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Pirfenidone (PFD) is a pharmacological compound with therapeutic efficacy in idiopathic pulmonary fibrosis. It has been chiefly characterized as an antifibrotic agent, although it was initially developed as an antiinflammatory compound because of its ability to diminish the accumulation of inflammatory cells and cytokines. Despite recent studies that have elucidated key mechanisms, the precise molecular activities of PFD remain incompletely understood. PFD modulates fibrogenic growth factors, thereby attenuating fibroblast proliferation, myofibroblast differentiation, collagen and fibronectin synthesis, and deposition of extracellular matrix. This effect is mediated by suppression of TGF-β1 (transforming growth factor-β1) and other growth factors. Here, we appraise the impact of PFD on TGF-β1 production and its downstream pathways. Accumulating evidence indicates that PFD also downregulates inflammatory pathways and therefore has considerable potential as a viable and innovative antiinflammatory compound. We examine the effects of PFD on inflammatory cells and the production of pro- and antiinflammatory cytokines in the lung. In this context, recent evidence that PFD can target inflammasome pathways and ensuing lung inflammation is highlighted. Finally, the antioxidant properties of PFD, such as its ability to inhibit redox reactions and regulate oxidative stress-related genes and enzymes, are detailed. In summary, this narrative review examines molecular mechanisms underpinning PFD and its recognized benefits in lung fibrosis. We highlight preclinical data that demonstrate the potential of PFD as a nonsteroidal antiinflammatory agent and outline areas for future research.

BACKGROUND.: Human metapneumovirus (hMPV) infection is implicated in exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Research into the pathogenesis of infection is restricted to animal models, and information about hMPV replication and inflammatory and immune responses in human disease is limited. METHODS.: Human primary bronchial epithelial cells (PBECs) from healthy and asthmatic subjects and those with COPD were infected with hMPV, with or without glucocorticosteroid (GCS) exposure. Viral replication, inflammatory and immune responses, and apoptosis were analyzed. We also determined whether adjuvant interferon (IFN) can blunt hMPV infection in vitro and in a murine model. RESULTS.: hMPV infected human PBECs and viral replication was enhanced in cells from patients with COPD. The virus induced gene expression of IFN-stimulated gene 56 (ISG56) and IFN-β, as well as IFN-γ-inducible protein 10 (IP-10) and regulated on activation, normal T cell expressed and secreted (RANTES), and more so in cells from patients with COPD. GCS exposure enhanced hMPV replication despite increased IFN expression. Augmented virus replication associated with GCS was mediated by reduced apoptosis via induction of antiapoptotic genes. Adjuvant IFN treatment suppressed hMPV replication in PBECs and reduced hMPV viral titers and inflammation in vivo. CONCLUSIONS.: hMPV infects human PBECs, eliciting innate and inflammatory responses. Replication is enhanced by GCS and adjuvant IFN is an effective treatment, restricting virus replication and proinflammatory consequences of hMPV infections.

Acridine dyes, including proflavine and acriflavine, were commonly used as antiseptics before the advent of penicillins in the mid-1940s. While their mode of action on pathogens was originally attributed to their DNA intercalating activity, work in the early 1970s suggested involvement of the host immune responses, characterized by induction of interferon (IFN)-like activities through an unknown mechanism. We demonstrate here that sub-toxic concentrations of a mixture of acriflavine and proflavine instigate a cyclic-GMP-AMP (cGAMP) synthase (cGAS)-dependent type-I IFN antiviral response. This pertains to the capacity of these compounds to induce low level DNA damage and cytoplasmic DNA leakage, resulting in cGAS-dependent cGAMP-like activity. Critically, acriflavine:proflavine pre-treatment of human primary bronchial epithelial cells significantly reduced rhinovirus infection. Collectively, our findings constitute the first evidence that non-toxic DNA binding agents have the capacity to act as indirect agonists of cGAS, to exert potent antiviral effects in mammalian cells.

Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development., IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans.

The innate immune response to virus must be balanced to eliminate infection yet limit damaging inflammation. A critical arm of the antiviral response is launched by the retinoic acid-inducible-gene I (RIG-I) protein. RIG-I is activated by viral RNA then associates with the mitochondrial antiviral signaling (MAVS) protein to subsequently induce potent inflammatory cytokines. Here, we demonstrate the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) is a crucial moderator of RIG-I signaling. MUL1 is localized to the mitochondria where it interacts with MAVS and catalyzes RIG-I post-translational modifications that inhibit RIG-I-dependent cell signaling. Accordingly, depletion of MUL1 potentiated RIG-I mediated nuclear factor-kappa B (NF-κB) and interferon (IFN) β reporter activity. Moreover, depletion of MUL1 boosted the antiviral response and increased proinflammatory cytokines following challenge with the RNA mimetic poly I:C and Sendai virus. We therefore submit that MUL1 is a novel regulator of the RIG-I-like receptor-dependent antiviral response, that otherwise functions to limit inflammation.

Glucocorticosteroids (GCS) are used on a daily basis to reduce airway inflammation in asthma and chronic obstructive pulmonary disease (COPD). This treatment is usually escalated during acute disease exacerbations, events often associated with virus infections. We examined the impact of GCS on anti-viral defences and virus replication and assessed supplementary interferon (IFN) treatment. Here, we report that treatment of primary human airway cells in vitro with GCS prior to rhinovirus (RV) or influenza A virus (IAV) infection significantly reduces the expression of innate anti-viral genes and increases viral replication. Mice given intranasal treatment with GCS prior to IAV infection developed more severe disease associated with amplified virus replication and elevated inflammation in the airways. Adjuvant IFN treatment markedly reduced GCS-amplified infections in human airway cells and in mouse lung. This study demonstrates that GCS cause an extrinsic compromise in anti-viral defences, enhancing respiratory virus infections and provides a rationale for adjuvant IFN treatment.

Individuals with asthma are prone to viral and bacterial infections, and most asthma exacerbations have been linked to viruses, particularly rhinovirus. Excess transforming growth factor (TGF)-beta present in asthmatic airways may cause immune suppression, as well as transdifferentiate fibroblasts to myofibroblasts, thereby augmenting proinflammatory responses after rhinovirus infection. After rhinovirus infection we examined virus replication and host cell immune responses in airway fibroblasts in the presence of TGF-beta1 and in myofibroblasts. Primary culture fibroblasts were pretreated with TGF-beta1 or transdifferentiated into myofibroblasts, and then infected with rhinovirus. Viral replication, virus release, chemokine production, and interferon (IFN) responses were measured over 72 hours. Rhinovirus replication and virus release into supernatants were enhanced in fibroblasts incubated with TGF-beta1 and in fibroblasts obtained from patients with asthma. Myofibroblasts also showed more rhinovirus replication, and infected myofibroblasts produced excess neutrophil chemokines. Examination of innate responses revealed blunting of type I IFN reactions with dissociated viral RNA and IFN mRNA responses. Addition of type I IFN restituted antiviral responses, and the effect of TGF-beta1 appeared to be mediated via actions on IFN regulatory factor-3 pathways. These data demonstrate that TGF-beta1 mediates enhanced virus replication and proinflammatory responses in airway cells. TGF-beta may act as an endogenous immunosuppressant promoting virus replication and inflammation during the evolution of acute severe asthma associated with rhinovirus infection.

Aims Viral upper respiratory tract infections caused by rhinovirus (RV) and influenza A virus (IAV) are associated with 50–80% of asthma exacerbations. Inhaled glucocorticosteroids (GCS) are routinely used to relieve asthma symptoms, and while it has been widely reported that GCS reduce inflammation associated with viral infection, the effects of GCS on viral replication has not been comprehensively examined. Methods Using primary human airway cells and a mouse model of IAV infection we examined the effect of GCS on the production of inflammatory mediators, anti-viral responses and viral replication following RV and/or IAV infection. Results GCS pre-treatment of epithelial cells significantly reduced IL-8, IL-6 and IP-10 production following RV and IAV infection, compared to untreated controls. The expression of anti-viral genes, including ISG56, ISG15, viperin and MxA, were significantly reduced in epithelial cells, fibroblasts and macrophages pre-treated with GCS prior to infection with RV or IAV. The impairment of anti-viral responses observed following GCS pre-treatment was associated with enhanced RV and IAV replication in both epithelial cells and fibroblasts. GCS pre-treatment of mice prior to IAV infection was associated with significant weight loss and severe disease progression compared with untreated mice. Viral loads were significantly elevated in both the lungs and nasal tissues of the GCS treated mice, and there were significantly elevated levels of IL-6, MCP-1 and KC in BAL fluid. Interferon and anti-viral ISG expression were significantly reduced in GCS treated mice. Treatment with recombinant interferon after infection delayed the onset of disease and improved survival. Conclusions Our data indicate that GCS at therapeutic doses enhanced virus replication through the suppression of innate anti-viral responses. Interferon treatment improved disease severity in the mouse model, suggesting this may be a viable therapeutic option to counteract the adverse effects of GCS treatment on viral replication and innate immune suppression.