Pirfenidone Mitigates Transforming Growth Factor-β–induced Inflammation after Viral Infection

Abstract

Infection by influenza A virus (IAV) and other viruses causes disease exacerbations in chronic obstructive pulmonary disease (COPD). Immune responses are blunted in COPD, a deficit compounded by current standard-of-care glucocorticosteroids (GCS) to further predispose patients to life-threatening infections. The immunosuppressive effects of elevated transforming growth factor-β (TGF-β) in COPD may amplify lung inflammation during infections while advancing fibrosis. In the present study, we investigated potential repurposing of pirfenidone, currently used as an antifibrotic for idiopathic pulmonary fibrosis, as a nonsteroidal treatment for viral exacerbations of COPD. Murine models of lung-specific TGF-β overexpression or chronic cigarette smoke exposure with IAV infection were used. Pirfenidone was administered daily by oral gavage commencing pre- or postinfection, and inhaled pirfenidone and GCS treatment preinfection were also compared. Tissue and BAL were assessed for viral replication, inflammation, and immune responses. Overexpression of TGF-β enhanced the severity of IAV infection, contributing to unrestrained airway inflammation. Mechanistically, TGF-β reduced innate immune responses to IAV by blunting IFN-regulated gene expression and suppressing production of antiviral proteins. Prophylactic pirfenidone administration opposed these actions of TGF-β, curbing IAV infection and airway inflammation associated with TGF-β overexpression and cigarette smoke–induced COPD. Notably, inhaled pirfenidone caused greater inhibition of viral loads and inflammation than inhaled GCS. These proof-of-concept studies demonstrate that repurposing pirfenidone and employing a preventative strategy may yield substantial benefit over antiinflammatory GCS in COPD. Pirfenidone can mitigate damaging viral exacerbations without attendant immunosuppressive actions and merits further investigation, particularly as an inhaled formulation.

Clinical Relevance

Overexpression of transforming growth factor-β in chronic lung diseases such as chronic obstructive pulmonary disease leaves patients susceptible to viral infections, which are further exacerbated by glucocorticosteroids. This study shows that pirfenidone mitigates virus-induced disease exacerbation without immunosuppressive actions, demonstrating that repurposing this approved drug may yield substantial benefit over the current gold standard treatment.

Pulmonary viral infections are common in healthy populations and typically have a self-limiting course. However, patients with preexisting inflammatory lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), are more susceptible to severe viral infections, often with devastating and prolonged consequences. Secondary bacterial infections are common (1, 2), a consequence of immunosuppressive effects associated with current standard-of-care treatment employing inhaled and oral glucocorticosteroids (GCS). However, other factors may play a role, with recent evidence linking endogenous mediators of airway inflammation and remodeling, such as transforming growth factor-β (TGF-β), to immune suppression (3).

TGF-β is an evolutionarily conserved pleiotropic factor regulating biological processes, including wound healing, immune responses, and tissue remodeling (4, 5). The role of TGF-β in the pathophysiology of COPD and viral exacerbations is likely to be complex and to lead to heterogeneity of pathogenic processes. Expression of TGF-β has been reported to be upregulated in the plasma, sputum, and airway walls of people with COPD and IPF (6–9), whereas a single report describes higher latent TGF-β binding protein 1 and TGF-β3 in the lamina propria but selective impairment of TGF-β signaling in the epithelia of small airways in patients with stable COPD (6). One key aspect of TGF-β biology that is often overlooked is its ability to suppress both innate and adaptive immune responses (3, 10). This attribute, coupled to overproduction of TGF-β in chronic lung diseases, may trigger enhanced susceptibility to severe viral infections causing amplified inflammation and tissue damage. To date, to our knowledge, this possibility has not been investigated in COPD, an ailment that affects more than 300 million people globally (11).

Pirfenidone is an antifibrotic, antiinflammatory compound approved internationally for the treatment of IPF (12, 13). Although its mechanisms of action are not fully defined, it is known that pirfenidone inhibits TGF-β–induced fibroblast differentiation, proliferation, and collagen production in vitro (14) and attenuates TNF-α and IL-1β expression, as well as collagen deposition in murine models of acute lung injury, asthma, and IPF (15–18). In phase 3 randomized controlled trials in IPF, pirfenidone reduced the rate of lung function decline (19, 20) and the risk of disease progression by 30% (21). Notably, a meta-analysis found that pirfenidone also reduces the frequency of episodes of acute worsening of IPF, often triggered by infections, by more than 40% (22). A recent clinical trial of pirfenidone in hospitalized adult patients with severe COVID-19 infection demonstrated antiinflammatory benefits of pirfenidone therapy (23), but, to date, to our knowledge, potential benefits of pirfenidone in virus-induced COPD exacerbations have not been assessed.

In the present study, we established the deleterious role of TGF-β in murine models of influenza A virus (IAV) infection. The reason for the use of this virus (IAV) rather than rhinovirus, despite the latter being a more common cause of COPD exacerbations, was to establish more robust infection and replication to demonstrate potential inhibitory effects of pirfenidone treatment.

Methods

Study Design

Our studies used a combination of in vivo murine models, in vitro assays, and human clinical studies. For further experimental details and ethics approvals, see the data supplement.

Mouse Models and IAV Inoculation

CC10-rtTA-tTS-TGF-B1 transgenic mice have been described (24). Mice (male and female Balb/c, 6–8 wk old) were administered doxycycline (Dox; 0.25 mg/ml in drinking water, refreshed every 3 d) for 48 hours or 8 weeks to induce TGF-β expression. Wild-type mice (male Balb/c, 8 wk old) were exposed to cigarette smoke (CS) for 8 weeks to induce a COPD phenotype (25).

After Dox administration or CS exposure, mice were anesthetized and infected intranasally with IAV (10² plaque-forming units, HKx31 strain, H3N2, in 50 μl of PBS). Drugs were administered either by oral gavage (30 or 100 mg/kg/d pirfenidone) or via intranasal inhalation under isoflurane anesthesia (13.2 mg/kg/d pirfenidone or 1 mg/kg/d fluticasone propionate [GCS]). Treatments commenced 2 days before IAV infection. The 30 mg/kg/d and 100 mg/kg/d oral doses of pirfenidone in mice were based on multiple published studies in animal lung disease models (reviewed in [26]) and are equivalent to or threefold higher than the clinical oral dose used in the treatment of IPF (2,403 mg/d). The inhaled dose of 13.2 mg/kg/d is based on bioavailability in patients in whom a 15-fold lower systemic pirfenidone exposure was achieved with nebulization compared with that reported with oral administration of the licensed oral dose (27). The 500 μM concentration used in vitro is consistent with previous studies showing antiinflammatory and antifibrotic efficacy in cell-based studies (28, 29).

Mice were weighed daily and assessed for visual signs of clinical disease. Animals that lost >15% of their original body weight were killed. Concentrations of infectious virus in lung tissue homogenates were determined by standard plaque assay on MDCK cells (30).

Histological Analysis of Lungs

Stained formalin-fixed, paraffin-embedded lung sections were scored for bronchitis and pneumonia severity, quantification of airspace enlargement, and measurement of epithelial and subepithelial thickness (see data supplement).

BAL Recovery and Characterization of Leukocytes and Cytokines

Mice were killed, and their lungs were flushed with 0.8 ml PBS three times via a 23-gauge tracheal catheter. Total BAL cells were counted using a hemocytometer with trypan blue exclusion. Differential cell counts were determined by flow cytometry (BD Biosciences) (30). Cytokine concentrations in mouse BAL were measured using a Cytometric Bead Array Mouse Inflammation Kit (BD Biosciences) or by ELISA (R&D Systems, Invitrogen).

Quantification of IFNs and IFN-regulated Genes

RNA was isolated from the lung right upper lobe, cDNA was prepared, and real-time PCR was performed on an Applied Biosystems 7900HT Fast Real-Time machine. Values were normalized to Gapdh. Primer sequences used for interferons and IFN-regulated genes (IRGs) have been published (31, 32).

Evaluation of Serum Cytokines in Patients with IPF

To determine the effect of pirfenidone on cytokine concentrations in IPF, patients were recruited to a prospective observational study. All patients had a blood sample taken at enrollment and after 3 months of pirfenidone treatment (801 mg thrice per day). Control subjects with no underlying lung disease were also recruited. TGF-β, C-reactive protein, vascular endothelial growth factor, periostin, and matrix metalloproteinase-7 levels were determined by ELISA (R&D Systems, Invitrogen).

Signaling in hTERT Fibroblasts

Human telomerase reverse transcriptase (hTERT)-immortalized fibroblasts were stimulated with IFN-β (Merck Serono Australia Pty Ltd), polyinosinic-polycytidylic acid (poly I:C), or LPS or transfected with ISD70 before pirfenidone treatment (see data supplement). IP-10 (IFN-γ–induced protein) and IL-6 concentrations were determined by ELISA (R&D Systems).

Western Blot Analysis

Frozen lung tissue was processed and prepared as detailed in the data supplement. Protein lysates were separated by SDS-PAGE using a 4–12% Bis-Tris gradient gel and transferred to a nitrocellulose membrane. Membranes were probed with antibodies for total and phosphorylated RIPK and MLKL (details in the data supplement), and chemiluminescence was captured using a Bio-Rad ChemiDoc MP Imaging System.

Statistical Analysis

Unpaired or paired t tests (two-tailed, parametric) and Wilcoxon or Mann-Whitney tests (two-tailed, nonparametric) were used to compare two sets of data. When comparing three or more data sets, one-way parametric or nonparametric ANOVA or two-way ANOVA with appropriate post hoc tests was used. The Mantel-Cox test was employed for survival analysis. Analysis was conducted in GraphPad Prism, and findings were considered statistically significant if P < 0.05.

Results

TGF-β Amplifies IAV-induced Disease Severity and Inflammation through Innate Immune Suppression

To investigate the effects of TGF-β on IAV infection, we used a transgenic murine model that selectively overexpresses active TGF-β1 in the lung after oral Dox administration (24). TGF-β concentrations in BAL fluid were below the level of detection in untreated transgenic mice but increased to 5.01 ± 2.15 ng/ml 48 hours after Dox, without further increases after prolonged administration (see Figure E1A in the data supplement). This transgenic model was combined with our established infection model using IAV (30).

Mice were administered Dox for 48 hours before IAV infection, then monitored daily for weight loss, signs of disease, and survival while Dox administration continued (Figure 1A). Dox alone did not cause significant weight loss or other clinical signs (Dox; Figure 1B). Dox-treated mice infected with IAV (Dox-IAV) lost significantly greater weight from Day 1 postinfection (Figure 1B). Although IAV-infected mice did not succumb to infection, Dox-IAV mice had reduced survival by Day 5, associated with increased viral replication at Day 3 postinfection (Figures 1C and 1D; P < 0.001).

Figure 1.

Transforming growth factor-β (TGF-β) overexpression increases disease severity after influenza A virus (IAV) infection. (A) CC10-rtTA-tTS-TGF-B1 transgenic mice were administered doxycycline (Dox) for 48 hours before infection with IAV, then culled at Days 2, 3, and 5 postinfection for analysis. (B) Daily weights, mean ± SEM, n = 8–31 per group, two-way ANOVA. (C) Survival curve for IAV (n = 10) and Dox-IAV (n = 24), Mantel-Cox test. (D) Viral titers by plaque assay, n = 8–9 per group, unpaired t test. (E) Representative hematoxylin and eosin–stained images from (i) control, (ii) IAV-infected, and (iii) Dox-IAV–infected mice. Dashed line = epithelial damage; solid line = inflammatory cell infiltration. Scale bars, 200 μm. (F) Lung tissue scored for bronchitis and pneumonia severity (0 = none, 1 = mild, 2 = moderate, 3 = severe). Individual scores and median values, n = 11–13 per group, Wilcoxon test. (G) Total cell number and inflammatory cell subtypes in BAL fluid, n = 6–23 per group, one-way ANOVA. n.d. = not detected. (H) TNF-α, MCP-1/CCL2, and IL-6 protein in BAL fluid by cytometric bead array (CBA) or ELISA, n = 6–23 per group except IL-6 in Dox group, n = 3, one-way ANOVA. (I) Ifnλ₂, Ifit1, Ifit2, and Isg15 mRNA expression by real-time PCR and normalized to Gapdh, n = 6–15 per group, one-way ANOVA. All data are representative of a minimum of two independent experiments. (D, H, I) are box-and-whisker plots showing median and interquartile range (IQR), with data points shown only for individual values greater than the 75th percentile plus 1.5 times the IQR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Lung pathology, inflammation, and other indices of disease severity were evaluated. Compared with control specimens [Figure 1E(i)], hematoxylin and eosin–stained lung sections from IAV-infected mice showed partial epithelial destruction (dashed circle), accompanied by mild inflammation confined to the respiratory bronchioles (solid line circle) [Figure 1E(ii)]. In Dox-IAV mice, the epithelial cell layer was largely destroyed, inflammatory exudate was present in the airways, and evidence of infection extended to adjacent lung parenchyma [Figure 1E(iii)]. Significantly higher severity of both bronchitis and pneumonia was observed in Dox-IAV mice (Figure 1F).

Inflammatory cell influx into the BAL fluid was measured by flow cytometry. Notable increases in inflammatory cells in both Dox (largely macrophages, some eosinophils) and IAV-infected groups (predominantly macrophages, inflammatory macrophages, and neutrophils) were enhanced in Dox-IAV mice (Figure 1G). All individual cell types, apart from inflammatory macrophages, were significantly elevated in Dox-IAV mice compared with IAV mice (Figure 1G). The Dox-induced increases were TGF-β dependent, because these changes were absent in nontransgenic mice treated with Dox (Figure E1B). Furthermore, at Day 3 postinfection, TNF-α, MCP-1/CCL2, and IL-6 concentrations in BAL fluid were significantly higher in Dox-IAV mice (Figure 1H).

Effects of TGF-β overexpression on host defense responses were investigated using quantitative PCR analyses of IFNs and IRGs. Induction of mRNA coding for Ifnλ₂, Ifit1, Ifit2, and Isg15 after IAV infection was strikingly reduced in Dox-IAV mice (Figure 1I). Nontransgenic mice that received Dox and infected with IAV did not exhibit similar immune suppression, confirming the specificity of responses (Figure E1C).

Prolonged Overexpression of TGF-β Alters Lung Structure with Persistent Enhancement of IAV Infection

Prolonged elevation of TGF-β concentrations in patients with chronic inflammatory lung diseases results in phenotypic changes within the lungs, including fibrosis and alveolar septal damage (5). To validate these structural changes in our transgenic murine model, we induced TGF-β expression for 8 weeks before infection (Figure 2A).

Figure 2.

Prolonged TGF-β exposure alters lung architecture and exacerbates IAV infection. (A) CC10-rtTA-tTS-TGF-B1 transgenic mice were administered Dox for 8 weeks before infection with IAV. Mice were culled at Day 3 postinfection for analysis. (B) Daily weights, mean ± SEM, n = 8–15 per group, two-way ANOVA. (C) Viral titers by plaque assay, n = 12 per group, unpaired t test. (D) Histological lung samples scored for bronchitis and pneumonia severity as outlined. Individual scores and median values, n = 5 per group, Wilcoxon test. (E) Total cell numbers in BAL fluid, n = 3–4 per group, one-way ANOVA. (F) TNF-α, MCP-1/CCL2, and IL-6 protein in BAL fluid by CBA or ELISA, n = 4–10 per group, one-way ANOVA. n.d. = not detected. (G) Representative Masson’s trichrome images from (i) control mice, (ii) Dox-treated mice, (iii) IAV-infected mice, and (iv) Dox-IAV–infected mice (scale bars, 200 μm) for (H) (i) mean epithelial thickness, (ii) mean subepithelial thickness, and (iii) mean linear intercept, n = 4–13 per group, one-way ANOVA. Data are representative of a minimum of two independent experiments. (C, E, F, H) are box-and-whisker plots showing median and IQR, with data points shown only for individual values greater than the 75th percentile plus 1.5 times the IQR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

With prolonged TGF-β overexpression, Dox-IAV mice lost significantly more weight (Figure 2B). Viral loads were 2.3-fold higher than in wild-type IAV-infected mice (Figure 2C); these findings were similar to short-term studies (Figure 1D).

Similar to short-term TGF-β induction, the influx of inflammatory cells after IAV infection was increased in Dox-IAV mice after 8 weeks (Figure 2E). Airway macrophages, neutrophils, lymphocytes, and eosinophils were all significantly elevated in Dox-IAV mice (data not shown). Consistent with detection of elevated inflammatory cell populations, TNF-α, MCP-1/CCL2, and IL-6 concentrations in BAL fluids were raised in Dox-IAV mice (Figure 2F).

Masson’s trichrome–stained lung sections showed a decline in mean epithelial thickness when TGF-β was present in excess [Figures 2G and 2H(i)]. Only the increase in mean subepithelial thickness, associated with collagen accumulation and airway fibrosis, was further enhanced by IAV infection [Figures 2G and 2H(ii)]. Alveolar enlargement, assessed using mean linear intercept measurements, was not evident [Figure 2H(iii)]. Taken together, these data confirmed that sustained TGF-β overexpression induced morphological changes accompanied by enhanced IAV infection and associated inflammation.

Pirfenidone Mitigates TGF-β–induced Severity of IAV Infection

Pirfenidone is an effective oral antifibrotic agent used for IPF but was initially developed as an antiinflammatory agent after showing considerable potential in preclinical models (18). We postulated that pirfenidone would counter the effects of TGF-β and mitigate immune suppression and disproportionate inflammation driven by excess TGF-β. Overexpression of TGF-β was induced, and pirfenidone (30 mg/kg/d [Figure E2A] or 100 mg/kg/d [Figure 3A]) was administered to evaluate benefits in the IAV infection model.

Figure 3.

Pirfenidone curbs IAV infection and reduces disease severity. (A) CC10-rtTA-tTS-TGF-B1 transgenic mice were administered Dox throughout the experiment starting 48 hours before pirfenidone treatment. Oral pirfenidone was administered in the morning and evening 48 hours before infection with IAV. On the day of IAV infection, pirfenidone was administered 30 minutes before IAV inoculation. Mice were culled at 2 and 3 days postinfection for analysis. (B) Daily weights, mean ± SEM, n = 8–23 per group, two-way ANOVA. (C) Virus titers by plaque assay, n = 10–11 per group, one-way ANOVA. (D) Histological samples scored for bronchitis and pneumonia severity. Individual scores and median values, n = 11–14 per group, Kruskal-Wallis test; ***P < 0.001 versus Dox-IAV; ^#P < 0.05 between groups. (E) Total cell number and inflammatory cell subtypes in BAL fluid, n = 10–13 per group, one-way ANOVA. (F) TNF-α, MCP-1/CCL2, and IL-6 protein in BAL fluid by CBA or ELISA, n = 9–13 per group, one-way ANOVA. (G) Ifnλ₂, Ifit1, Ifit2, and Isg15 mRNA expression by real-time PCR and normalized to Gapdh, n = 3 for control and Dox, n = 12–19 per IAV groups, one-way ANOVA. Data are representative of a minimum of two independent experiments. (C, E, F, G) are box-and-whisker plots showing median and IQR, with data points shown only for individual values greater than the 75th percentile plus 1.5 times the IQR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Infected mice treated with either dose of pirfenidone lost significantly less weight than the Dox-IAV mice (Figures E2B and E3B). Only the higher dose of pirfenidone reduced viral replication in lung homogenates compared with untreated Dox-IAV mice (Figure 3C). The inhibitory effects of pirfenidone on replication were not evident in nontransgenic IAV-infected mice (Figure E2C). Decreased viral loads after treatment with 100 mg/kg/d pirfenidone were matched by reduced disease severity as reflected by lower bronchitis scores (Figure 3D). The lower dose of pirfenidone had no impact on pathology scores (Figure E2D).

Total cell numbers in BAL from Dox-IAV mice were significantly lower with pirfenidone treatment, chiefly because of lower macrophage and eosinophil numbers (Figure 3E). BAL concentrations of TNF-α, MCP-1/CCL2, and IL-6 were also reduced (Figure 3F). Notably, pirfenidone did not prevent suppression of antiviral immune responses by TGF-β in Dox-IAV mice (Figure 3G).

To determine whether therapeutic administration of pirfenidone had similar effects, we conducted further experiments, beginning pirfenidone treatment 1 day after IAV infection (Figure E3A). Administered in this fashion, pirfenidone did not appreciably reduce weight loss, viral loads, inflammatory cell accumulation, or mediator production in response to infection (Figures E3B–E3E), demonstrating that early administration of pirfenidone is required for efficacy in this model.

Pirfenidone Treatment Does Not Suppress TGF-β Production in Mice or in Human Disease

A key question is whether the actions of pirfenidone are mediated by direct effects on TGF-β production. To examine this aspect, the effect of pirfenidone on TGF-β protein concentrations in BAL fluid was measured in transgenic mice. The Dox-induced elevation in TGF-β protein was previously established (Figure E1A), and pirfenidone did not reduce BAL TGF-β in infected transgenic mice (Figure E4A).

Given that pirfenidone is in clinical use for IPF, we also evaluated the effect of treatment on concentrations of TGF-β in peripheral blood from patients with verified IPF. Venesection was performed before and 3 months after commencement of oral pirfenidone. Serum concentrations of TGF-β were not raised in IPF relative to healthy people and were not altered by pirfenidone (Figure E4B). Protein concentrations of other biomarkers implicated in inflammation and fibrosis (33) were also measured before and after pirfenidone treatment (Figure E4). Periostin and matrix metalloproteinase-7, but not C-reactive protein or vascular endothelial growth factor, were elevated in IPF at baseline compared with healthy controls; however, all were unchanged after treatment with pirfenidone (Figures E4C–E4F).

Mechanistically, Pirfenidone Inhibits Both NF-κB and IRF3 Pathways but Has No Effect on Necroptosis

We next investigated the effect of pirfenidone on cytokine release mediated via type I IFN and pathogen-sensing signaling pathways in vitro (Figure 4A). hTERT-immortalized fibroblasts were incubated with IFN-β to activate the transcriptional activator IFN-stimulated gene factor 3 (ISGF3) via STAT1/2 and IFN regulatory factor 9 (IRF9). Alternatively, cells were treated with synthetic double-stranded RNA (poly I:C) or bacterial LPS to activate Toll-like receptor 3 (TLR3) or TLR4 or were transfected with immunostimulatory DNA to activate the cGAS-STING (cyclic GMP-AMP synthase stimulator of IFN genes) signaling pathway. Production of both IL-6 and IP-10, which predominantly results from the activation of NF-κB, IRF3, and ISGF3 signaling pathways by innate immune agonists in hTERT cells (Figure 4A), was markedly attenuated by pirfenidone (Figures 4B and 4C), establishing the broad antiinflammatory and immune-modulatory effects of pirfenidone on innate immune signaling.

Figure 4.

Mechanistically pirfenidone inhibits both NF-κB and IFN regulatory factor 3 (IRF3) pathways, but not necroptosis. (A) Signaling pathways and potential sites of inhibition by pirfenidone. Human telomerase reverse transcriptase immortalized fibroblasts stimulated with 250 IU/ml IFN-β, 10 μg/ml polyinosinic-polycytidylic acid (polyI:C or pI:C), or 100 ng/ml LPS or transfected with 2.5 μg/ml immunostimulatory DNA (ISD) for 1 hour before the addition of 0.5 mg/ml pirfenidone. IL-6 and IP-10 were measured by ELISA in conditioned media collected either (B) 6 hours post-treatment or (C) 24 hours post-transfection, n = 3. (D–H) CC10-rtTA-tTS-TGF-B1 transgenic mice were administered Dox throughout the experiment starting 48 hours before pirfenidone treatment. Oral pirfenidone was administered in the morning and evening 48 hours before infection with IAV. On the day of IAV infection, pirfenidone was administered 30 minutes before IAV inoculation. Mice were culled at 3 days postinfection, and lung homogenates were used for Western blots. (D) Western blot analysis of signaling components in the necroptosis pathway. Each lane represents an individual mouse. Densitometry for (E) total RIPK3, (F) pRIPK3:RIPK3, (G) total MLKL, and (H) pMLKL:MLKL, mean ± SEM, n = 3 per group. *P < 0.05, ***P < 0.001, (B, C) unpaired t test or (E–H) one-way ANOVA.

Necroptosis is a proinflammatory form of caspase-independent cell death that is believed to have evolved to combat infections (34) and can be triggered by TLR ligation, TNF-α receptor activation, or pathogen sensing by ZBP1 (35). After activation, RIPK1 and RIPK3 assemble to form a complex with MLKL, with RIPK3-mediated phosphorylation of MLKL leading to lytic cell death. Because both IAV and COPD have been linked to RIPK1 and RIPK3 activity (36–38) and TNF-α itself is elevated after IAV infection, we sought to determine whether pirfenidone-induced reductions in TNF-α were attenuating this necroptotic signaling.

Examination of total and phosphorylated RIPK3 and MLKL by Western blot analysis in lung homogenates from control, IAV, Dox-IAV, and Dox-IAV-Pirf mice (Figure 4D) revealed no major changes in RIPK3 or pRIPK3 concentrations (Figures 4D–4F). In comparison, total MLKL protein was higher in mice infected with IAV than in control animals, with no significant induction of pMLKL (Figures 4D, 4G, and 4H). Importantly, pirfenidone had no effect on the total or phosphorylated forms of either RIPK3 or MLKL compared with the Dox-IAV group, suggesting that despite reducing TNF-α concentrations, pirfenidone is not curbing infection by limiting necroptosis (Figures 4D–4H).

Inhaled Pirfenidone Confers Benefits Comparable with Oral Administration and May Have Advantages over GCS Treatment

Having demonstrated that early administration of oral pirfenidone blunts the response to IAV infection, we next examined an inhaled formulation of pirfenidone (developed by Avalyn Pharma [27, 39]) to minimize potential side effects. To mirror clinical use, prophylactic inhaled pirfenidone was compared with inhaled daily GCS treatment currently used by many patients with COPD who experience frequent exacerbations (Figure 5A).

Figure 5.

Attenuation of disease severity by inhaled pirfenidone is superior to inhaled glucocorticosteroid (GCS) treatment. (A) CC10-rtTA-tTS-TGF-B1 transgenic mice were administered Dox for 48 hours, then inhaled pirfenidone (PFD) or inhaled GCS were administered for 48 hours before infection with IAV. Mice were culled 3 days postinfection. (B) Daily weights, mean ± SEM, n = 5–18 per group, two-way ANOVA, *P < 0.05 Dox-IAV versus Dox-IAV-GCS. (C) Virus titers were measured by plaque assay, n = 5–12 per group, presented as a box-and-whisker plot showing median and IQR, one-way ANOVA, ***P < 0.001 for Dox-IAV-PFD versus both groups. (D) Disease severity scored for bronchitis, n = 6 per group, Kruskal-Wallis, then Dunn’s multiple comparisons test, P < 0.005 for Dox-IAV-PFD versus Dox-IAV. (E) Disease severity scored for pneumonia, n = 11–14 per group. (F) IL-6, KC, TNF-α, and MCP-1/CCL2 protein in BAL fluid measured by ELISA, presented as box-and-whisker plot showing median and IQR, n = 5–11 per group, one-way ANOVA. Data are representative of a minimum of two independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001.

Before infection, only mice receiving inhaled GCS lost weight, with similar IAV-induced weight loss observed in all groups by 3 days postinfection (Figure 5B). Notably, mice treated with inhaled pirfenidone had significantly lower IAV replication than both the untreated and GCS-treated Dox-IAV mice (Figure 5C). Significant reductions in bronchitis severity scores were observed with inhaled pirfenidone but not GCS (Figure 5D). Pneumonia scores tended to be numerically lower in the pirfenidone-treated group but were not statistically different (Figure 5E). Finally, concentrations of inflammatory mediators, including IL-6, KC, TNF-α, and MCP-1, were significantly reduced in the pirfenidone-treated mice but not in the GCS-treated group (Figure 5F).

Pirfenidone Reduces Disease Severity after IAV Infection in a CS Exposure Model of COPD

Our initial studies established therapeutic effects of pirfenidone in models of TGF-β overexpression. Because excess TGF-β production has been reported in COPD (40), we next evaluated possible benefits of the compound in an established model of chronic CS exposure (25). The experimental groups were no IAV infection (CS), no pirfenidone treatment (CS-IAV), and oral administration of pirfenidone starting 2 days before infection (CS-IAV-Pirf; Figure 6A).

Figure 6.

Pirfenidone reduces viral replication and disease severity in a murine cigarette smoke (CS)-induced model of chronic obstructive pulmonary disease. (A) Balb/c mice were exposed to CS for 8 weeks before infection with IAV. Pirfenidone treatment (100 mg/kg/d, administered in two doses) began 2 days before IAV infection and continued throughout the time course. Mice were culled at Day 3 postinfection for analysis. (B) Masson’s trichrome–stained sections for mean linear intercept, n = 4–8 per group, unpaired t test. (C) Viral titers by plaque assay, n = 7–8 per group, one-way ANOVA. (D) Representative Masson’s trichrome images from (i) control, (ii) CS-exposed, (iii) CS-IAV–infected, and (iv) CS-IAV-Pirf mice. Scale bars, 200 μm. (E) Disease severity scored for bronchitis (0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe). Individual scores and median values, n = 4–8 per group, Wilcoxon test. (F) n = 7–16 per group, one-way ANOVA. (G) MCP-1/CCL2 and IL-6 protein in BAL fluid by CBA, n = 7–8 per group, one-way ANOVA. (H) (i) Ifnλ₂, (ii) Ifit1, (iii) Ifit2, and (iv) Isg15 mRNA expression by real-time PCR and normalized to Gapdh, n = 5–11 per group, one-way ANOVA. All data are representative of a minimum of two independent experiments. (B, C, F–H) are box-and-whisker plots showing median and IQR, with data points shown only for individual values less than the 25th percentile minus 1.5 times the IQR or greater than the 75th percentile plus 1.5 times the IQR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

The mean linear intercept was significantly greater in mice exposed to CS (Figure 6B), consistent with classic phenotypic lung manifestations of COPD. TGF-β mRNA in lung tissue of CS-exposed mice was elevated compared with nonexposed control animals (280.12 ± 19.4 vs. 172.9 ± 9.6, ΔΔCT normalized to Gapdh; P < 0.001), but TGF-β protein in BAL fluid remained below detection levels.

Viral loads in CS-exposed mice were significantly reduced by pirfenidone (Figure 6C). Increased bronchitis scores, reflecting epithelial damage and inflammatory cell accumulation, were also attenuated by the compound (Figures 6D and 6E). Pirfenidone marginally reduced IAV-induced increases in total inflammatory cell influx in BAL (Figure 6F; P = 0.09) and significantly decreased proinflammatory cytokines, including MCP-1/CCL2 and IL-6 (Figure 6G). IAV-induced increases in TNF-α were more variable in CS mice, and although an inhibitory effect of pirfenidone was observed, it did not reach statistical significance (data not shown). Pirfenidone treatment was not associated with increased expression of either Ifnλ2, Ifit1, Ifit2, or Isg15 gene expression compared with CS-IAV (Figure 6H).

Discussion

Inflammatory and fibrotic lung conditions, including COPD, are characterized by overproduction of TGF-β. We used murine models of TGF-β overexpression and CS-induced COPD to demonstrate damaging consequences associated with this cytokine, including enhanced infection, inflammation, and fibrosis, after viral infection. Treatment with pirfenidone, a known inhibitor of TGF-β–mediated activities, mitigated these detrimental effects, and inhaled pirfenidone was superior to inhaled GCS treatment. Collectively, our current studies reveal a key role for TGF-β in the pathogenesis of exacerbated lung disease caused by viral infection and indicate that pharmacological treatment directed at blunting activities of TGF-β can diminish these pathologies.

TGF-β is active in a spectrum of human airway diseases characterized by inflammation with accompanying mild to severe fibrosis (40, 41, 42). The potential contribution of TGF-β to increased susceptibility to infections, chiefly viral infections such as IAV, is therefore worthy of consideration. Once initiated, a pattern of repeated exacerbations occurs with increasing frequency, severely compromising patient quality of life with declining lung function caused by inflammation, tissue damage, and fibrosis (43, 44). We and others have shown that TGF-β blunts IFN production and IRG responses in epithelial cells and fibroblasts, leading to amplified inflammation and worsened infection (31, 45, 46). However, to date, the putative role of TGF-β as a central regulator of damaging lung tissue responses in vivo has not been characterized, to our knowledge, and there are no known preventative or other therapeutic strategies.

We first evaluated responses to IAV infection in a transgenic murine model of TGF-β overexpression. Although elevated concentrations of TGF-β have been reported in lung diseases (24, 47–49), BAL data for COPD are lacking. The Dox-induced concentrations in mouse BAL are in the range of exogenous TGF-β concentrations shown to induce fibrosis or inhibit rhinovirus-induced immune responses in cell culture studies (31, 50). Disease severity was greater with excess TGF-β (Dox-IAV mice), including greater weight loss, higher mortality, and enhanced viral loads. Worsened bronchitis- and pneumonia-like changes with TGF-β overexpression were accompanied by increased inflammatory cytokines. Innate immune responses to IAV as reflected by Ifnλ₂ and IRGs were suppressed. TGF-β has been implicated in both the development and homeostasis of alveolar macrophages (51). However, this was not evident in the present model, where macrophage numbers in BAL were significantly elevated only in mice that were infected with IAV either without or with Dox treatment. These in vivo findings confirming enhanced IAV infection with increased TGF-β were concordant with amplified rhinovirus and respiratory syncytial virus infections that we and others have demonstrated using in vitro cell culture models (31, 45, 46).

Overexpression of TGF-β leads to progressive airway structural changes in addition to compromised immune responses to infection (5). We therefore extended TGF-β overexpression to 8 weeks, followed by IAV infection. Airway structural alterations attributable to TGF-β alone were evident as reduced epithelial thickness and increased subepithelial fibrosis. The negative impacts of prolonged TGF-β overexpression on virus infection, disease severity, and inflammation were similar to our findings in short-term studies. Notably, the effects of TGF-β and IAV infection on airway fibrosis appeared to be additive, consistent with reports of bidirectional interactions whereby IAV infection of mice mediated greater degrees of fibrosis via increased TGF-β activation (52).

Pirfenidone is an orally available synthetic compound approved worldwide for the treatment of IPF (12, 13, 28). The mechanism of action of pirfenidone is not fully understood, but its antifibrotic and antiinflammatory activities may be exerted through inhibition of TGF-β signaling (28) and by dampening the activity of NF-κB and other transcription factors (53). The benefits of pirfenidone in the context of infection have not been evaluated, although clinical studies have reported reductions in the frequency of acute IPF exacerbations, often linked to intercurrent viral infections (22). We demonstrate, for the first time, to our knowledge, that prophylactic treatment with pirfenidone can diminish the severity of disease after IAV infection in both TGF-β–overexpressing mice and in a model of COPD characterized by excess TGF-β. The compound blunted viral replication and improved bronchitis and pneumonia scores, as well as reducing other parameters of lung inflammation. We also demonstrate that both inhaled and oral pirfenidone administration are chiefly successful if administered before viral infection.

Employing inhaled pirfenidone to blunt inflammation may arguably be superior to current practice, avoiding the marked immunosuppressive effects of GCS (54, 55) that reduce their efficacy in virus-induced exacerbations (56). We have established the relative benefits of inhaled pirfenidone compared with inhaled GCS, including greater inhibition of viral loads and inflammation. An inhaled formulation of pirfenidone has been reported to have few side effects in patients with IPF (27, 39), further supporting its potential clinical utility as a daily “preventer” for COPD exacerbations.

Potential mechanisms fundamental to the inhibitory effects of pirfenidone were investigated. Pirfenidone treatment had no effect on TGF-β in either BAL from transgenic mice after in vivo induction using Dox or serum of patients receiving oral pirfenidone for IPF. This suggests a mechanism of action for pirfenidone involving inhibition of inflammation independent of regulation of TGF-β production but potentially inhibiting actions of TGF-β via reduced signaling through its receptors or further downstream in its signaling pathway. Pirfenidone had no antiviral activities unless TGF-β was overexpressed, but its efficacy was not associated with reduced concentrations of the cytokine or restoration of innate immune responses. In serum samples from patients with IPF, TGF-β was not elevated compared with control healthy individuals, consistent with previous reports (47, 57). Pirfenidone also had no impact on systemic TGF-β concentrations in this clinical context, indicating that inhibition of TGF-β signaling pathways locally within the lung itself may be yielding benefits in IPF and suggesting that a local inhaled medication could have therapeutic advantages.

Inflammatory pathways impacted by pirfenidone in vitro were IRF3/9 and NF-κB regulated, attesting to its broad molecular activities that may contribute to robust efficacy of the compound noted in current studies. In the in vitro system here—that is, in the context of innate immune agonists and the hTERT fibroblasts—IL-6 production is mediated more strongly by NF-κB than by ISGF3, as evidenced by greater responses to LPS (TLR4) than poly I:C (TLR3) compared with IFN-β (IFNAR1/2). Conversely, similar IP-10 production in response to poly I:C and LPS is consistent with similar IRF3 responses mediating this induction. We acknowledge that these infection-relevant stimuli activate additional signaling pathways not addressed in the present study, but we propose that in demonstrating the broad antiinflammatory and immune-modulatory effects of pirfenidone on innate immune signaling in vitro, we have identified some potential mechanisms contributing to its demonstrated antiinflammatory actions in the context of infection in vivo.

Our further investigations examining protein expression in lungs from transgenic mice overexpressing TGF-β and infected with IAV failed to demonstrate an inhibitory effect of pirfenidone on the RIPK/MLKL signaling pathways implicated in necroptosis associated with viral infection and elevated TNF-α (36, 38). Although the mechanisms underlying the reduction in viral loads with pirfenidone remain to be defined, the substantial antiinflammatory benefits accompanying demonstrable reductions in viral replication make pirfenidone a uniquely suitable therapy for virus-associated disease exacerbations in COPD.

The propensity of patients with COPD to contract viral infections leads to repeated exacerbations, an inexorable decline in lung function, and premature death (58). Because TGF-β is overexpressed in COPD (59), we conducted proof-of-concept investigations to test pirfenidone in a validated murine model of CS-induced COPD (25). Chronic CS exposure induced TGF-β gene expression that was associated with inflammation and airway remodeling, including fibrosis and emphysema, as previously shown (25). Pirfenidone attenuated IAV-induced weight loss and reduced both viral loads and bronchitis severity. As noted in our studies with excess TGF-β, pirfenidone diminished other inflammatory indices, such as total BAL cell counts and cytokine production induced by IAV infection. Chronic CS exposure has previously been shown to suppress the IFN response to IAV infection (60). Consistent with our findings in the TGF-β overexpression model, the reduction in viral loads observed with pirfenidone treatment in IAV-infected mice after chronic CS exposure was not due to increased IFN or IRG gene expression, at least not at the 3-day postinfection time point.

Limitations

There are several important caveats to our study. First, in our novel proof-of-concept studies, we demonstrated dose-dependent benefits of oral pirfenidone (30 mg/kg and 100 mg/kg) in the context of excess TGF-β. Only the higher dose, approximately two to three times higher than currently used in IPF (30–50 mg/kg), was assessed in the chronic COPD model. Because the dose of inhaled pirfenidone tested was much lower (13.2 mg/kg) than the clinical oral dose, and because there is emerging strong evidence that an inhaled formulation is practicable (27, 39), further studies using an inhaled treatment strategy and extended to the CS model are warranted. Second, we did not investigate combined use of inhaled pirfenidone (preventer use) followed by oral pirfenidone (therapeutic use). The use of this protocol in the context of IAV infection in COPD models would mirror current clinical practice using inhaled GCS for prophylaxis and oral GCS for exacerbations. Third, we were unable to determine the exact mechanism(s) whereby pirfenidone curbs IAV replication. The inhibitory effects of pirfenidone treatment could be due to reduced viral entry that would inhibit subsequent inflammation, reduced inflammation that could inhibit subsequent viral replication, or earlier boosting of type I IFN to increase viral clearance. Future studies to assess effects of pirfenidone on viral uptake and replication in vitro and regulation of type I IFN responses at earlier time points postinfection may help clarify the mechanisms underlying the beneficial actions of pirfenidone. The compound had no direct antiviral effects and did not restore reduced IFN production caused by TGF-β. Finally, the relevance of current studies to human COPD requires verification in appropriate human trials.

Conclusions

In conclusion, viral infections are a cardinal cause of poor outcomes in lung conditions such as COPD characterized by chronic inflammation. We provide evidence that TGF-β mediates endogenous immune suppression and contributes to severe infections, subsequent lung damage, and fibrosis. Targeting TGF-β by repurposing pirfenidone as a prophylactic broad-spectrum antiinflammatory may yield substantial benefit over GCS, without attendant steroid-related side effects or detrimental immune suppression.