key: cord-0930213-rhgzd3hd authors: Abusalamah, Hazar; Reel, Jessica M.; Lupfer, Christopher R. title: Pyruvate Affects Inflammatory Responses of Macrophages During Influenza A Virus Infection date: 2020-07-04 journal: Virus Res DOI: 10.1016/j.virusres.2020.198088 sha: 16e7b5c36baca4a0eafc420ed2040012924e6507 doc_id: 930213 cord_uid: rhgzd3hd Pyruvate is the end product of glycolysis and transported into the mitochondria for use in the tricarboxylic acid (TCA) cycle. It is also a common additive in cell culture media. We discovered that inclusion of sodium pyruvate in culture media during infection of mouse bone marrow derived macrophages with influenza A virus impaired cytokine production (IL-6, IL-1β, and TNF-α). Sodium pyruvate did not inhibit viral RNA replication. Instead, the addition of sodium pyruvate alters cellular metabolism and diminished mitochondrial reactive oxygen species (ROS) production and lowered immune signaling. Overall, sodium pyruvate affects the immune response produced by macrophages but does not inhibit virus replication. Pyruvate (Pyr) (C3H4O3) is a central molecule in cellular metabolism. In addition to the typical glycolysis-to-TCA pathway (Barnett 2003) , Pyr can be derived from lactate taken up from outside the cells or synthesized intracellularly from amino acids (Halestrap and Price 1999; Karmen, Wroblewski, and Ladue 1955) . Instead of entering the TCA cycle, anaerobic glycolysis can occur (fermentation) where Pyr is reduced into lactate in order J o u r n a l P r e -p r o o f to regenerate NAD + . In rapidly dividing cells, like some immune cells or cancer cells, this also occurs even when oxygen is present (aerobic glycolysis/Warburg effect) (Roiniotis, et al. 2009 ). Although energetically less favorable, aerobic glycolysis facilitates metabolite production necessary for rapid cell division, such as amino acid and nucleic acid synthesis (Vander Heiden, Cantley, and Thompson 2009) . Reports have shown that IAV infection severely alters metabolism including amino acid and lipid metabolism (Chandler, et al. 2016 ). The innate immune system has germline-encoded pattern-recognition receptors (PRRs). These sensors are capable of recognizing microorganisms that invade the host (Akira, Uematsu, and Takeuchi 2006) . PRRs can bind to pathogen-associated molecular patterns (PAMPs) such as RNA from viral genomes (Crozat and Beutler 2004) . Detection of PAMPs by PRRs activates a variety of immune signaling pathways resulting in cytokines production, increased phagocytosis and cell death. However, these responses can be modulated by metabolic processes. When retinoic acid inducible gene-I (RIG-I) is activated by cytoplasmic viral RNA, it moves to the mitochondria, where it interacts with Mitochondrial Antiviral Signaling protein (MAVS) (Kato, et al. 2006) . MAVS then recruits adaptors proteins at the mitochondria forming the MAVS signalosome, which activates the transcription factors IRF3/7 and NF-κB (Seth, et al. 2005) . However, lactate can inhibit this pathway, thus dampening inflammation during viral infection (Zhang, et al. 2019 ). The inflammasome is another immune signaling pathway that forms a multiprotein complex, which activates the cysteine protease caspase-1 (Agostini, et al. 2004 ). Active caspase-1 then activates the inflammatory cytokines interleukin (IL)-1β and IL-18 (Sutterwala, et al. 2006) . Inflammasome activation by NOD-like receptor containing a J o u r n a l P r e -p r o o f pyrin 3 (NLRP3) is somewhat unique, as its main activation signals are cellular damage including oxidative stress and potassium efflux (Allen, et al. 2009; Petrilli, et al. 2007 ). Intriguingly, NLRP3 appears to be tuned-in to the metabolic state of cells through glycolysis (Moon, et al. 2015; Xie, et al. 2016) . Pyr is well studied in metabolism, but its role in the immune response is not. During the course of infecting macrophages with IAV, we noted that different brands of cell culture media with different nutrient compositions affected the magnitude of the immune response. In particular, the inclusion of sodium pyruvate (NaPyr) in culture media inhibited immune signaling during IAV infection. Here we show that NaPyr added to BMDM cell culture media inhibits the release of important pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. In addition to these findings, we observed that addition of NaPyr does not inhibit viral replication, rather it suppresses the immune response to IAV through altering metabolism and ROS production. WT C57BL/6J mice were bred and raised in the Temple Hall Vivarium at Missouri State University. Mice were euthanized via CO2 asphyxiation and cervical dislocation and bone marrow collected for differentiation into macrophages. All breeding and experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines (protocols 16.009 and 19.019), the AVMA Guidelines on Euthanasia, NIH J o u r n a l P r e -p r o o f regulations (Guide for the Care and Use of Laboratory Animals), and the U.S. Animal Welfare Act of 1966. Bone Marrow Derived Macrophages (BMDM) were produced by harvesting bone marrow from the femur and tibia of 7-14-week-old C57BL/6J mice. Bone marrow cells were then grown for 5 days in bone marrow differentiation media, which consisted of Dulbecco's Modified Eagle Medium (DMEM) + 10% FBS + 1% Pen/Strep + 1% Non-essential amino acids (NEAA) and supplemented with L929 cell conditioned media. L929 cell conditioned medium contains Macrophage colony-stimulating factor (M-CSF) and was produced by growing L929 cells in DMEM+ 10% FBS+ 1% Pen/Strep for 10 days and then filtering the media via a 0.2μm filter. On day 5 of BMDM growth, cells were scraped and re-plated into 12-well plates at 1x10 6 cells/well in 1ml BMDM media and incubated overnight to allow cells time to adhere to the plates. Macrophages were used the following day for experiments. The strain of IAV used in all experiments is influenza A/PR/8/34 H1N1. In order to generate virus, we inoculated pathogen-free hen's eggs with 1000 PFU of IAV. 3 days post inoculation, the allantoic fluid was harvested, centrifuged to remove debris, and frozen at -80°C for later use. J o u r n a l P r e -p r o o f The IAV plaque assay was performed using MDCK cells seeded at 3x10 5 cells/well in 12well plates in DMEM+ 5% FBS+ 1%Pen/Strep. 10-fold dilutions of the virus were prepared in MEM without FBS. MDCK cells were washed with PBS twice and 100µl of each virus dilution were added to duplicate wells in 12-well plates and incubated at 37 o C and 5% CO2 for one hour. Semisolid overlay was prepared as previously described (Lupfer, et al. 2008 ). TPCK-trypsin was added to a final concentration of 1.0 µg/ml. After the full hour of incubation, infection medium was removed from 12-well plates, and 2ml of the warm overlay with TPCK trypsin was added to each well and allowed to solidify. Plates were turned upside down and incubated for 3 days. After 3 days, the overlaid agar was removed and plaques counted after staining with 1% crystal violet in methanol. Cell culture supernatants collected from infected and control BMDM were analyzed for IL-1β, TNF-α, and IL-6. ELISA kits were purchased from Ebioscience (88-7013-88, 88-7324-88, 88-7064-88) and assays performed according to the manufacturer's recommendations. Plates were read at 450nm on a microplate reader (BioTek ELx808). Cell lysates were collected by adding RIPA buffer with protease and phosphatase inhibitors (Thermo Scientific, PIA32959, PIA32957) to BMDM treated or infected as indicated. 4x SDS loading dye was then added to samples, which were boiled for 20 minutes and resolved by SDS-PAGE and then transferred to PVDF membranes. Western blotting for caspase-1 (caspase-1 p45 and p20), phosphorylated IκBα and total IκBα were then performed by incubating membranes in primary antibody diluted in 5% milk in TBST J o u r n a l P r e -p r o o f overnight at 4 0 C (See table 1 for a list of antibodies). The next day, membranes were washed 3x in TBST buffer and incubated for 45 minutes in secondary antibody diluted in 5% milk in TBST (Table 1) . Membranes were washed again, and images obtained using Super Signal West Fempto substrate (ThermoFisher, A53225) and an Azure C300 digital imaging system. Anti-Rabbit-HRP secondary Jackson Immuno Res.111-035-144 Anti-mouse-HRP secondary BioRad, HAF007 Macrophages were plated in 12-well plates and infected and/or treated as indicated. After 24 hours, and 30 minutes before collecting samples, cells were stained with a mitochondrial specific ROS sensitive dye (2.5nM Mitosox; ThermoFisher, M36008) or a cell death stain (5 mM SYTOX-red; ThermoFisher, S34859). After 30 minutes, the media was removed and 1ml of PBS was added to each well and the macrophages were scraped off the wells. Cells were analyzed on an ACURI C6 or Attune NxT flow cytometer. 10,000 cells per sample were analyzed for fluorescence intensity and percentage of cells positive for each dye. J o u r n a l P r e -p r o o f BMDM were infected and treated as indicated and samples were collected at 6, 12, and 24 hours after infection. Media was removed and 500µl Trizol (Invitrogen, AM9738) was added to samples and incubated for 5 minutes at room temperature. RNA was then isolated according to the manufactures protocol. All samples were normalized to 200ng/μl RNA in nuclease-free water. The High Capacity cDNA Reverse Transcriptase kit (Thermo Fisher Scientific 436881) was then used to convert 1ug RNA into cDNA. Then, cDNA was diluted 1:5 in nuclease-free water, and 5 µl cDNA was used per reaction to perform qRT-PCR with the DyNAmo HS SYBR Green qPCR master mix (Thermo Scientific 00596849) according to the manufacturer's instructions using a STRATAGENE-Mx3005P PCR machine. (See table 2 for Primer Sequences). To test the effects of NaPyr on the immune response of BMDM to other stimuli, 1x10 6 BMDM were plated per well in 12-well plates. The next day, BMDM were washed 2x with PBS and 400µl of RPMI 1640 +10% FBS and L-glutamine but without NaPyr was added to each well. Some wells were treated with 1µg/ml LPS for 4 hours with inclusion of 5mM ATP (Sigma, L3129 and Acros, 102800100) for the last 30 minutes. Some wells were also treated with 1, 2 or 5mM NaPyr. Samples were collected at the end of 4 hours of treatment. Poly I:C samples were treated with 25µg/ml as indicated above and supernatants collected 24 hours post treatment. Statistical analysis was performed using GraphPad PRISM6. Comparison of 2 conditions was performed using the 2-sided student's t-test. Comparison of multiple conditions was performed using the One-Way ANOVA with Tukey's post-hoc test. A p-value <0.05 was considered statistically significant. In discussion with other researchers ( . We also noted that bone marrow derived dendritic cells (BMDC) generally produce higher cytokine levels than BMDM in response to IAV infection, but BMDC are typically cultured in RPMI 1640. We examined the composition of these media and determined that NaPyr was associated with lower immune responses. Therefore, we infected BMDM with IAV in RPMI1640 medium with and without NaPyr. Our data demonstrate that addition of NaPyr significantly impaired cytokine production by BMDM infected with IAV ( Figure 1A-C) . We next examined virus replication by collecting cell culture media from infected BMDMs 24h after infection and performing viral plaque assays. BMDM are refractory to infection with some strains of IAV (Cline, Beck, and Bianchini 2017) , but similar levels of functional virions were recovered from BMDMs in our model with or without NaPyr treatment ( Figure 1D ). As virion production was low overall, we further confirmed that NaPyr did not affect J o u r n a l P r e -p r o o f virus growth or its ability to infect macrophages by examining viral RNA levels (IAV M1 and NP genes). NaPyr did not inhibit the replication of virus RNA, demonstrating that NaPyr does not affect cytokine responses by inhibiting IAV replication (Figure 1E-F) . In addition, NaPyr treatment of IAV infected BMDM had no effect on cell death ( Figure 1G ). We examined cytokine gene expression by RT-PCR at 6, 12, and 24h after IAV infection. NaPyr did have an inhibitory effect on gene expression in IAV infected BMDM compared to virus infected BMDM cultured in the absence of NaPyr (Figure 2A-D) . We performed western blotting on cell lysates from BMDM infected with IAV and treated with NaPyr but did not observe any significant differences in the activation of NF-κB (phospho-IκBα) ( Figure 2E-F) . However, NaPyr inhibited caspase-1 activation in BMDM infected with IAV ( Figure 2G-H) . To determine if NaPyr treatment broadly inhibited immune signaling, we treated BMDM with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), which is a potent activator of the NLRP3 inflammasome (Mariathasan, et al. 2006) . Intriguingly, LPS+ATP treated BMDM cultured with NaPyr produced similar amounts of IL-1β, IL-6, and TNF compared to control infected cells (Figure 3A-C) . Furthermore, caspase-1 activation was not inhibited, even at higher doses of NaPyr than used with IAV ( Figure 3D-E) . To determine if NaPyr's anti-inflammatory properties were linked to viral ligands as opposed to bacterial ligands, we stimulated BMDM with the TLR3 ligand poly I:C (PIC). Interestingly, BMDM treated with PIC and cultured with NaPyr produced similar amounts of IL-6 compared to the untreated controls ( Figure 3F ). Anti-inflammatory effects of NaPyr are associated with altered metabolism in BMDM Previously, NaPyr was reported to be an antioxidant with potential therapeutic uses in a variety of inflammatory diseases (Ramos-Ibeas, et al. 2017; Votto, et al. 2008 ). NLRP3 activation in many instances is dependent on reactive oxygen species (ROS) and mitochondrial damage (Lupfer, et al. 2013; Lupfer, et al. 2014; Heid, et al. 2013 (Figure 4A-B) . During LPS+ATP treatment, mitochondrial ROS was elevated, but NaPyr had no effect on ROS in this setting (Figure 4A, C) . These results indicate that NaPyr inhibits ROS in a context specific manner. IAV replication requires a massive metabolic burst to produce not only the viral nucleotides and proteins for virus replication, but also the antiviral immune responses of the cell. Previous research has shown that IAV induces a unique and elevated catabolic profile including increased lactate production (Smallwood, et al. 2017) . Elevated lactate levels have been reported to inhibit RIG-I signaling, which could explain our observations (Zhang, et al. 2019) . We examined lactate production from BMDM and observed a significant increase in lactate caused by IAV infection alone, but this was not enhanced by the addition of NaPyr ( Figure 4D ). We thus hypothesized that NaPyr may fulfil a metabolic need during IAV infection, as opposed to the formation of a byproduct. We examined intracellular ATP production by BMDMs infected with IAV and found that IAV J o u r n a l P r e -p r o o f infection results in increased ATP levels over uninfected BMDM or LPS+ATP treated BMDM ( Figure 4E) . Importantly, NaPyr treatment was able to transiently boost ATP output from BMDM to match the need seen in IAV infected cells ( Figure 4E ). As the ATP needs of IAV infected cells were not copied by LPS+ATP, NaPyr may specifically decrease mitochondrial ROS during IAV infection by balancing metabolic stress. The ability of metabolites to affect the immune response to infection is an important area of research with implications for preventing and treating disease. Recent research shows that changes in metabolism in cells of the immune system can affect diseases such as influenza, cancer, diabetes and more (Matarese, Procaccini, and De Rosa 2008; Shi, et al. 2011; Diers, et al. 2012) . Our data clearly indicate that treatment of IAV infected macrophages with NaPyr can reduce cytokines production (IL-1β, TNF-α, and IL-6). However, NaPyr does not affect virus titer or RNA replication in macrophages. Instead, NaPyr alters the immune function of the macrophages. Antioxidants that can prevent mitochondrial damage also prevent NLRP3 inflammasome activation and release of IL-1β from infected cells (Lupfer, et al. 2013; Lupfer, et al. 2014) . Previous reports indicate that NaPyr can decrease inflammation by its antioxidant potential (Ramos-Ibeas, et al. 2017; Das 2006; Xia, et al. 2016) . Although NaPyr may function as a ROS scavenger, we further propose that NaPyr reduces metabolic stress and ROS generation in an infection or disease specific manner. Specifically, pyruvate is taken into cells and bypasses many of the regulatory checkpoints for energy metabolism J o u r n a l P r e -p r o o f such as glucose transporters and phosphofructokinase (Schell and Rutter 2013; Trompette, et al. 2018) . It can be directly transported into the mitochondria for use in the TCA cycle and ATP production or used in anabolic pathways (Brand and Nicholls 2011) . Thus, addition of NaPyr to cells increases ATP production, as we observed, and likely affects additional metabolic pathways. In our model, we propose that decreased mitochondrial ROS is thus a secondary, but important, anti-inflammatory effect of NaPyr treatment. There are also additional factors that may affect the ability of NaPyr to inhibit other stimuli. In the case of LPS+ATP treatment, the treatment duration is much shorter than IAV (only 4 hours for LPS+ATP instead of 24 hours for IAV). Thus, intrinsic differences in the timing and pathways of the different stimuli may further impact the effects of NaPyr and should be examined further. In conclusion, NaPyr affects cytokine production by inhibiting inflammatory signaling pathways and not by affecting virus growth or cell death in macrophages. Metabolic pathways are important for cellular activation and have documented roles in immune signaling and immune cell function (Chandler, et al. 2016; Roiniotis, et al. 2009; Moon, et al. 2015; Xie, et al. 2016; Smallwood, et al. 2017) . Understanding the effects NaPyr has on the immune response to IAV and other infections will help elucidate the immune response in general and determine if certain nutrients can improve the immune response. Furthermore, severe IAV infection in human patients is associated with a metabolic crisis (especially depleted ATP) resulting in multi organ failure (Kido, et al. 2016) . As pyruvate clearly increases ATP production, decreases ROS and limits inflammation during IAV infection, it is worth examining as a potential therapeutic option in this and other diseases. Finally, severe infections with IAV and the current COVID19 pandemic are both J o u r n a l P r e -p r o o f associated with a "cytokine storm" that results in severe immunopathology (Han, et al. 2020; Huo, et al. 2018) . Corticosteroids are used in severe cases to suppress this overt inflammation. Importantly, the only drug to date that has demonstrated clinical benefit for COVID19 is dexamethasone (Horby, et al. 2020; Selvaraj, et al. 2020 ). Unfortunately, corticosteroids may leave the host susceptible to outgrowth of the initial pathogen or to secondary infection (Theoharides and Conti 2020). As we observed no significant change in virus replication in this model, NaPyr may have therapeutic benefit for severe IAV and other infections where excessive inflammation is a key factor. N115 is an FDA approved NaPyr based nasal spray (Emphycorp, Inc) currently used for the treatment of patients with COPD and Idiopathic Pulmonary Fibrosis, that has been shown safe and effective in phase I/II/III clinical trials. Importantly, N115 significantly decreased nasal and lung inflammation including IL-6 and improved measures of lung function including FEV-1, SaO2, FVC, FEV-1/FVC ratios (Votto, et al. 2008 , and personal communication with Dr. Alain Martin, Emphycorp, Inc.). Clinical trials using inhaled sodium pyruvate (N115) for COVID-19 infected patients are currently in progress to determine if the inhalation of sodium pyruvate can reduce the severity of lung inflammation in this disease. HA, JMR helped to conceptualize the project, collect and analyze the data and helped write the manuscript. CRL conceptualized the project, developed methodology, managed the project and resources, analyzed the data, wrote and approved all drafts of the manuscript. Data are representative of 2-4 independent experiments with n=2-3 wells per experiment. 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Not So Fast Dietary Fiber Confers Protection against Flu by Shaping Ly6c(-) Patrolling Monocyte Hematopoiesis and Cd8(+) T Cell Metabolism Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation Inhaled Sodium Pyruvate Improved Fev1 and Decreased Expired Breath Levels of Nitric Oxide in Patients with Chronic Obstructive Pulmonary Disease Addition of Sodium Pyruvate to Stored Red Blood Cells Attenuates Liver Injury in a Murine Transfusion Model Pkm2-Dependent Glycolysis Promotes Nlrp3 and Aim2 Inflammasome Activation Lactate Is a Natural Suppressor of Rlr Signaling by Targeting Mavs We thank Teneema Kurikose, St Jude Children's Research Hospital, for helpful discussion in the initial stages of this research. We also thank Missouri State University for research funding through Faculty Startup funds to CRL and Graduate Student Research funds to HMA and JMR.