Abstract
Background and Purpose
Severe influenza A virus (IAV) infections are associated with damaging hyperinflammation that can be fatal. There is an urgent need to identify new therapeutic agents to treat severe and pathogenic IAV infections. Repurposing of drugs with an existing and studied pharmacokinetic and safety profile is a highly attractive potential strategy. We have previously demonstrated that the NLRP3 inflammasome plays time‐dependent roles during severe IAV infection with early protective responses and later dysregulation leading to excessive inflammation, contributing to disease severity.
Experimental Approach
We tested two existing drugs, probenecid and AZ11645373, to target P2X7 receptor signalling and dampen NLRP3 inflammasome responses during severe IAV infection. In vitro, the drugs were assessed for their ability to limit NLRP3 inflammasome‐dependent IL‐1β secretion in macrophage cultures. In vivo, their effects were assessed on hyperinflammation and disease during severe IAV infection in C57BL/6 mice.
Key Results
Treatment of macrophages with probenecid or AZ11645373 in vitro diminished NLRP3 inflammasome‐dependent IL‐1β secretion. Intranasal therapeutic treatment of mice displaying severe influenza disease with probenecid or AZ11645373 reduced pro‐inflammatory cytokine production, cellular infiltrates in the lung, and provided protection against disease. Importantly, these drugs could be administered at either early or late stage of disease and provide therapeutic efficacy.
Conclusions and Implications
Our study demonstrates that the anti‐inflammatory drugs probenecid and AZ11645373, which have documented pharmacokinetics and safety profiles in humans, are effective at dampening hyperinflammation and severe influenza disease providing potentially new therapeutic strategies for treating severe or pathogenic IAV infections.
Abbreviations
- BAL
- bronchoalveolar lavage
- COPD
- Chronic Obstructive Pulmonary Disease
- DC
- dendritic cell
- IAV
- influenza A virus
- iBMDM
- immortalised bone‐marrow derived macrophages
- PFU
- plaque‐forming units
1. INTRODUCTION
The year 2018 marked the 100th anniversary of the Spanish influenza A virus (IAV) pandemic that caused significant worldwide mortality. The emergence of a novel or pandemic virus poses a constant threat to global health. In particular, H7N9 IAV infections are associated with mortality rates of approximately 40% in humans, and experts predict a pandemic is inevitable (Lam et al., 2015). Hyperinflammation is a characteristic feature of severe and fatal IAV infections with aberrant production of pro‐inflammatory cytokines and excessive immune cell infiltration, the so‐called “cytokine storm” that contributes to lethality (Short, Kroeze, Fouchier, & Kuiken, 2014; H. Wang & Ma, 2008). There is a critical unmet medical need to develop new and effective treatment strategies to reduce IAV‐induced hyperinflammation.
NLRP3 inflammasomes are innate cytoplasmic complexes activated during IAV infection, maturing the inactive pre‐cursors cytokines, pro‐IL‐1β and pro‐IL‐18, into their bioactive forms IL‐1β and IL‐18 through caspase‐1 processing. These potent pro‐inflammatory cytokines induce inflammation, including trafficking of immune cells (e.g., neutrophils and T cells), activation of epithelial and endothelial cells, and autocrine/paracrine cytokine production (e.g., TNFα, IL‐6, and IL‐1β; Dinarello, 2009; Kaplanski, 2018). Inflammasome responses require two signals: (a) priming of cells by activating the prototypic inflammatory transcription factor NF‐κB that mediates synthesis of pro‐IL‐1β, pro‐IL‐18, and up‐regulation of components of the NLRP3 inflammasome and (b) triggering of inflammasome formation, which results in IL‐1β and pro‐IL‐18 maturation and secretion. A number of cellular processes/stimuli trigger this second signal such as potassium efflux that typically involves extracellular ATP activation of the P2X7 receptor, ROS, and lysosomal maturation (reviewed in Ong, Mansell, & Tate, 2017; Tate & Mansell, 2018). IAV infection is initially recognised by innate detection of viral RNA via the RIG‐I sensor and toll‐like receptor 3 (TLR3) and toll‐like receptor 7 (TLR7) to prime the inflammasome via increasing expression of pro‐IL‐1β and pro‐IL‐18, as well as NLRP3 (De Nardo, De Nardo, & Latz, 2014; Ong et al., 2017). Subsequently, viral RNA and the M2 protein from seasonal and pathogenic IAV are sensed by NLRP3 to trigger inflammasome assembly (Allen et al., 2009; Ichinohe, Pang, & Iwasaki, 2010). We have previously shown that the IAV PB1‐F2 protein from pathogenic PR8 H1N1 and H7N9 activates NLRP3 (McAuley et al., 2013; Pinar et al., 2017), contributing to severe disease pathology.
Initial studies demonstrated mice lacking components of the inflammasome complex (NLRP3, ASC, and caspase‐1) display increased susceptibility to infection (Allen et al., 2009; Ichinohe et al., 2010; Thomas et al., 2009). However, using the small molecule NLRP3 inhibitor MCC950, we recently demonstrated the temporal role of the NLRP3 inflammasome in IAV disease: inducing protective immunity in the initial period of infection, as treatment of mice from day 1 post‐infection with MCC950, accelerated weight loss and mortality (Tate et al., 2016). However, delaying commencement of MCC950 treatment until mice display signs of severe disease identified for the first time that NLRP3 promotes hyperinflammation and thus constitutes a major pathogenic factor in disease.
While our studies identified the benefits of delayed targeting of the NLRP3 inflammasome, an ideal therapy could be administered at any stage of infection without causing adverse effects. While MCC950 is highly potent at concentrations as low as 1 μM (Coll et al., 2015; Pinar et al., 2017) and provides protection later in infection, it renders mice susceptible early in infection. We hypothesised that less potent NLRP3 inhibitors would dampen inflammation sufficiently to balance protective versus detrimental inflammation and therefore protect from IAV‐induced pathology without the need for a diagnostic test to initiate treatment. The present study aimed to identify and repurpose drugs that have a well‐documented pharmacokinetic and safety profile in humans (e.g., FDA approved for treatment of inflammatory conditions or undergone clinical trials), as novel inhibitors of NLRP3 responses to treat severe IAV infection.
2. METHODS
2.1. In vitro stimulation of murine macrophages
Immortalised wild‐type C57BL/6 bone‐marrow derived macrophages (iBMDMs; Hornung et al., 2008) were grown in DMEM (Gibco) supplemented with 10% heat inactivated FBS and 2‐mM glutamine. iBMDMs were seeded in 96‐well plates 24 hr prior to incubation with LPS (O114:B5; 100 ng·ml−1, InvivoGen) for 3 hr. Cells were then incubated with probencid (water soluble; ThermoFisher) or AZ11645373 (Tocris) at the indicated concentrations 1 hr prior to stimulation with NLRP3 inflammasome activators silica (250 μg·ml−1), nigericin (6 μM), ATP (5 mM), or influenza virus PR8 PB1‐F2 peptide (100 μg·ml−1; McAuley et al., 2013; Pinar et al., 2017). After a further 6 hr, cell supernatants were collected, and levels of IL‐1β were quantified by ELISA according to the manufacturer's instructions (R&D Systems).
2.2. Influenza virus infection of mice
All animal care and experimental procedures complied with the approved guidelines and were approved by the Monash Medical Centre Animal Ethics Committee. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology. Six‐ to 8‐week‐old C57BL/6 male and female mice were maintained in the Specific Pathogen‐Free Physical Containment Level 2 (PC2) Animal Research Facility at the Monash Medical Centre. IAV strains used in this study were A/PR/8/34 (H1N1), as well as HKx31 (H3N2), which is a high‐yielding reassortant of PR8 that carries the surface glycoproteins of A/Aichi/2/1968 (H3N2). Viruses were grown in 10‐day embryonated chicken eggs by standard procedures and titrated on Madin‐Darby Canine Kidney (MDCK) cells (RRID:CVCL_0422).
For virus infection studies, groups of eight male and female C57BL/6 mice were randomised into treatment groups. Mice were lightly anaesthetised and infected intranasally with 105 plaque‐forming units (PFU) of HKx31 (H3N2) or 50 PFU PR8 (H1N1) in 50‐μl PBS (previously shown to induce severe disease in C57BL/6 mice; Mansell & Tate, 2018; Tate et al., 2016). Following infection, mice were treated at the time points indicated (every 48 hr; according to ethically approved guidelines) with 40 mg·kg−1 probenecid (water soluble; ThermoFisher) or 20 mg·kg−1 AZ11645373 (Torcis) via the intranasal route in 50‐μl PBS. Control mice were treated with PBS alone. Mice were weighed daily and assessed for visual signs of clinical disease, including inactivity, ruffled fur, laboured breathing, and huddling behaviour. Animals that lost ≥20% of their original body weight or displayed severe clinical signs of disease were killed. Due to the nature of the interventions, research staff administering the treatments were not blinded to group allocation.
Bronchoalveolar lavage (BAL) fluid was immediately obtained following killing by flushing the lungs three times with 1 ml of PBS. The lungs were then removed and frozen immediately in liquid nitrogen. Titres of infectious virus in lung homogenates were determined by standard plaque assay on MDCK cells.
2.3. Quantification of mouse pro‐inflammatory cytokines in BAL fluid
To detect cytokines, BAL fluid was collected and stored at −80°C. IL‐1β was quantified by ELISA according to the manufacturer's instructions (R&D Systems). Levels of IL‐6, CCL2, IFN‐γ, IL‐10, IL‐12p70, and TNF‐α proteins were determined by cytokine bead array and mouse inflammation kit (Becton Dickinson).
2.4. Recovery and characterisation of leukocytes from mice
For flow cytometric analysis, BAL cells were treated with red blood cell lysis buffer (Sigma Aldrich), and cell numbers and viability were assessed via Trypan blue exclusion using a haemocytometer. BAL cells were incubated with Fc block (2.4G2; eBiosciences), followed by staining with fluorochrome‐conjugated monoclonal antibodies to Ly6C, Ly6G, CD11c, and I‐Ab (MHC‐II; BD Biosciences, USA). Neutrophils (Ly6G+), airway macrophages (CD11c+ I‐Ab low), dendritic cells (DC; CD11c+ I‐Ab high), inflammatory macrophages (Ly6G− Ly6C+) were quantified by flow cytometry, as described previously (Tate et al., 2016). Live cells (propidium iodide negative) were analysed using a BD FACS Canto II flow cytometer (BD Biosciences) and FlowJo software (RRID:SCR_008520). Total cell counts were calculated from viable cell counts performed via trypan blue exclusion.
2.5. Data and statistical analysis
The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). When comparing three or more sets of values, a one‐way ANOVA was used with Tukey's post hoc analysis. A Student's t test was used when comparing two values (two‐tailed, two‐sample equal variance). Survival proportions were compared using the Mantel–Cox log‐rank test. A P value <.05 was considered statistically significant.
2.6. Nomensclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Fabbro et al., 2017a, b; Alexander, Kelly et al., 2017; Alexander, Peters et al., 2017).
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