12,13], we evaluated whether BI-1 overexpression provided protection against virus-induced ROS production. Virus-induced ROS production was significantly suppressed by BI-1 overexpression, but not by BI-1 ∆C overexpression ( Figure 4A). These observations were confirmed by flow cytometric analysis of the cells stained with Redox Red sensor CC-1 ( Figure 4B). In the virus-infected mock control and BI-1 ∆C-overexpressing cells, ROS production obviously increased following virus infection, but the virus-induced ROS production was significantly suppressed in the BI-1-overexpressing MDCK cells. These data indicated that BI-1 would act as anti-influenza virus factor by protecting cells against virus-induced ROS production and apoptosis.

BI-1 Overexpression Suppresses ROS-Mediated Influenza Virus Infection in MDCK Cells
As influenza virus infection modulates ROS production in infected cells [12,13], we evaluated whether BI-1 overexpression provided protection against virus-induced ROS production. Virus-induced ROS production was significantly suppressed by BI-1 overexpression, but not by BI-1 ∆C overexpression ( Figure 4A). These observations were confirmed by flow cytometric analysis of the cells stained with Redox Red sensor CC-1 ( Figure 4B). In the virus-infected mock control and BI-1 ∆C-overexpressing cells, ROS production obviously increased following virus infection, but the virus-induced ROS production was significantly suppressed in the BI-1-overexpressing MDCK cells.
These data indicated that BI-1 would act as anti-influenza virus factor by protecting cells against virus-induced ROS production and apoptosis.

BI-1 May Independently Act as an Antiviral Protein through the Suppression of ROS Production by Activating the Expression of HO-1 with Additional Effect of the ROS Scavenging Agent NAC
Next, we treated the virus-infected cells with N-acetyl cysteine (NAC) to investigate the effect of ROS on the antiviral function of BI-1. NAC treatment led to the significant suppression of virus-induced ROS production in the mock control and BI-1 ∆C-overexpressing MDCK cells ( Figure  5A). The additional suppression of ROS production was also observed by BI-1 overexpressing cells following NAC treatment. These observations were then confirmed by flow cytometric analysis of the intracellular ROS production ( Figure 5B). In the virus-infected mock MDCK and BI-1 ∆C-overexpressing cells, ROS production was significantly increased about two-fold compared to the uninfected cells, while BI-1 overexpression led to impede the suppression of ROS production. The treatment of NAC in virus treated cells led to a significant reduction in ROS production in mock control and BI-1 ∆C-overexpressing MDCK cells, while BI-1 overexpressing MDCK cells became like untreated cells after 12 h ( Figure 5A,B). After 24 h of virus treatment, ROS level was augmented dramatically in both control and BI-1 ∆C-overexpressing MDCK cells which could be diminished by NAC treatment (Figure 5A,B). In addition, BI-1 overexpressing MDCK cells showed a reduction in virus induced ROS level and the reduction was slightly more but statistically significant in NAC treatment group ( Figure 5A,B). The expression of viral genes, such as HA, NA, or NP, was also significantly inhibited by BI-1 overexpression, and the additional reduction of the viral genes expression was also detected by NAC treatment ( Figure 5C). Of note, the expression of heme oxygenase-1 (HO-1), which counteracts the accumulation of ROS in stressed cells, was significantly induced following BI-1 overexpression, but the enhanced HO-1 expression in BI-1-overexpressing cells was not suppressed following NAC treatment, suggesting that the BI-1-induced enhancement of HO-1 expression may play an important role in the BI-1 overexpression-induced antiviral activity, probably in a ROS-independent manner which need to be studied in detail. The cell viability and virus titer production results also showed that BI-1 may act as an antiviral protein through the   Figure 5A). The additional suppression of ROS production was also observed by BI-1 overexpressing cells following NAC treatment. These observations were then confirmed by flow cytometric analysis of the intracellular ROS production ( Figure 5B). In the virus-infected mock MDCK and BI-1 ∆C-overexpressing cells, ROS production was significantly increased about two-fold compared to the uninfected cells, while BI-1 overexpression led to impede the suppression of ROS production. The treatment of NAC in virus treated cells led to a significant reduction in ROS production in mock control and BI-1 ∆C-overexpressing MDCK cells, while BI-1 overexpressing MDCK cells became like untreated cells after 12 h ( Figure 5A,B). After 24 h of virus treatment, ROS level was augmented dramatically in both control and BI-1 ∆C-overexpressing MDCK cells which could be diminished by NAC treatment (Figure 5A,B). In addition, BI-1 overexpressing MDCK cells showed a reduction in virus induced ROS level and the reduction was slightly more but statistically significant in NAC treatment group ( Figure 5A,B). The expression of viral genes, such as HA, NA, or NP, was also significantly inhibited by BI-1 overexpression, and the additional reduction of the viral genes expression was also detected by NAC treatment ( Figure 5C). Of note, the expression of heme oxygenase-1 (HO-1), which counteracts the accumulation of ROS in stressed cells, was significantly induced following BI-1 overexpression, but the enhanced HO-1 expression in BI-1-overexpressing cells was not suppressed following NAC treatment, suggesting that the BI-1-induced enhancement of HO-1 expression may play an important role in the BI-1 overexpression-induced antiviral activity, probably in a ROS-independent manner which need to be studied in detail. The cell viability and virus titer production results also showed that BI-1 may act as an antiviral protein through the suppression of ROS production and ROS-independent regulation of HO-1 expression in addition to the effect of the ROS scavenging agent NAC ( Figure 5D,E).   The emergence of new strains influenza virus and influenza infections with high morbidity and mortality have been reported in humans, which have a pandemic threat. Although vaccines are used to prevent influenza infections, they take time to impart protection but the viruses may escape immunity induced by vaccination due to antigenic discrepancy between the vaccine strain and the circulating strain or due to antigenic drift [6,7]. Therefore, developing effective antiviral drugs is urgently needed to combat the disease. Influenza virus infection of susceptible host cell causes virus propagation, cell death, and viral gene expression. Notably, following influenza virus infection in the MDCK cell line, the increase in the titer of the influenza virus increases following ROS production [12]. In our previous studies, we found that influenza virus infection, replication, and propagation were prevented by several natural products [31,32]. In this study, we investigated the cell membrane protein BI-1 and found that anti-apoptotic protein BI-1 regulated the cellular responses to influenza virus infection. Importantly, the overexpression of BI-1 in MDCK cells The emergence of new strains influenza virus and influenza infections with high morbidity and mortality have been reported in humans, which have a pandemic threat. Although vaccines are used to prevent influenza infections, they take time to impart protection but the viruses may escape immunity induced by vaccination due to antigenic discrepancy between the vaccine strain and the circulating strain or due to antigenic drift [6,7]. Therefore, developing effective antiviral drugs is urgently needed to combat the disease. Influenza virus infection of susceptible host cell causes virus propagation, cell death, and viral gene expression. Notably, following influenza virus infection in the MDCK cell line, the increase in the titer of the influenza virus increases following ROS production [12]. In our previous studies, we found that influenza virus infection, replication, and propagation were prevented by several natural products [31,32]. In this study, we investigated the cell membrane protein BI-1 and found that anti-apoptotic protein BI-1 regulated the cellular responses to influenza virus infection. Importantly, the overexpression of BI-1 in MDCK cells impaired influenza virus production, propagation, and the synthesis of viral genes. BI-1 prevented virus adsorption into the MDCK cells by interfering with viral membrane fusion to the cells. BI-1 overexpression also suppressed NA activity to inhibit virion release, viral RNA synthesis by inhibiting NP, and virus titer production in MDCK cells. Previous studies have reported that BI-1 functions as an anti-apoptotic protein and ROS scavenger [14,20,22], recently in another study we had reported that in human embryonic kidney cells, BI-1 overexpression significantly suppressed ROS production, while BI-1∆C did not make any significant effect on ROS production [19], indicating that the C-terminal mutant plays in dominant negative fashion. In our studies, we found that the overexpression of BI-1 significantly reduced apoptotic cell death and ROS production in influenza-infected MDCK cells. Thus, BI-1 overexpression in MDCK cells suppressed ROS production and thereby inhibited viral gene and viral titer production, propagation, and cytopathogenicity ( Figure 6). Moreover, our study revealed that BI-1-overexpressing MDCK cells were more resistant to influenza virus infection and virus-induced cell death. The anti-oxidant molecule NAC is known to inhibit ROS production, viral replication, and virus-induced apoptosis in influenza-infected cells [33]. As BI-1 had shown a significant anti-influenza effect against A/PR/8/34 including substantial reduction of ROS production, in our studies we used NAC as ROS scavenger whether it had any notable effect in BI-1 overexpressing cell. In this study, in BI-1-overexpressing MDCK cells, NAC had an additional effect on reducing ROS and viral replication but it was not significant, although, in the mock control and BI-1 ∆C overexpressing cells, it had a significant effect on reducing ROS and inhibiting virus-induced cell death and viral replication and propagation. Moreover, in our studies, we had observed activated expression HO-1 in influenza infected BI-1 overexpressing cells. Although ROS production was significantly reduced but, the BI-1-induced increase in HO-1 expression was not suppressed by NAC treatment, possibly because of the enhanced expression of HO-1 protein in BI-1-overexpressing cells and because HO-1 may play an important role in the suppression of virus-induced ROS production, cell death, virion release from host cells, and the replication and propagation of the viral HA, NA, and NP genes.
BI-1 has been shown to regulate ROS accumulation during cellular stress [22,30,34,35], and here, we present evidence that influenza virus-induced ROS accumulation was suppressed following BI-1 overexpression. HO-1 is known to be activated during cellular stress and influence pro-oxidant signals such as ROS. Interestingly, in our study, we found that HO-1, which is a ROS scavenger, was strongly upregulated in BI-1-overexpressing cells. Thus, highly activated cellular HO-1 in BI-1-overexpressing MDCK cells reduced ROS accumulation following influenza infection. HO-1 may be important for the cytoprotective function of BI-1 as BI-1 overexpressing cells, which had a higher basal level of HO-1, had a survival advantage against influenza virus infection.
Taken together, our findings indicate that the overexpression of BI-1 in the MDCK cell line played a key role in the regulation of host cellular defense mechanisms against influenza virus infection. The overexpression of BI-1 slowed down the ROS-dependent propagation of influenza virus and virus-induced pathogenicity and induced increased expression of HO-1. Our study adds to previous efforts to identify small molecules for use against influenza infection, and our results regarding the anti-apoptotic protein BI-1 as an anti-influenza agent that can suppress ROS production will facilitate the development of novel mechanism-based therapeutics against influenza virus infection, but further studies are needed to elucidate the mechanisms involved. It is known that MDCK cells are the best choice to study the influenza virus infection due to rapid virus growth capability and great adaptation in serum free media [36]. BI-1 is naturally present in cells to protect cellular death. According to the result of amino acid sequence alignment, more than 97% of amino acid sequence was same in both human and canine BI-1 protein. In this study, our main purpose is to find the role of BI-1 in influenza virus induced cell death. Thus, we used our available human BI-1 lentivirus expressing construct [19]. Analysis of the amino acid sequences of BI-1 protein was performed using data obtained from Gen Bank (NCBI). The accession number of the protein sequence of BI-1 was NP_003208.1. Scan Prosite (http:www.expasy.org/tools/ scanprosite/PROSITE) and NCBI RPS-BLAST (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi) were used for conserved domain database screening. The lentiviral construct containing wild type Bax inhibitor-1 (BI-1) or the non-functional deletion mutant form (BI-1 ∆C; deletion of 9 amino acids (EKDKKKEKK) from the C-terminal region of BI-1) was prepared, and the lentiviral vector particles were purified from the supernatant of transiently transfected 293T cells. The cell supernatant was collected 72 h post-transfection, filtered through polyether sulfone membranes (0.45-µm pore size), and concentrated 120-fold by ultracentrifugation (50,000× g for 90 min at 4 • C). The pellet was resuspended in the cell culture medium and subsequently added to the cells. After 1 week of culture, the cells were centrifuged and resuspended in PBS and sorted with a FACSVantage™ flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The sorted MDCK cells were cultured in virus growth medium (MEM without FBS) supplemented with 10% bovine serum albumin, 100 U/mL penicillin/streptomycin, and 2 µg/mL trypsin TPCK.

Cell Viability and Cell Cycle Analysis
The mock MDCK, BI-1-overexpressing MDCK, and BI-1 ∆C-overexpressing MDCK cells were seeded in 96-well plates for the measurement of cell viability at a concentration of 2 × 10 4 cells per well. Twenty-four hours after seeding, when the cells were confluent, they were washed twice with PBS and infected with the virus at 100 TCID 50 and incubated at 37 • C for 2 h in a 5% CO 2 incubator. The virus-containing media were replaced with virus growth media 2 h after infection. MTT assays (Sigma-Aldrich, St. Louis, MI, USA) were performed 24 and 48 h after infection according to the manufacturer's instructions using a spectrophotometer (Bio-Rad, Hercules, CA, USA) at a wavelength of 490 nm to determine the total cell metabolic activity. For cell cycle analysis, mock, BI-1-overexpressing, and BI-1 ∆C-overexpressing MDCK cells were harvested in 24, 36, and 48 h time frame after virus infection by trypsin-EDTA (Gibco), and stained with propidium iodide (PI) (Sigma-Aldrich) following manufacturer protocol and analyzed by a Beckman Coulter FC-500 flow cytometer (Beckman Coulter, Fullerton, CA, USA) using Cell Quest 3.2 software. The percentage of the apoptotic sub-G0/G1 populations of BI-1-overexpressing MDCK, and BI-1 ∆C-overexpressing MDCK cells were counted and compared to the control.

Virus Yield Reduction Assay
The mock control cells, overexpressing MDCK-BI-1, and BI-1 ∆C-overexpressing MDCK cells were seeded in 6-well cell culture plates at a concentration of 2-3 × 10 5 cells per well. When the cells were confluent, the cell growth media were removed, and the cells were washed twice with PBS and infected with the virus at 1000 TCID 50 diluted in virus-containing media. The virus-containing media were replaced by virus growth media, and HA assays were performed 48 h after virus infection. Briefly, 25 µL of PBS was added to each well of the 96-well round bottom plates. Then, 25 µL of the cell supernatant containing the virus was diluted by two-fold serial dilution in each well, and 25 µL of PBS was added again to each well. Then, 50 µL of 1% chicken RBCs was added to each well followed by incubation for 1 h at room temperature to observe hemagglutination activity. HA titers were calculated as hemagglutination units/50 µL (HAU/50 µL).

Intracellular ROS Detection
The mock MDCK, MDCK-BI-1 overexpressing, and MDCK-BI-1 ∆C overexpressing cells were seeded at a concentration of 2-3 × 10 5 cells per well in 6-well cell culture plates. The media were removed when the cells were confluent, and the cells were washed twice with PBS and infected with the virus at 1000 TCID 50 in virus-containing media. Then, 12 h, 24 h and 48 h after virus infection, the cells were exposed to Redox Red sensor CC-1 (Molecular Probes, Inc., Eugene, OR, USA) for 30 min in the dark according to the manufacturer's instructions to determine ROS levels. The cells were then washed and trypsinized, and the fluorescence intensity was quantified and analyzed with a Beckman Coulter FC-500 flow cytometer (Beckman Coulter, Fullerton, CA, USA) using Cell Quest 3.2 software. At least 10,000 cells were analyzed from each sample. For the measurement of intracellular ROS levels, fluorescence-stained cells were imaged with an inverted fluorescence microscope. To determine ROS scavenging activity, 2 h after virus infection, the cells were treated with 10 mM NAC. Intracellular ROS levels were measured in control MDCK, BI-1-overexpressing MDCK, and BI-1 ∆C-overexpressing MDCK cells using Redox Red sensor CC-1. Fluorescence-stained cells were imaged with an inverted fluorescence microscope to determine the intensity of ROS production, and flow cytometry was performed for ROS quantification.

RT-PCR
Confluent mock MDCK, BI-1-overexpressing MDCK, and BI-1∆C-overexpressing MDCK cells grown in 6-well plates were infected with influenza A/PR/8/34 virus at 100 TCID50. The cells were scraped off 24 h after infection, and cell pellets were collected by centrifugation (1500× g for 5 min). Cell pellets were washed two times with PBS, and total cellular and viral RNAs were isolated using Easy Blue total RNA extraction kits (Intron Biotechnology). First-strand cDNA was synthesized from 3 µg of total RNA. PCR reactions were performed using primers for HA, (

Western Blot Analysis
The MDCK, over expressed MDCK-BI-1 and over expressed MDCK-BI-1∆C cells seeded in 60 mm dishes and infected with influenza A/PR/8/34 virus at 1000 TCID 50 , were washed 3 times in ice-cold PBS, scraped from the dishes, and collected in extraction buffer (1% TritonX-100, 100 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10% glycerol, 50 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). After the cells had been incubated on ice for 30 min, the lysates were centrifuged and protein containing cleared lysates was quantified using the Bradford Protein Assay Reagent (Bio-Rad). Equal amounts of protein were then separated on 10-12% SDS PAGE gels and electrophoretically transferred on to nitrocellulose membranes (0.2 mm). These membranes were blocked with 3-5% non-fat dry milk and Tris-buffered saline (TBS) and subsequently probed for HA and Actin in TBS containing 3% non-fat dry milk. Antibody-antigen complexes were detected using goat anti-mouse IgG or goat anti-rabbit IgG peroxidase conjugates, followed by use of an enhanced chemiluminescence (ECL) detection kit from Amersham (Buckinghamshire, UK).

Statistical Analysis
The results were expressed as the mean ± SE. Each value is the mean of at least three independent experiments in each group. The statistical significance of the differences between the two cell populations was determined using the two-tailed Student's t-test (Origin), and p values equal to or less than 0.05 and 0.01 were considered significant.


s2fieldsofstudy: Biology Medicine

text:
The TLR7 ligand R848 prevents mouse graft-versus-host disease and cooperates with anti-interleukin-27 antibody for maximal protection and regulatory T-cell upregulation

In spite of considerable therapeutic progress, acute graft-versus-host disease still limits allogeneic hematopoietic cell transplantation. We recently reported that mouse infection with nidovirus lactate dehydrogenase elevating virus impairs disease in non-conditioned B6D2F1 recipients of parental B6 spleen cells. As this virus activates TLR7, we tested a pharmacological TLR7 ligand, R848, in this model and observed complete survival if donor and recipients were treated before transplantation. Mixed lymphocyte culture performed 48 h after R848-treatment of normal mice demonstrated that both T-cell allo-responsiveness and antigen presentation by CD11b+ and CD8α+ dendritic cells were inhibited. These inhibitions were dependent on IFNAR-1 signaling. In the B6 to B6D2F1 transplantation model, R848 decelerated, but did not abrogate, donor T-cell implantation and activation. However, it decreased interferon-gamma, tumor necrosis factor-alpha and interleukin-27 while upregulating active transforming growth factor-beta 1 plasma levels. In addition, donor and recipient Foxp3+ regulatory T-cell numbers were increased in recipient mice and their elimination compromised disease prevention. R848 also strongly improved survival of lethally irradiated BALB/c recipients of B6 hematopoietic cells and this also correlated with an upregulation of CD4 and CD8 Foxp3+ regulatory T cells that could be further increased by inhibition of interleukin-27. The combination of anti-interleukin-27p28 mono -clonal antibody and R848 showed strong synergy in preventing disease in the B6 to B6D2F1 transplantation model when recipients were sublethally irradiated and this also correlated with upregulation of regulatory T cells. We conclude that R848 modulates multiple aspects of graft-versus-host disease and offers potential for safe allogeneic bone marrow transplantation that can be further optimized by inhibition of interleukin-27.

Introduction
Allogeneic hematopoietic cell transplantation (HCT) is an important therapeutic option for a wide range of malignant and non-malignant disorders, including acquired and genetic anomalies. [1][2][3] However, it remains plagued by bone marrow failure or graft-versus-host disease (GvHD), which develop in approximately 50% of allogeneic HCT recipients. 4 With a death rate in the range of 15%, 5 acute GvHD thus remains a major medical challenge, especially in older patients and in steroid-resistant individuals in whom mortality reaches 90%. 6 A hallmark of acute GvHD is the release of inflammatory cytokines and a pathogenic contribution has been demonstrated for tumor necrosis factor-alpha (TNFα), 7 interleukin(IL)-6, 8 IL-23 9 and more recently IL-27. 10,11 This last, a member of the IL-6 and IL-12 families, is a cytokine with multiple activities 12 that both promotes Th1 type immune reactions 13 and restrains excessive inflammation. 14 IL-27 is formed by noncovalently linked p28 (also called IL- 30) 15 and Epstein-Barr virus-induced gene 3 product (EBI3), which are structurally related to IL-12p35 and IL-12p40, respectively. 13   I n spite of considerable therapeutic progress, acute graft-versus-host disease still limits allogeneic hematopoietic cell transplantation. We recently reported that mouse infection with nidovirus lactate dehydrogenase elevating virus impairs disease in non-conditioned B6D2F1 recipients of parental B6 spleen cells. As this virus activates TLR7, we tested a pharmacological TLR7 ligand, R848, in this model and observed complete survival if donor and recipients were treated before transplantation. Mixed lymphocyte culture performed 48 h after R848-treatment of normal mice demonstrated that both T-cell allo-responsiveness and antigen presentation by CD11b + and CD8α + dendritic cells were inhibited. These inhibitions were dependent on IFNAR-1 signaling. In the B6 to B6D2F1 transplantation model, R848 decelerated, but did not abrogate, donor T-cell implantation and activation. However, it decreased interferon-gamma, tumor necrosis factor-alpha and interleukin-27 while upregulating active transforming growth factor-beta 1 plasma levels. In addition, donor and recipient Foxp3 + regulatory T-cell numbers were increased in recipient mice and their elimination compromised disease prevention. R848 also strongly improved survival of lethally irradiated BALB/c recipients of B6 hematopoietic cells and this also correlated with an upregulation of CD4 and CD8 Foxp3 + regulatory T cells that could be further increased by inhibition of interleukin-27. The combination of anti-interleukin-27p28 monoclonal antibody and R848 showed strong synergy in preventing disease in the B6 to B6D2F1 transplantation model when recipients were sublethally irradiated and this also correlated with upregulation of regulatory T cells. We conclude that R848 modulates multiple aspects of graft-versus-host disease and offers potential for safe allogeneic bone marrow transplantation that can be further optimized by inhibition of interleukin-27.

The TLR7 ligand R848 prevents mouse graft-versus-host disease and cooperates with anti-interleukin-27 antibody for maximal protection and regulatory T-cell upregulation
binds to a heterodimeric receptor, comprising WSX-1/IL-27Rα and gp130. This receptor signals through STAT1 and STAT3 and is expressed on hematopoietic stem cells, all lymphoid and myeloid cells, vascular endothelium and keratinocytes. 16 IL-27 is produced by macrophages, monocytes and dendritic cells (DC) and its receptor is expressed on memory CD4 and CD8 T cells. 13,17,18 In the context of GvHD, IL-27 inhibition confers protection by downregulating interferon-gamma (IFNγ) and upregulating Foxp3-expressing regulatory T cells (Treg) that represent a major control point of GvHD severity, as reported previously. [19][20][21] We recently made the surprising observation that lactate dehydrogenase elevating virus (LDV), a single-stranded positive-sense RNA enveloped mouse nidovirus, enhanced survival in the lethal form of acute GvHD induced by B6 spleen cell injection into non-conditioned B6D2F1 recipients. 22 In this model, which is mainly characterized by severe bone marrow failure 23 and liver destruction, LDV protection correlated with a transient impairment of antigen presentation by DC and with alterations in T-cell allo-responsiveness, both dependent on IFNAR-1 signaling, in agreement with the reported GvHD inhibition by type I interferons. 24 As LDV activates Toll-like receptor 7 (TLR7), 25 we considered the possibility that the TLR7 agonist resiquimod (R848), 26 an imidazoquinoline originally developed as an antiviral agent 27 and an immune adjuvant, 28 could replicate the protective effects of the virus. This idea is somewhat counterintuitive since R848 has adjuvant activity and promotes Th1 immune reactions. 29 However, prolonged exposure of mice to the compound was reported to induce a disruption of the lymphoid system resembling that induced by human immunodeficiency virus. 30 We here report that a transient treatment of mice with R848 just before allogeneic HCT conferred strong protection against GvHD which correlated with inhibition of inflammatory cytokine production, upregulation of transforming growth factor-beta (TGF-β) and Treg and could be further enhanced by anti-IL-27 monoclonal antibody (anti-IL-27) therapy.

Mice
Mice were bred under specific pathogen-free conditions at the animal facility of the Ludwig Cancer Research Brussels Branch under the direction of Guy Warnier (DVM). Experimental protocols and animal handling were approved by the ethical committee of the Medical Faculty of the Université de Louvain (accreditation n.: 2016/UCL/MD/010). IFN-α/βR -/-129/Sv mice (IFNAR-1 -/-) were a gift from Dr. M. Aguet. 31

Other detailed methods
All other methods are described in the Online Supplementary Methods.

R848 prevents lethal parent to F1 non-conditioned graftversus-host disease
The effect of R848 was first tested in non-conditioned (nc) GvHD to avoid the inflammatory cytokine burst induced by host irradiation. The agent was administered to recipient B6D2F1 and/or donor B6 mice 24 and 48 h before donor spleen cell injection. Data pooled from five experiments showed 100% mortality in control mice by day 25. R848 treatment of either recipient or donor resulted in survival rates of 40% and 60%, respectively, while combined treatment of both resulted in 100% long-term survival ( Figure 1A). Morbidity evaluated by weight loss was also totally suppressed after donor and recipient treatments ( Figure 1B). Timing of R848 administration was important since treatments given too early (6-5 days before transplantation) or too late (5-6 days after transplantation) were not protective ( Figure 1C). R848 treatment also minimized hepatocyte destruction but mononuclear cell infiltration was still present ( Figure 1D). This tissue protection was confirmed by suppression of serum amyloid A (SAA)1/2 and SAA3 mRNA expression in liver cells ( Figure 1E).
Finally, after 14 days, host spleen cell numbers had dropped from ±50x10 6 to ±2x10 6 in the control GvHD group but remained unchanged or even slightly increased after R848 treatment, demonstrating host spleen cell protection by R848. Spleen cell implantation by R848-treated B6 donors was low after 14 days, approximately 4x10 6 cells, but increased with time, resulting in a permanent chimerism reaching 20x10 6 B6 cells per spleen after 100 days ( Figure 1F).

R848 treatment inhibits production of IFNγ, TNFα and IL-27 but stimulates active TGF-β1 secretion in non-conditioned graft-versus-host disease
B6→ncB6D2F1 GvHD was characterized by high concentrations of plasma IFNγ and IL-27 that reached maxima at day 10 and returned to nearly undetectable levels by day 14. R848 treatment essentially abolished these cytokine peaks ( Figure 2A). RNA analysis confirmed upregulation of Ifng expression in spleen and liver of untreated transplanted mice and its complete silencing by R848 ( Figure 2B). A similar inhibition was seen for TNFα ( Figure 2A), a cytokine that also contributes to GvHD pathology. 34 Given the potential implication of TGF-β in the control of GvHD, 35,36 we also measured TGF-β1 and TGF-β3 by enzyme-linked immunosorbent assays selectively detecting the active forms of these cytokines and observed a strong upregulation of the former (Figure 2A), but not the latter (data not shown) after R848 treatment. As shown in Figure 2A, active TGF-β1 was upregulated from day 6 to day 14, but was no longer detectable at day 50 (data not shown).

R848 treatment before hematopoietic cell transplantation inhibits T-cell allo-responsiveness and major histocompatibility complex presentation by conventional dendritic cells through type I interferon signaling
Optimal prevention of B6→ncB6D2F1 GvHD required treatment of both donor and recipient with R848. Since host T cells are not reactive against the implanted parental cells, the action of R848 on the F1 partner is likely on antigen presentation. To assess the influence of R848 on this process and on T-cell responsiveness, normal B6D2F1 and B6 mice were injected with R848 or phosphate-buffered saline for two consecutive days before spleen cell collection. B6 spleen cells were then incubated with B6D2F1 irradiated adherent spleen cells as a source of antigen-presenting cells (APC). In control mice, this combination induced strong proliferation and IFNγ production. R848 treatment of either B6D2F1 stimulating or B6 responder mice inhibited proliferation and IFNγ production ( Figure 3A), indicating that 48 h of treatment with R848 of otherwise non-manipulated mice impaired both antigen presenting and responder cells.
TLR7 activation leads to production of type I interferons. 26 We, therefore, tested the implication of IFN in R848mediated T-cell suppression in allogeneic mixed lymphocyte cultures using spleen cells from 129/Sv (H-2D b ) or 129/Sv-IFNAR-1 -/mice as responder cells and B6D2F1 irradiated adherent spleen cells as APC. This experiment confirmed the inhibitory effect of R848 on the allogeneic responder cells in a different mouse strain and showed that, in 129/Sv-IFNAR-1 -/responder cells, proliferation and IFNγ production were not inhibited ( Figure 3B). R848-induced inhibition was not correlated with an increase in Foxp3 + Treg ( Figure 3C) and depletion of Treg with anti-CD25 PC61 antibody before R848 treatment did not prevent the inhibition of IFNγ production during the allogeneic mixed lymphocyte culture ( Figure 3D).
We previously showed that TLR7 viral stimulation transiently inhibits conventional DC (cDC), the only splenic cells able to activate an allogeneic T-cell response in vitro. 22 To evaluate the effect of R848 on allogeneic antigen presentation in the allogeneic mixed lymphocyte culture, CD11b + cDC, CD8a + cDC and pDC were purified from 129/Sv mice 48 h after R848 treatment and co-cultured with FVB (H-2 q ) haematologica | 2019; 104(2) CD4 T cells. CD11b + and CD8α + cDC were the main stimulating cells in the mixed lymphocyte culture and this capacity was impaired in mice treated with R848 ( Figure  3E). This suppression of APC was also dependent on IFNAR-1 ( Figure 3F). These data indicate that at the time of donor cell transfer, R848 administration inhibited both antigen stimulation by cDC and T-cell responsiveness in a type 1 interferon-dependent process. This impairment of T-cell allo-responsiveness induced by R848 after 48 h did not involve Treg.

R848 treatment impairs effector donor T cells and increases regulatory T cells in a non-conditioned graft-versus-host disease model
To evaluate the effect of R848 on responder T cells during GvHD, we first used CFSE-labeled CD5 B6 cells. Six days after transplantation, staining dropped considerably in B6 cells transplanted into B6D2F1 but not into B6 recipients. This loss of CFSE staining, although diminished, still occurred in R848-treated mice with GvHD, indicating that donor cell division was not completely suppressed by R848 ( Figure 4A). The slower expansion of donor T cells after R848 treatment was confirmed by the decrease in implanted donor CD4 and CD8 T cells 14 days after transplantation ( Figure 4B). However, ultimately R848 did not abrogate B6 donor cell engraftment as chimerism was still detected up to 100 days after transplantation (see Figure 1G). Donor CD8 T cells were also five times less abundant in R848-protected GvHD than in control ncGvHD. These effects were not the consequence of R848 toxicity for T cells since host CD4 T cells remained unchanged ( Figure 4B).
The persistence of donor T cells in the R848-protected mice raised the question of the activation state of these cells. We evaluated the proportion of naïve and memory cells with CD62L and CD44 labeling, respectively. The massive loss of CD62L expression by CD4 T cells observed in control ncGvHD was partially inhibited by R848 (67% in control mice, 3.9% in control GvHD and 26% in R848-protected GvHD) and the same trend was seen for CD8 T cells, indicating that donor T-cell activation was partially inhibited by R848 ( Figure 4C). In contrast, the upregulation of CD44 on CD4 T cells was not significantly modified by R848 (2.50-fold upregulation in control and 2.45 fold with R848, Figure 4C). With regards to the activation marker CD69, its expression was unchanged by R848 on CD8 T cells and even somewhat enhanced on CD4 T cells ( Figure  4D). In contrast to B6 donor cells, there was no significant change in CD44/CD62L T-cell proportions in control and R848 recipient groups. However, in control GvHD, as the B6D2F1 splenocytes were destroyed, their numbers dramatically dropped (Online Supplementary Figure S1A) whereas in the R848 group, the numbers of CD44 + and CD62L + T cells were significantly more important than in control GvHD but remained in the same order as in naïve mice. In

A B
addition, an increase was observed for CD69 + CD4 T cells in R848-protected mice as compared to control B6D2F1 mice (Online Supplementary Figure S1B). We next examined the influence of R848 treatment on Treg in the same experiments. First, we observed a 10-fold decrease in the proportion of donor and recipient Foxp3 + CD4 T cells 14 days after B6 spleen cell transplantation. In contrast, in R848-treated GvHD mice, recipient Foxp3 + CD4 T cells increased 4-fold while donor Treg returned to normal B6 levels ( Figure 4E).
Together, these data indicate that during ncGvHD R848 affected donor CD4 and CD8 T-cell implantation and activation but the inhibition was only partial and did not prevent the establishment of permanent chimerism. On the other hand, R848 prevented GvHD-induced loss of donor and recipient Treg and even increased the latter above normal levels.

Regulatory T cells contribute to R848-mediated prevention of non-conditioned graft-versus-host disease
The contribution of Treg to the protective effect of R848 was tested by depletion of Treg using anti-CD25 PC61 antibody treatment of B6 donors and B6D2F1 recipients. PC61 antibody was injected 4 days before R848 treatment and a second injection was administered 1 day after B6 spleen cell transplantation. As compared to donor CD4 T cells from R848-treated mice with GvHD, B6 CD4 T cells from the PC61-R848 GvHD group engrafted host spleen faster and their numbers were significantly higher at 14 and 20 days after B6 cell transfer and continued to expand up to day 50. PC61 antibody completely depleted their Foxp3 + Treg population which remained totally absent during the course of GvHD in contrast to that in the R848-treated mice, in which they expanded more than 6-fold from day 14 to day 50 ( Figure 5A). Regarding host Treg, in PC61-R848-treated haematologica | 2019; 104(2)  Figure S2A,B).
PC61 treatment of the R848 GvHD mice resulted in weight loss starting from day 17, a few days later than in control GvHD mice. The percentage weight loss was finally the same in both groups, suggesting a significant contribution of Treg in the prevention of ncGVHD morbidity by R848 ( Figure 5B). However, PC61 treatment only partially decreased the survival of R848-treated mice (70% versus 90%) ( Figure 5C). This trend was observed in two additional experiments.
In order to test whether Treg depletion affected the level of donor T-cell activation, we evaluated CD44 and CD69 expression levels 14 and 20 days after ncGVHD induction. When Treg were depleted in R848-treated mice, CD44 + and CD69 + B6 CD4 and CD8 T cells were significantly increased and CD69 levels even exceeded those of the control ncGVHD group. Compared to day 14 levels, the B6 CD69 + T-cell population tripled at day 20, indicating that an absence of Treg increased expansion of memory and activated donor T cells ( Figure 5D). However, Treg depletion by PC61 did not seem to influence early cytokine production since no significant differences in IFNγ, IL-27p28 and active TGF-β1 plasma concentrations were observed between R848-and PC61-R848-treated mice ( Figure 5E).
Together, the data suggest that Treg from donors and recipients contributed to R848-mediated GvHD prevention. However, despite the depleting treatment, a small population of host Treg remained present, which could explain why R848 protection was not completely abrogated and resulted in death of only 30% of PC61-R848-treated mice. As shown previously, R848 GvHD protection correlates with a strong drop in pro-inflammatory cytokines and this was still observed after Treg depletion, which could also explain why the protective effect of R848 was not completely suppressed by Treg depletion.

R848 cooperates with anti-interleukin-27p28 monoclonal antibody in regulatory T-cell upregulation and graft-versus-host disease prevention in conditioned models
B6 spleen cell transfer into ncB6D2F1 recipients is an optimal strategy for dissecting the mechanisms involved in immune-mediated hematopoietic cell destruction and can be used as a model of induced bone marrow failure 23 but does not fully replicate allogeneic HCT. To test R848 in full allogeneic HCT, BALB/c mice were injected with R848 24 h before irradiation (8 Gy) and immediately after B6 spleen and bone marrow cell transfer (B6→8Gy-BALB/c). All untreated mice rapidly developed acute GvHD and started to die from day 7 whereas 90% of the mice injected with R848 survived more than 40 days ( Figure 6A).
However, R848 did not completely prevent the GvHD reaction since IL-27p28 and IFNγ were still upregulated in R848-protected mice ( Figure 6B). This incomplete suppression of a GvHD reaction was even more striking in sublethally irradiated (5 Gy) B6D2F1 recipients of B6 spleen cells, in which R848 pre-treatment of host and donor before transplantation, as in the experiments described above in non-conditioned mice, solely delayed disease and no longer increased survival significantly ( Figure 6C), contrary to the total protection seen in non-irradiated recipients. This also correlated with the persistence of pro-inflammatory cytokines IL-27p28 and IFNγ, which R848 treatment failed to decrease (data not shown). As IL-27, which has been shown to contribute to GvHD pathology, 10,11 was still present in the irradiated models, we tested the combination of R848 and anti-IL-27 and observed that this resulted in 100% survival of the 5 Gy-irradiated B6D2F1 recipients ( Figure 6C).
The anti-IL-27-R848 combination also operated in B6→8Gy-BALB/c GvHD as it significantly decreased the concentration of plasma IFNγ, which was not the case when either agent was used separately ( Figure 6B). However, it did not prevent T-cell engraftment since we observed the same numbers of CD4 and CD8 T cells ( Figure 7A) and total B6 donor cell implantation ( Figure  7B). Already after 6 days, the number of B6 CD4 T cells recovered from the BALB/c spleen was equivalent to the total number injected and for CD8 T cells the number of cells had increased ±10-fold. These numbers were not modified by R848, anti-IL-27 or the anti-IL-27-R848 combination ( Figure 7A). Similarly, upregulation of the memory marker CD44 and the activation marker CD69 in B6 CD4 and CD8 T cells recovered 6 days after transplantation were not modified by R848. The only difference was observed for the CD62L naïve cell marker that was completely lost in B6 CD4 and CD8 T cells recovered from control GvHD mice but remained significantly higher in the group treated with the anti-IL-27-R848 combination (Online Supplementary Figure S3A).
As we previously showed that Treg contribute to the R848 protection in B6→ncB6D2F1 GvHD, Treg popula- A B C tions were also analyzed 6 days after B6→8 Gy-BALB/c GvHD induction. The numbers of Foxp3 + CD4 T cells were significantly higher in the R848-treated group than in the control group and adding anti-IL-27 to the R848 treatment further increased the Treg population 2-fold ( Figure 7C). Sixty-five percent of these Foxp3 + CD4 cells were positive for latent TGF-β1-associated protein (LAP), attesting their state of activation ( Figure 7D). The stimulating effect of the anti-IL-27-R848 combination on Foxp3 expression was even more striking for CD8 Foxp3 + T cells, which were barely detectable in the donor B6 spleen but reached levels equivalent to their CD4 counterparts in the anti-IL-27-R848 group. This was not seen in mice treated with either R848 or anti-IL-27 ( Figure 7C). Interestingly, following anti-IL-27 treatment, B6 Foxp3 + CD4 T cells increased 3-fold more than in the control GvHD group and 75% of these cells were activated. In contrast, R848 administration did not significantly amplify these cells, which correlated with the failure of the TLR7 ligand to increase survival in B6→5Gy-B6D2F1 mice. The anti-IL-27-R848 combination induced a strong increase of Foxp3 + CD4 T cells, which were 5-fold more numerous than in control GvHD mice and 85% were LAP + . In this irradiated model, the Foxp3 + CD8 population, although present, was substantially less abundant (Online Supplementary Figure S3B).
Thus, R848 treatment failed to provide complete protection against conditioned GvHD but full protection was restored when anti-IL-27 was added. The combination was necessary to abrogate IFNγ production and to induce maximal Foxp3 + CD4 and CD8 cell responses.

Discussion
Innate pattern recognition receptors, including TLR, are implicated in the control of GvHD. 37 Most often TLR stimulation aggravates disease, as reported for TLR4 38 and TLR9, 39 but the consequences may differ depending on the time of TLR stimulation with respect to allogeneic HCT as observed for the TLR7/8 agonist 3M-001. 40,41 The present study demonstrates the prevention of murine GvHD by the TLR7 ligand resiquimod/R848 and further improvement of protection by inhibition of IL-27. 10,11 Our results indicate that R848 administration to recipients 48 h before and at the time of allogeneic HCT promoted survival in lethally irradiated recipients of fully allogeneic hematopoietic cells and in the lethal form of GvHD induced in ncB6D2F1 recipients of B6 parental spleen cells. In this model, R848 inhibited IFNγ, IL-27 and TNFα production while upregulating TGF-β1, thus altering the cytokine balance. However, this suppression of Th1 cytokine produchaematologica | 2019; 104(2) D A B C tion was not observed in mice with conditioned GvHD which were also protected by R848, suggesting that inhibition of IFNγ, IL-27 and TNFα was not the only underlying mode of action. This conclusion was indirectly confirmed in the sublethally irradiated B6→B6D2F1 model in which R848 was not protective on its own but still enhanced protection by anti-IL-27. Together, these results indicate that the protective activity of R848 involves but is not limited to IL-27 inhibition as its effect could still be improved by antibody-mediated IL-27 inhibition.
In the B6→ncB6D2F1 GvHD model, complete protection required treatment of both the donor and recipient. As the contribution of F1 recipients to GvHD is limited to antigen presentation, these results suggest that R848 impaired both APC and T-cell responders. Analyses performed just before GvHD induction confirmed this hypothesis since DC antigen presentation and T-cell allo-responsiveness were inhibited in spleen cells collected from R848-treated mice. In agreement with the literature, 42 we observed that R848 induced a transient loss and functional inhibition of splenic cDC (both CD11b + and CD8α + ), the main cells able to induce allogeneic responses in vitro and that are known to play an important role in GvHD induction in vivo. 43,44 This drop in splenic DC was very transient and antigen presentation returned to normal after 7 days (unpublished observation), suggesting that a short inactivation of host DC is sufficient to alter the initiation of GvHD. R848 induced a similar inhibition of the capacity of T cells to respond to allogeneic in vitro stimulation. Type I interferons seem to be critical in the suppression of DC and T-cell allo-responsiveness by R848 as both remained unaltered in R848-treated IFNAR-1 -/mice. This observation is in line with reported inhibition of DC and CD4 T cells by type I interferons. 24 Importantly, the inhibition of T-cell allo-responsiveness by R848 in vivo treatment, demonstrated by ex vivo mixed lymphocyte cultures, did not prevent their implantation as chimerism was maintained for months. Moreover, the implanted T cells completely lost naïve T-cell marker CD62L and showed only partial inhibition of CD44 and CD69 memory and activation marker upregulation. This implies the existence of other regulatory mechanisms permitting the persistence of donor T cells in the host with reduced GvHD manifestations. A likely explanation was the effect of R848 on donor and recipient Foxp3 + Treg. The number of these cells dropped dramatically during GvHD but not in R848-treated mice in which their numbers even increased compared to basal control levels. This was particularly striking for Foxp3 + CD8 T cells. Moreover, the presence of LAP on their surface demonstrated that these cells were in an activated state. These results are in agreement with the upregulation of Treg by R848 reported in an asthma model. 45 Given the implication of Treg in the control of GvHD, 19,20 this Treg stimulation probably contributed to the protective effect of R848. This conclusion was substantiated by the observation that depletion of Treg by anti-CD25 antibody treatment restored morbidity to levels equivalent to those of control GvHD mice although survival was only partly impaired. The importance of Treg upregulation for GvHD prevention by R848 was further demonstrated in the B6→5Gy-B6D2F1 model in which R848 treatment failed to induce a robust Treg response which correlated with poor survival. Interestingly, adding anti-IL-27p28 monoclonal antibody to R848 restored the Treg response and resulted in complete protection. These results are in line with reported GvHD impairment by in vitro R848 stimulation of Treg 46 and reveal the R848-anti-IL-27 combination as a new option for maximal Treg upregulation and prevention of GvHD without inhibiting donor cell implantation.
R848 upregulated plasma levels of active TGF-β1 during GvHD, which probably contributed to the observed increase in Treg and to the inhibition of allogeneic T-cell responsiveness that characterized T cells recovered from R848-treated mice. These results are in agreement with the reported severity of murine GvHD induced by donors lacking SMAD3 35 as well as the predictive impact of TGF-β expression in human allogeneic HCT donors on GvHD occurrence. 47 The beneficial effect of high TGF-β1 levels could be related not only to Foxp3 + Treg cell expansion but also to the capacity of TGF-β to inhibit cytotoxic T-cell development. 48 The complete and long-lasting donor Treg elimination by anti-CD25 antibody only partly suppressed GvHD prevention by R848. This phenomenon could be explained by the presence of a small host Treg population, which recovered after 14 days and remained stable during the course of the GvHD. This partial suppression of R848 protection could also be explained by the other suppressive mechanisms induced by the drug, such as type I interferon-mediated inhibition of DC and T cells and the downregulation of Th1 cytokines mentioned earlier.
In summary, our results demonstrate that, in the context of allogeneic HCT, R848 alters both alloantigen presentation by cDC and Th1-cell responsiveness in a process dependent on type I interferons and correlating with increased TGF-β1 as well as Treg expansion and diminished IFNγ, TNFα and IL-27 production. When not sufficient on its own, R848 protection can be further enhanced by anti-IL-27p28 monoclonal antibody leading to maximal expansion of Foxp3 + Treg cells. A remarkable feature of this novel GvHD preventive procedure is that it needs to be applied only just before and at the time of allogeneic HCT and results in permanent coexistence of the host and allogeneic T cells with a significant reduction in GvHD symptoms.
Transposing these data to human conditions raises a number of issues. One is that R848 activation could be more complex in humans as it activates both TLR7 and TLR8, the latter being inactive in mice. Should this raise problems one could, however, use other agents that activate only TLR7 in humans, such as CL264, a 9-benzyl-8 hydroxyadenine derivative. Another issue is the expression of TLR7 by human not murine CD4 T cells. 49,50 This could in fact further improve the efficacy of TLR7-based prevention of GvHD since engaging TLR7 in human CD4 T cells induces a NFATc2-dependent anergy. 50


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Protective Effects of L-902,688, a Prostanoid EP4 Receptor Agonist, against Acute Blood-Brain Barrier Damage in Experimental Ischemic Stroke

Ischemic stroke occurs when a clot forms in the brain vasculature that starves downstream tissue of oxygen and nutrients resulting in cell death. The tissue immediately downstream of the blockage, the core, dies within minutes, but the surrounding tissue, the penumbra is potentially salvageable. Prostaglandin E2 binds to four different G-protein coupled membrane receptors EP1–EP4 mediating different and sometimes opposing responses. Pharmacological activation of the EP4 receptor has already been established as neuroprotective in stroke, but the mechanism(s) of protection are not well-characterized. In this study, we hypothesized that EP4 receptor activation reduces ischemic brain injury by reducing matrix metalloproteinase (MMP)-3/-9 production and blood-brain barrier (BBB) damage. Rats underwent transient ischemic stroke for 90 min. Animals received an intravenous injection of either the vehicle or L-902,688, a highly specific EP4 agonist, at the onset of reperfusion. Brain tissue was harvested at 24 h. We established a dose-response curve and used the optimal dose that resulted in the greatest infarct reduction to analyze BBB integrity compared to vehicle-treated rats. The presence of IgG, a blood protein, in the brain parenchyma is a marker of BBB damage, and L-902,688 (1 mg/kg; i.v.) dramatically reduced IgG extravasation (P < 0.05). Consistent with these data, we assessed zona occludens-1 and occludin, tight junction proteins integral to the maintenance of the BBB, and found reduced degradation with L-902,688 administration. With immunoblotting, qRT-PCR, and/or a fluorescence resonance energy transfer (FRET)-based activity assay, we next measured MMP-3/-9 since they are key effectors of BBB breakdown in stroke. In the cerebral cortex, not only was MMP-3 activity significantly decreased (P < 0.05), but L-902,688 treatment also reduced MMP-9 mRNA, protein, and enzymatic activity (P < 0.001). In addition, post-ischemic administration of the EP4 agonist significantly reduced pro-inflammatory cytokines IL-1β (P < 0.05) and IL-6 (P < 0.01) in the ischemic cerebral cortex. Most importantly, one injection of L-902,688 (1 mg/kg; i.v) at the onset of reperfusion significantly reduces neurological deficits up to 3 weeks later (P < 0.05). Our data show for the first time that pharmacological activation of EP4 with L-902,688 is neuroprotective in ischemic stroke by reducing MMP-3/-9 and BBB damage.

INTRODUCTION
Stroke is listed as the fifth leading cause of death in the USA and about 87% of strokes are ischemic (Mozaffarian et al., 2016). Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved drug for ischemic stroke. Only a small proportion of stroke patients are eligible to receive rtPA because it carries a high risk of bleeding/hemorrhagic transformation in addition to direct neurotoxicity (Kaur et al., 2004) and has a short effective time window of only 4.5 h after stroke onset (Del Zoppo et al., 2009). It is therefore essential to search for alternative pharmaceutical interventions to reach a larger percentage of ischemic stroke patients.
An ischemic stroke occurs when a major cerebral artery is occluded, and cells just downstream in the core of the stroke necrotically die within minutes. Cell death is perpetuated into the surrounding penumbra over the course of hours to days later. Reactive oxygen/nitrogen species (ROS) formation further compromises the integrity of an already degraded blood-brain barrier (BBB) by activating matrix metalloproteinases (MMP) i.e., MMP-3, MMP-9 that cleave the basement membrane of the neurovascular unit and the tight junction proteins (TJPs) between endothelial cells (Rosell et al., 2008;Sood et al., 2008;Candelario-Jalil et al., 2011;Turner and Sharp, 2016;Hafez et al., 2018). This triggers an inflammatory response and infiltration of immune cells which have been associated with increased cell death, formation of free radicals/ROS, and secondary injury (Yilmaz et al., 2006;Jin et al., 2010;Benakis et al., 2014).
In ischemic stroke, the increase in COX-2-derived PGE 2 formation correlates with BBB opening and infiltration of peripheral immune cells (Candelario-Jalil et al., 2007). Moreover, in vivo data show that direct injection of PGE 2 into the rat brain leads to increased permeability of the BBB (Schmidley et al., 1992;Messripour et al., 2015). In the context of focal cerebral ischemia, previous studies have shown that activation of EP1 and EP3 PGE 2 receptors significantly exacerbate stroke injury (Manabe et al., 2004;Kawano et al., 2006;Ahmad et al., 2007Ahmad et al., , 2008Abe et al., 2009;Fukumoto et al., 2010;Zhen et al., 2012;Shimamura et al., 2013). We recently showed that genetic deletion or pharmacological blockade of the EP1 receptor results in a dramatic reduction in stroke injury and BBB permeability, which correlated with reduced levels of MMP-3 and MMP-9 (Frankowski et al., 2015). Stroke-induced BBB damage is significantly reduced in EP3 deficient mice or in wild-type animals treated with an EP3 receptor antagonist (Ikeda-Matsuo et al., 2011).
Unlike EP1 and EP3 receptors, activation of EP2 and EP4 receptors has previously been shown to be neuroprotective in stroke (McCullough et al., 2004;Ahmad et al., 2005;Liang et al., 2011;Akram et al., 2013). Although several studies have provided strong evidence of a protective role of EP4 in neuroinflammation and cerebral ischemia, nothing is known of the effects of EP4 activation on BBB permeability after stroke. In this study, our objective was to investigate whether EP4 receptor activation would impact BBB permeability and neurobehavioral outcomes in a clinically relevant animal model of transient focal cerebral ischemia. We hypothesized that EP4 receptor agonism with L-902,688 reduces infarct size and neurological deficits by reducing MMP-3, MMP-9, and BBB damage.

Animals
Adult male rats (10-12 weeks, ∼280-320 g, Sprague Dawley from Charles River Laboratories International, Wilmington, MA, US) were allowed to acclimatize for 1 week before experiments in housing facilities on a 12 h light/dark cycle with free access to food and water with two rats per cage. All animal procedures were performed in accordance with approved guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals, the ARRIVE guidelines (https://www.nc3rs. org.uk/arrive-guidelines), and the guidelines approved by the Institutional Animal Care and Use Committee at the University of Florida (protocol #201406503). Experiments were planned to reduce the total number of animals used and to reduce potential pain and suffering.

Intraluminal Filament Model of Transient Focal Ischemia and Drug Treatment
To mimic ischemic stroke, rats were subjected to 90 min of transient middle cerebral artery occlusion (MCAO) using the intraluminal filament model of focal ischemia, described in detail in our previous publications (Candelario-Jalil et al., 2007;Hawkins et al., 2013Hawkins et al., , 2017. Rats were deeply anesthetized with 2-2.5% isoflurane in medical grade oxygen and maintained at a constant 37 • C throughout surgery on a heated platform (Cat # TP-700 T/Pump; Stryker Global Industries, Kalamazoo, MI, USA). A midline ventral cut was made, and the common carotid artery (CCA) was separated from the vagus nerve and ligated with a 4-0 silk suture (Cat # SP116; Harvard Apparatus, Holliston, MA, USA). The external carotid artery (ECA) and pterygopalatine arteries were temporarily clipped with a microvascular clip to prevent incorrect placement of the occluding filament. An arteriotomy was performed on the CCA a few millimeters above the ligation to allow for a 4-0 silicone-coated filament (Cat # 403523PK10; Doccol Corporation, Sharon, MA, USA) insertion through the internal carotid artery up into the middle cerebral artery until detection of a slight resistance. After temporarily closing the ventral incision, rats were allowed to recover in a temperature controlled heated chamber (Cat # ICS DW-1 Warmer, Thermo-Care, Paso Robles, CA, USA) for about 80 min to prevent hypothermia before re-anesthetizing the animal to remove the filament. At the onset of reperfusion, animals randomly received an intravenous injection of vehicle (saline; n = 10), 0.3 mg/kg L-902,688 (n = 8), or 1.0 mg/kg L-902,688 (n = 8). L-902,688 (5-[(1E,3R)-4,4difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone) was obtained from Cayman Chemical (Ann Arbor, MI, USA; Cat # 10007712). L-902,688 is a potent EP4 agonist with a K i value of 0.38 nM and an EC 50 value of 0.6 nM. It displays >4,000-fold selectivity for EP4 over other prostanoid receptors and has a half-life in vivo of ∼12 h in rats (Young et al., 2004). Treatment schedule was determined by simple randomization using a coin flip to determine the initial treatment and then treatment was alternated. Visual confirmation of occlusion was demonstrated by curling and circling behavior during the 90-min occlusion period. In this stroke model, the core of the stroke is represented by subcortical cell death, and the potentially viable penumbra is represented by the cortex in which cell death occurs mainly by apoptosis at later time points.

Tissue Collection and Homogenization
Rats were deeply anesthetized with 150 mg/kg i.p. pentobarbital and perfused with ice-cold physiological saline. Brains were extracted and sliced at 2 mm intervals in a rat brain matrix (Zivic Instruments, Pittsburgh, PA, USA). The fourth slice (anterior to posterior), which roughly corresponds to bregma and represents the core of the stroke in this model, was dissected into ipsilateral and contralateral cortex and striatum/subcortex, and immediately frozen on dry ice for molecular analyses. The remaining slices were used for infarct calculation. Tissue was weighed and homogenized with a Tissue-Tearor in radioimmunoprecipitation buffer containing 1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl pH 7.6, and 1% IGEPAL R CA-630 at 10 µL/mg of tissue and HALT Protease Inhibitor Cocktail, HALT Phosphatase Inhibitor Cocktail and 0.5 M EDTA (Cat. No. 78430;Cat. No. 78428;and Cat. No. 1860851, respectively; Thermo Fisher Scientific, Waltham, MA, USA) at 10 µL/mL of total volume. Samples were sonicated with a Vibra-Cell TM sonicator (Sonics & Materials Inc., Newtown, CT, USA) twice for 15 s separated by 15-min incubations on ice before centrifugation at 14,000× g for 20 min at 4 • C. The resulting supernatants were stored at −80 • C until use.

Infarct Calculation
To measure the infarct size, brain slices 1-3 and 5-6 were incubated in the dark in 2% 2,3,5-triphenyltetrazolium chloride in phosphate-buffered solution (PBS) for 30 min at room temperature, and placed in 4% paraformaldehyde. Live tissue stains red, and dead tissue remains white. Sections were scanned with an HP Scanjet 8300 (Palo Alto, CA, USA) at 600 dpi rostral side down except for the 3rd slice which was also scanned caudal side down to represent the rostral side of the 4th slice. Due to the significant edema produced by this stroke model, infarcts were calculated indirectly (Swanson et al., 1990;Frankowski et al., 2015). Using Adobe Photoshop CS5, the red tissue was delineated for each slice and the stroke surface area (mm 2 ) was calculated by subtracting live, red tissue on the ipsilateral side from the red tissue on the contralateral side. To calculate total infarct volume, the surface area (mm 2 ) of dead tissue was summed for each slice and multiplied by the thickness of the slice (2 mm).

ELISA and MMP Activity Assay
To measure BBB permeability, we performed ELISA analyses for immunoglobulin G (IgG). Blood proteins like IgG are not present in the brain parenchyma unless the BBB was compromised, providing an indirect method of BBB permeability; we therefore measured IgG in 100 µg protein from brain lysates prepared from thoroughly perfused rat brains. We used a commercially available rat IgG ELISA kit (Cat# E101, Bethyl Laboratories, Inc., Montgomery, TX, USA).
Two large contributors of BBB degradation in stroke are MMP-3 and MMP-9 which proteolytically cleave tight-junction proteins between endothelial cells and collagen IV in the basement membrane along the endothelium (Candelario-Jalil et al., 2009). Using a fluorometric immunocapture assay that our team developed (Hawkins et al., 2013), we measured MMP-3 and MMP-9 activity in 50 µg of brain lysate. Briefly, 96well plates were coated with Protein A/G to stably orient and immunocapture antibodies, coated with either an MMP-3 antibody (Cat # SC-6839-R, Santa Cruz Biotechnology, Dallas, TX, USA) or an MMP-9 antibody (Cat # SC-6841-R, Santa Cruz Biotechnology), and was incubated with 50 µg of total protein, then probed with a specific FRET peptide substrate (For MMP3: Substrate XIII, Cat # 60580-01 or MMP-9: Substrate III, Cat # 60570-01). The substrate (5-FAM/QXL TM 520) is bound to a quencher molecule that can be cleaved by either MMP-9 or MMP-3 to allow fluorescence. Values were normalized to 1 ng of recombinant rat MMP-9 or MMP-3.

Immunoblotting
We probed 40 µg of total protein for the tight junction proteins occludin (Cat # ab167161; AbCam) at 1:1,000 reduced in 5% β-mercaptoethanol and denatured with 10 min of boiling, and zonula occludens-1 (Cat # 61-7300; Life Technologies, Carlsbad, CA, USA) (ZO-1) at 1:500 reduced in 2% β-mercaptoethanol without boiling. Because it is known to degrade tight-junction proteins, so we also probed 50 µg of total protein for MMP-9 (Cat # ab76003, Abcam) at 1:5,000 reduced in 5% β-mercaptoethanol and denatured with 10 min of boiling. To separate proteins, we ran samples through 4-20% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA) at 200 V for 45 min in 0.1% SDS Tris-glycine buffer. Gels were equilibrated in Tris-glycine buffer containing 10% methanol for 10 min, then transferred at 25 V for 30 min onto either a nitrocellulose (Cat # 926-31092, Li-Cor, Lincoln, NE, USA) or PVDF (Immobilon-FL, Millipore, Billerica, MA, USA) membrane using the semi-dry Trans-Blot Turbo transfer apparatus. Membranes were blocked in 5% milk in TBS for 1 h at room temperature, then incubated overnight at 4 • C with primary antibody in 5% milk in TBST. Membranes were washed 4 times with TBST, and incubated with goat anti-rabbit IRDye 800CW (1:30,000; Li-Cor) in 5% milk in TBST containing 0.01% SDS for 1 h at room temperature. Excess antibody was removed with four more TBST washes, and incubated with primary antibody against β-actin (1:10,000, Cat # A1978, Sigma-Aldrich, Saint Louis, MO, USA) for 1 h at room temperature to ensure equal protein loading. Membranes were washed four times with TBST, incubated with donkey anti-mouse IRDye 680LT (1:40,000; Li-Cor) in 5% milk in TBST containing 0.01% SDS for 1 h at room temperature, and scanned with an Odyssey infrared scanning system (Li-Cor). Target protein signal was divided by actin signal to obtain densitometric values, and normalized across blots by dividing by a control sample.

qRT-PCR
Tissue (3 mm) corresponding to the core of the stroke near bregma was dissected into ipsilateral and contralateral hemispheres and further divided into striatal and cortical sections and placed in RNAlater RNA Stabilization Reagent (Cat. No. 76106, Qiagen, Germany) at 10 µL/ mg of tissue. RNA was isolated with the Aurum Total RNA Fatty and Fibrous Tissue kit  according to the manufacturer's instructions. One microgram of RNA was reverse-transcribed into cDNA with iScript TM Reverse Transcription Supermix (Cat # 1708841, BioRad), and diluted to 10 ng/µL with IDTE buffer pH 8.0 (Cat #11-05-01-13; Integrated DNA Technologies, Coralville, IA, USA). Twenty nanograms of cDNA from ipsilateral and contralateral cortical and subcortical tissue were run in triplicate, probed with exon-exon spanning primers (500 nM, Integrated DNA Technologies) for IL-1β, IL-6, Mmp-9, or Mmp-3, and normalized to the housekeeping gene Ywhaz (Frankowski et al., 2015; Table 1 for primer sequences) with PerfeCTa R SYBR R Green Fastmix R (Cat # 95072-012; Quanta Biosciences, Beverly, MA, USA) using a Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following procedure: polymerase activation/DNA denaturation phase at 95 • C for 30 s, then 40 cycles of denaturing at 95 • C for 5 s and annealing at 60 • C for 30 s. Specificity of each primer was confirmed using nontemplate controls and melt curves. The normalized expression shown in the bar graphs (Figure 4) was calculated using the CFX Manager TM software (Bio-Rad) and represent the relative quantity of the target gene normalized to the reference gene (Ywhaz), and further normalized to the biological control (contralateral sample of the vehicle-treated group).

Assessment of Long-Term Neurological Deficits
Rats were trained on tasks 24 and 48 h before inducing MCAO and tested 48 h, and at 1, 2, and 3 weeks post-ischemia. To measure long-term sensory and fine motor control deficits with the adhesive removal test, rats were trained to remove a small
Frontiers in Neuroscience | www.frontiersin.org sticker placed on the front, contralateral paw, and the latency to remove was recorded for 3 trials, and the average of the two lowest values was chosen for analysis. To measure motor deficits, rats were trained to stay on a rotarod that accelerated from 4 to 40 rpm. Latency to fall off was measured for 5 trials and the average of the two highest values were normalized to each animal's baseline values. Experimental details of the adhesive removal and accelerating rotarod tests have been described by our group in recent publications (Hawkins et al., 2017;Yang et al., 2017).

Statistics
Infarct measurement was performed with a one-way ANOVA with a Dunnett's multiple comparison post-test. PCR data was analyzed with a Student's t-test comparing vehicle-treated ipsilateral data to L-902,688-treated ipsilateral data. Behavioral performance was analyzed using a t-test between vehicle-treated rats and L-902,688-treated rats at each time point. Statistics were analyzed with GraphPad Prism version 6.0, and a p-value of less than 0.05 was considered statistically significant. Data are reported as mean ± SEM.

RESULTS
After 24 h of reperfusion, infarct volume was significantly reduced in the cortex ( * P < 0.05, * * P < 0.01, Figure 1A) and subcortex ( * P < 0.05, * * P < 0.01, Figure 1B) with 0.3 and 1.0 mg/kg L-902,688, respectively compared to the vehicle-treated group. Because only 1.0 mg/kg L-902,688 significantly reduced total infarct volume ( * P < 0.05, n = 8-10, Figure 1C), this dose was used for the rest of the study. Representative TTC-stained brain sections are shown in Figure 1D for both treatment groups, which help to better appreciate the reduction in infarct size in stroked rats receiving the EP4 agonist, L-902,688, at the onset of reperfusion (after 1.5 h of stroke onset).
To elucidate the mechanism of protection with 1.0 mg/kg L-902,688, IgG extravasation into the brain parenchyma was measured with an ELISA. Because IgG is a blood protein, there is minimal amount detected in a thoroughly perfused brain unless the BBB integrity is compromised. With 1.0 mg/kg L-902,688, we found significant reduction of IgG in the ipsilateral cortex ( * P < 0.05, Figure 2A) and the ipsilateral subcortex ( * P < 0.05, Figure 2B) compared to the ipsilateral vehicle cortex and subcortex.
Because MMP-3 and MMP-9 are major contributors to BBB damage after stroke, we wanted to see if the reduced BBB damage evidenced by reduced IgG extravasation was associated with reduced MMP-3/MMP-9 activity and protein levels. Densitometric analysis of immunoblots showed reduced MMP-9 levels in the ipsilateral cortex ( * P < 0.05, Figure 3A) and a non-significant reduction in the ipsilateral subcortex (p = 0.0792, Figure 3B) compared to ipsilateral vehicle values. This effect was mirrored in our MMP-9 activity assay data in the cortical ( * P < 0.05, Figure 3C), but not subcortical (Figure 3D) tissue. Because MMP-3 can activate MMP-9, we also measured MMP-3 activity and found 1.0 mg/kg L-902,688 reduced MMP-3 activity in the ipsilateral cortex ( * P < 0.05, Figure 3E), but not in the subcortex ( Figure 3F).
Reduced IgG extravasation and reduced MMP-9 and MMP-3 activity suggested that 1.0 mg/kg L-902,688 reduced stroke-induced BBB damage. We therefore measured levels of the tight junction proteins ZO-1 and occludin in cortical tissue with immunoblotting. We found a non-significant preservation of ZO-1 in the ipsilateral cortex in L-902,688treated rats compared to vehicle-treated rats ( Figure 5A). Additionally, degradation of the 125-kDa occludin dimer was significantly reduced in the ipsilateral cortex with  EP4 receptor activation. This is associated with reduced injury-induced low molecular weight 65-kDa occludin ( Figure 5B).
Finally, we wanted to confirm that the reduction in infarct size was associated with reduced neurological deficits long-term. EP4 activation with one intravenous injection of 1.0 mg/kg L-902,688 at the onset of reperfusion showed sustained improvement in neurological function. Animals receiving 1.0 mg/kg L-902,688 performed better at the adhesive removal test at 1, 2, and 3 weeks after stroke ( * P < 0.05, * * P < 0.01, Figure 6A) and were also able to stay on the rotarod longer than vehicle-treated rats at 1, 2, and 3 weeks after stroke ( * P < 0.05, Figure 6B). There was a non-significant (P = 0.0888), but substantial decrease in IL-1β in the ipsilateral subcortex. (C) IL-6 is significantly downregulated in the ipsilateral cortex (**P = 0.0089) and slightly reduced in the subcortex (D) (P = 0.1186). (E) L-902,688 decreased MMP-9 in the ipsilateral cortex (***P < 0.001) (F) There was a trend toward MMP-9 downregulation with treatment in the ipsilateral subcortex, but it did not reach significance (P = 0.2335). (G) EP4 receptor activation with L-902,688 potently reduces MMP-3 mRNA expression in the ischemic cerebral cortex (**P = 0.0083). (H) No significant reduction in MMP-3 expression was found in the ischemic subcortical region between treatment groups (P = 0.5104). Student's t-test comparing vehicle and L-902,688 ipsilateral groups. CXI, ipsilateral cortex; CXC, contralateral cortex; STI, ipsilateral subcortex; STC, contralateral subcortex. Vehicle (n = 10) and 1.0 mg/kg L-902,688 (n = 8).
Frontiers in Neuroscience | www.frontiersin.org FIGURE 5 | Effect of L-902,688 on Tight Junction Proteins. We performed immunoblots to measure (A) ZO-1 in vehicle (n = 10) and L-902,688 (n = 8) groups and found a non-significant trend toward ZO-1 preservation in the ipsilateral hemisphere of the treated group compared to the vehicle in cortical samples. (B) Degradation of the dimeric form of occludin (∼125 kDa) was reduced in the ipsilateral cortex of L-902,688 and this was associated with decreased induction of the low molecular weight occludin (∼65 kDa). Data are reported as ipsilateral divided by contralateral due to the wide variability in the occludin content in both the contralateral and the ipsilateral hemispheres of vehicle-treated rats. CXI, ipsilateral cortex; CXC, contralateral cortex; STI, ipsilateral subcortex; STC, contralateral subcortex. *P < 0.05 compared with vehicle-treated animals.

DISCUSSION
Here, we show for the first time that activation of the EP4 receptor with L-902,688 given at the onset of reperfusion significantly reduces infarct size, blood-brain barrier (BBB) breakdown, MMP-3 and MMP-9 levels, degradation of tight junction proteins, and stroke-induced increase in the expression of the pro-inflammatory cytokines IL-1β and IL-6. More importantly, post-ischemic treatment with L-902,688 resulted in an improved long-term neurological recovery as assessed using the adhesive removal and accelerating rotarod tests.
Expressed in cardiovascular, neuronal, and immune cells (Sando et al., 1994;Hata and Breyer, 2004;Yokoyama et al., 2013) the EP4 receptor is uniquely suited to influence infarct outcome in ischemic stroke. The BBB can be conceptually dissected into neurovascular units comprised of neurons, astrocytic endfeet, pericytes, and endothelial cells. Typically, the neurovascular unit synergistically maintains homeostatic levels of permeability for vasculature to parenchyma substance exchange. During an ischemic event, the BBB undergoes biphasic opening at 3 and 48 h after reperfusion which is correlated with MMP levels in this stroke model (Rosenberg et al., 1998). This is relevant because MMP-9-induced BBB opening is detrimental in the acute phase of stroke (Sood et al., 2008;Yang et al., 2010).
Because endothelial cells are on the front line, they are the first cell type to be affected by hypoxia. One mechanism of neuroprotection may be via EP4 receptor-induced vasodilation, altering cerebral blood flow in response to ischemia (Taniguchi  , 2014). This makes sense in light of the fact that EP4 receptor activation increases eNOS and phospho-Ser 1177 eNOS, increasing local NO levels. Direct protection may also shield neurons from against stroke insult, as well because neuronal EP4 receptor activation reduces cell death in vitro and ex vivo after excitotoxic challenge and in vitro after hypoxic/hypoglycemic challenge (Liang et al., 2011). These effects likely translate into reduced infarct size with a single bolus of EP4 receptor agonist L-902,688 at the onset of reperfusion in our stroke model of transient focal ischemia. This reduction is correlated with decreases in several measures of BBB permeability. EP4 receptor activation reduces IgG extravasation in both the core of the stroke represented by the ipsilateral subcortex and in the penumbra represented by the ipsilateral cortex. EP4 receptor activation similarly reduced stroke-induced MMP-9 and MMP-3 activity, particularly in the cortex, and strokeinduced MMP-9 mRNA levels. MMP-9 and MMP-3 are key contributors to BBB disruption in the context of ischemic stroke since these proteases degrade the basal lamina and tight junction proteins essential to the barrier function of the neurovascular unit (Rosenberg et al., 1998;Asahi et al., 2001;Rosell et al., 2008;Candelario-Jalil et al., 2009;Turner and Sharp, 2016).
Not surprisingly, EP4 receptor agonism reduced proinflammatory gene transcription along with reduced infarct and BBB damage. IL-1β activates microglia/macrophages, stimulating more IL-1β release and triggering immune cell infiltration that contribute to increased BBB permeability and apoptotic death in penumbral neurons (Yamasaki et al., 1995;Hawkins and Davis, 2005;McColl et al., 2007;Clausen et al., 2008;Sandoval and Witt, 2008). We found robust reductions in IL-1β gene expression in the cortex and the subcortex with EP4 receptor activation. L-902,688 further reduced acute phase IL-6 expression, and increased IL-6 is correlated with larger stroke volume and worse outcome (Waje-Andreassen et al., 2005). These data are interesting because EP4 activation has shown to upregulate IL-6 expression in some cell types (Hata and Breyer, 2004;Zhou et al., 2016). L-902,688-dependent IL-6 reductions are likely reflective of reduced cell damage/infarction (Tarkowski et al., 1995;Suzuki et al., 1999Suzuki et al., , 2009Smith et al., 2004). In a model of subarachnoid hemorrhage in rats, a very recent study found that AE1-329, an EP4 receptor agonist, significantly reduced BBB damage, edema, and expression of IL-1β, IL-6, and TNF-α (Xu et al., 2017).
EP4 agonism protects the tight junction proteins between endothelial cells that are vital determinants of BBB permeability. In the cortex, we found a non-significant trend toward ZO-1 preservation with EP4 receptor activation, and significant preservation of dimeric occludin (125 kDa) in the ipsilateral cortex which was associated with reduced stroke-induced 65-kDa occludin, a likely phosphorylated form of occludin. Increased levels of the 65-kDa band of occludin have been detected in models of ischemic stroke and are associated with BBB disruption (Kago et al., 2006;Takenaga et al., 2009;Fukumoto et al., 2010;Muthusamy et al., 2014;Frankowski et al., 2015). No change in the lower molecular weight occludin band (50 kDa) was observed between ipsilateral and contralateral hemispheres irrespective of the treatment group. Our model of focal ischemia typically induces such occludin alterations (Frankowski et al., 2015).
Oxidative stress is a key mechanism of BBB disruption and neuronal death in ischemic stroke (Chan, 2001;Li et al., 2017). Free radicals/ROS directly damage endothelial cells composing the BBB and indirectly activate MMPs, which lead to proteolytic breakdown of basal lamina proteins and TJPs resulting in injury to the neurovascular unit (Gürsoy-Ozdemir et al., 2004;Kahles et al., 2007;Hafez et al., 2018). One potential mechanism through which EP4 receptor activation could reduce stroke-induced BBB opening is reduction of oxidative damage during the reperfusion phase. This notion is supported by previous studies showing that EP4 agonists reduce free radical formation in neurons and microglia exposed to amyloid β (Echeverria et al., 2005) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) (Pradhan et al., 2017). It remains to be determined whether treatment with L-902,688 or other EP4 agonists reduces oxidative stress in vivo after stroke.
The EP4 receptor is expressed in many cell types including endothelium, neurons, microglia, astrocytes, and peripheral immune cells (Sando et al., 1994;Hata and Breyer, 2004;Yokoyama et al., 2013;Bonfill-Teixidor et al., 2017). Permeability of the BBB after stroke can be altered by complex cellular and molecular interactions between cells of the neurovascular unit and the peripheral immune system. We found that EP4 receptor activation with L-902,688 potently reduces levels of some of the key mediators of stroke-induced BBB damage including IL-1β, IL-6, MMP-3, and MMP-9. The main cellular sources of these pro-inflammatory mediators after stroke include activated microglia and astrocytes, as well as infiltrating neutrophils and macrophages (Benakis et al., 2014;Amantea et al., 2015). It has been shown that EP4 signaling decreases the activation of nuclear factor-kB (NF-kB), a master regulator of pro-inflammatory gene transcription, in activated microglia (Shi et al., 2010;Woodling et al., 2014), as well as in peripheral immune cells (Takayama et al., 2002(Takayama et al., , 2006Minami et al., 2008). Based on our data and previous reports, suppression of immune cell activation and production of pro-inflammatory mediators are suggested as mechanisms by which EP4 agonism confers neurovascular protection in ischemic stroke.
Most importantly, we found that a single administration of L-902,688 at the onset of reperfusion reduced sensorimotor deficits in the adhesive removal test and the rotarod assessment up to 3 weeks after stroke. Although infarct size is not always a consistent indicator of stroke outcome, sensitive behavioral assessments like the adhesive removal and rotarod tests are relatively reliable indicators of functional neurological deficits that can detect changes at least up to 3 weeks after injury (Yang et al., 2017).
Limitations of our study include the utilization of one type of transient stroke model that includes reperfusion injury, and these data would be strengthened by confirming neuroprotection in other stroke models. We only used young healthy male rats, which is another limitation of our study. Since age, diabetes, hypertension, and hypercholesterolemia are among the most important risk factors for stroke, future studies should investigate the effects of EP4 agonism in animals of both sexes with these comorbid conditions. Furthermore, because we were limited to pharmacological intervention in rats, it would be of interest to subject transgenic conditional knockout mice lacking EP4 specifically in myeloid cells, endothelial cells, or neurons to stroke to determine the relative contribution of EP4 activation from different cells of the neurovascular unit. Future studies will determine whether delayed administration of L-902,688 (several hours after stroke onset) also confers sustained, longterm neuroprotection as was found with a different EP4 receptor agonist in mice (Liang et al., 2011). This will provide further support that EP4 receptor activation has clinical relevance if it proves to be effective up to 4.5 h after stroke, the current therapeutic window for tPA.
To our knowledge, we are the first group to establish that EP4 agonism with a single administration of L-902,688 at the onset of reperfusion is neuroprotective in a transient MCAO stroke model in rats up to 3 weeks after ischemia. This neuroprotection is due to the dynamic crosstalk between inflammation and BBB degradation. EP4 activation reduces pro-inflammatory IL-1β gene transcription and matrix metalloproteinases MMP-3 and MMP-9, major contributors of BBB damage. These effects culminate in reduced tight junction protein degradation that maintain the integrity of the BBB and reduced long-term neurological deficits.

FUNDING
Funding for this project was provided by the National Institute of Health (NIH), grant number R01-NS065849 to EC-J. Publication of this article was funded in part by the University of Florida, Open Access Publishing Fund.