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Abstract
Rationale: Necroptosis, mediated by RIPK3 (receptor-interacting protein kinase 3) and MLKL (mixed lineage kinase domain-like), is a form of regulated necrosis that can drive tissue inflammation and destruction; however, its contribution to chronic obstructive pulmonary disease (COPD) pathogenesis is poorly understood.
Objectives: To determine the role of necroptosis in COPD.
Methods: Total and active (phosphorylated) RIPK3 and MLKL were measured in the lung tissue of patients with COPD and control subjects without COPD. Necroptosis-related mRNA and proteins as well as cell death were examined in lungs and pulmonary macrophages of mice with cigarette smoke (CS)-induced experimental COPD. The responses of Ripk3−/− and Mlkl−/− mice to acute and chronic CS exposure were compared with those of wild-type mice. The combined inhibition of apoptosis (with the pan-caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone [qVD-OPh]) and necroptosis (with deletion of Mlkl in mice) was assessed.
Measurements and Main Results: The total MLKL protein in the epithelium and macrophages and the pRIPK3 and pMLKL in lung tissue were increased in patients with severe COPD compared with never-smokers or smoker control subjects without COPD. Necroptosis-related mRNA and protein levels were increased in the lungs and macrophages in CS-exposed mice and experimental COPD. Ripk3 or Mlkl deletion prevented airway inflammation upon acute CS exposure. Ripk3 deficiency reduced airway inflammation and remodeling as well as the development of emphysematous pathology after chronic CS exposure. Mlkl deletion and qVD-OPh treatment reduced chronic CS-induced airway inflammation, but only Mlkl deletion prevented airway remodeling and emphysema. Ripk3 or Mlkl deletion and qVD-OPh treatment reduced CS-induced lung-cell death.
Conclusions: Necroptosis is induced by CS exposure and is increased in the lungs of patients with COPD and in experimental COPD. Inhibiting necroptosis attenuates CS-induced airway inflammation, airway remodeling, and emphysema. Targeted inhibition of necroptosis is a potential therapeutic strategy in COPD.
Emphysema and chronic obstructive pulmonary disease (COPD) are likely underpinned by aberrant cell death that leads to airway inflammation, airway remodeling, and emphysema. Apoptotic cell death can be programmed and controlled during apoptosis and is considered to be noninflammatory, whereas necroptosis, a form of regulated necrosis, is highly proinflammatory. The roles of these pathways in COPD are poorly understood.
We used combination analysis of human COPD lung tissue, mouse models of experimental COPD, mice deficient in key necroptotic pathway mediators (RIPK3 [receptor-interacting protein kinase 3] or MLKL [mixed lineage kinase domain-like]), and inhibitors to define the roles of cell death pathways. Necroptosis signaling is increased in the lungs in human and experimental COPD and correlates with disease severity. Genetic necroptosis inhibition suppresses airway inflammation and remodeling and emphysema in experimental COPD, whereas pharmacological caspase inhibition reduces inflammation only. Inhibiting necroptosis may be a new therapeutic approach for COPD.
Chronic obstructive pulmonary disease (COPD) is a common lung disease characterized by persistent respiratory symptoms and impaired lung function (1). It is underpinned by a varied and complex set of pathologies, including airway inflammation, airway remodeling, and emphysema (destruction and enlargement of alveoli). The major risk factor is exposure to cigarette smoke (CS). Inhaled corticosteroids combined with long-acting β2-agonists can partially suppress inflammation and provide symptomatic relief in patients with COPD (1). However, there are currently no treatments that halt or reverse progression of the disease or symptomatic burden, highlighting the need for mechanistic studies to identify new therapeutic targets.
Aberrant cell death correlates with the development of emphysema in COPD (2–4). Cell death can occur through apoptosis, various types of regulated necrosis, or “unregulated” necrosis. The mechanism by which cells die has important implications for inflammatory responses during disease (5). Apoptosis is the best characterized form of cell death and has been previously linked to increased lung-cell death in emphysema (6). However, critical evidence of a causal relationship between apoptosis and emphysema is lacking; the role of apoptosis in COPD is poorly understood.
Necroptosis is a genetically encoded mechanism of necrotic cell death defined by rupture of the plasma membrane. It involves 4 RHIM domain-containing proteins, including RIPK1 (receptor-interacting protein kinase 1) and RIPK3, which eventually converge on RIPK3-mediated phosphorylation of MLKL (mixed lineage kinase domain-like) , which executes necroptosis. It is a form of regulated cell death but is highly proinflammatory because of the release of damage-associated molecular patterns (DAMPs) into the extracellular environment after MLKL-mediated plasma membrane rupture (7). Selected TLR (Toll-like receptor) ligands, TNFα, and IFNs can induce necroptosis, although the pathway is endogenously inhibited by proapoptotic caspase-8 (7, 8). These necroptosis stimuli are also implicated in COPD pathogenesis (9–11). Inhibiting RIPK1 kinase activity can prevent cell death by apoptosis and/or necroptosis. In the latter, RIPK1 is an upstream activator of RIPK3 and MLKL, leading to necroptosis. RIPK1 inhibition can suppress airway inflammation induced by acute CS exposure and prevent decreases in the metabolic activity of epithelial-cell cultures induced by toxic doses of CS extract (CSE) in vitro (12). However, the role of RIPK3 and MLKL and the relative contributions of necroptosis and apoptosis in COPD pathogenesis have not been assessed.
Here, we define for the first time the contribution of necroptosis- and apoptosis-related signaling to COPD pathogenesis by using lung tissue samples from patients with COPD, never-smokers, and smoker control subjects without COPD and mouse models of CS-induced experimental COPD. Some of the results of these studies have been previously reported in the form of an abstract (13–15).
Detailed descriptions of the human and mouse ethics statements and studies, the induction of CS-induced experimental COPD, the quinoline-Val-Asp-difluorophenoxymethylketone (qVD-OPh) intervention, BAL fluid (BALF) staining, immunohistochemistry and immunofluorescence, the RNA sequencing of fluorescence-activated cell sorter–sorted alveolar macrophages and data processing, and the statistical analysis are as previously described (16–44) and are included the online supplement.
Human studies were approved by the medical ethics committee of Ghent University Hospital (2016/0132) and the University Hospital Gasthuisberg (S51577). Descriptions of the study cohorts and sampling are provided in the online supplement (see Tables E1 and E2 in the online supplement).
Lung tissues from our biobank at Ghent University Hospital and explants from patients with end-stage COPD from the University Hospital Gasthuisberg (Leuven, Belgium) were analyzed. On the basis of preoperative spirometry findings and results from diffusion capacity tests and questionnaires, subjects were categorized as never-smokers and smokers without airflow limitation or as patients with COPD. COPD severity was defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification. No patients were treated with neoadjuvant chemotherapy. Lung tissue from patients with solitary pulmonary tumors was obtained at the maximum distance from the pulmonary lesions and was confirmed by a pathologist to have no signs of retroobstructive pneumonia or tumor invasion.
Mouse studies were approved by the University of Newcastle Animal Ethics Committee and the Faculty of Medicine and Health Sciences (Ghent University).
At the Hunter Medical Research Institute, mice were exposed to CS twice per day and 5 d/wk for up to 8 weeks by using a custom-built, nose-only system, which used 12 cigarettes per run (12–20). At Ghent University (alveolar macrophage RNA sequencing), mice underwent whole-body exposure to the smoke of five cigarettes without a filter four times per day and 5 d/wk for 4 weeks (21–24). Control mice were exposed to normal air.
For human studies, data are presented as the median ± interquartile range (n = 8–16 per group). The Shapiro-Wilk test was used to assess normality, which was followed by one-way ANOVA with a Bonferroni post hoc correction for multiple comparisons or a Kruskal-Wallis test with a Dunn test for multiple comparisons. The Spearman rank correlation (RS) was used to test for correlations, with a P value of <0.05 being considered to indicate statistical significance. Linear regression analyses were performed by using SPSS software (IBM) to correct for potential confounders (age, sex, current smoking). For mouse experiments, data are presented as the mean ± SEM, and n = 4–6 mice per group. Student’s t tests were performed to compare two groups, and for multiple comparisons of more than two groups, Bonferroni corrections were done.
We performed immunohistochemical staining for total RIPK3 and MLKL and immunoblotting for total RIPK3 and MLKL as well as pRIPK3 (phosphorylated RIPK3) and pMLKL on lung tissue from two cohorts (clinical characteristics are shown in Tables E1 and E2), comparing never-smokers and smokers without airflow obstruction and participants with COPD at GOLD stages II and III–IV. Immunohistochemistry revealed positive RIPK3 and MLKL staining in the airway epithelium (Figure 1A and see Figure E1A). The quantification of epithelial RIPK3 (see Figure E1B) and immunoblot analysis for total RIPK3 and MLKL (see Figures E1C–E1F) showed no differences in total RIPK3 or MLKL across the different patient groups. The quantification of epithelial MLKL showed significantly higher levels in patients with COPD at GOLD stages III–IV than in never-smokers and smokers without airflow limitation (Figure 1B), which remained significant after adjustment for age, sex, and smoking status (β = 13.7 ± 5.9, P = 0.0251). We also detected MLKL protein in alveolar macrophages (Figure 1C) and observed a significant increase in MLKL staining intensity in alveolar macrophages of patients with COPD at GOLD stages III–IV compared with never-smokers (Figure 1D). pRIPK3 and pMLKL were significantly increased in lung tissue homogenates from patients with severe to very severe COPD (GOLD stages III–IV) compared with smokers without airflow limitation (Figures 1E–1H, when normalized to GAPDH, or Figures E1G and E1H, when normalized to total RIPK3 or MLKL protein). After adjustment for age, sex, and current smoking by linear regression analyses, the associations of pRIPK3 (β = 0.90 ± 0.42, P = 0.038) and pMLKL protein levels (β = 1.58 ± 0.46; P = 0.002) with COPD at GOLD stages III–IV remained significant. Moreover, we found a strong, positive correlation between pRIPK3 and pMLKL protein levels (Figure 1I; RS = 0.7287, P = 0.0007). pRIPK3 and pMLKL protein levels had a significant negative correlation with the DlCO (Figure 1J and 1K; RS = −0.5493, P = 0.0004 and RS = −0.5470, P = 0.0004). Thus, the elevated expression and activation of necroptosis proteins are associated with increasing COPD severity.
Figure 1. Protein levels and activation of RIPK3 (receptor-interacting protein kinase 3) and MLKL (mixed lineage kinase domain-like) are increased in human chronic obstructive pulmonary disease (COPD) lung resections. (A–D) Immunohistochemistry for MLKL in clinical samples. (A) Representative images of immunohistochemical staining for MLKL in lung tissue sections from never-smokers (NS), smokers without airflow limitation, and patients with COPD at Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages II and III–IV. (B) Quantification of MLKL+ staining in the airway epithelium (n = 8–16). (C) Micrographs of alveolar macrophages stained for MLKL. (D) Semiquantitative assessment of the MLKL signal in macrophages from NS, smokers without COPD, and patients with COPD at GOLD stages II and III–IV (n = 8–16). (E) Immunoblot of pRIPK3 (phosphorylated RIPK3) of lung tissue homogenates from NS, smokers without airflow limitation, and patients with COPD at GOLD stages II and III–IV. (F) Quantification of pRIPK3 immunoblotting of human lung tissue homogenates, as assessed by using densitometry; band intensities were normalized to GAPDH (n = 10/group). (G) Immunoblot of pMLKL on lung tissue homogenates from NS, smokers without airflow limitation, and patients with COPD at GOLD stages II and III–IV (n = 10/group). (H) Quantification of pMLKL immunoblotting of human lung tissue homogenates, as assessed by using densitometry; band intensities were normalized to GAPDH (n = 10/group). (I) Spearman rank correlation (RS) between pRIPK3 and pMLKL protein levels. (J and K) RS of pRIPK3 and pMLKL protein levels with DlCO (percentage). Statistical analyses were conducted by using the Shapiro-Wilk test to test for normality and were then conducted by using one-way ANOVA with Bonferroni post hoc correction for multiple comparisons (in D) or the Kruskal-Wallis test with Dunn multiple comparisons (in B, F, and H). RS was used to test for correlations (in I–K). Results are shown as the median ± interquartile range. *P < 0.05 and **P < 0.01 compared with other groups indicated. Pbm = perimeter of the basement membrane.
[More] [Minimize]To assess the potential role of necroptosis in COPD pathogenesis, we employed both nose-only (1, 4, 8, or 12 wk of exposure) and whole-body (4 wk of exposure) mouse models of CS-induced lung inflammation and COPD-like pathology. We performed TUNEL staining to assess cell death and found an increase in the number of positive cells in the lungs, in the airways, in the parenchyma, and among immune cells in experimental COPD, but positivity was not more prominent in a particular cell type (8 and 12 wk; Figure 2A). We next examined the mRNA transcript levels of the core necroptosis-related factors Ripk1, Ripk3, and Mlkl through quantitative PCR (qPCR) analysis of whole-lung tissue from mice exposed to CS at time points that preceded (4 wk) and were concomitant with (8 and 12 wk) increased cell death (TUNEL staining) in the lung parenchyma. Ripk1 mRNA expression was upregulated at 4 weeks but not at 8 or 12 weeks of nose-only CS exposure (see Figures E2A–E2C), whereas Ripk3 mRNA was not differentially expressed in the whole lungs of CS-exposed mice (see Figures E2D–E2F) compared with normal air–exposed mice. Mlkl mRNA was partially increased at 4 weeks (Figure 2B) and was significantly increased at 8 and 12 (P = 0.0644) weeks of CS exposure (Figure 2C). The qPCR comparison of blunt dissected airway tissue versus parenchymal tissue revealed increased airway mRNA expression of Mlkl after 8 weeks of CS exposure (Figure 2E). RNA sequencing of alveolar macrophages sorted by flow cytometry from the BALF of mice that underwent whole-body CS exposure for 4 weeks revealed significant increases in Ripk3 and Mlkl mRNA but not in Ripk1 mRNA, as compared control animals that underwent exposure to normal air (Figures 2F–2H). Ripk1 and Ripk3 protein levels were significantly increased after 8 weeks of nose-only CS exposure, at which point emphysematous pathology had developed (Figures 2I–2K) (17). Immunoblot analysis of total Mlkl protein in the lungs of mice exposed to CS or air for 8 weeks revealed two bands, the lower of which (∼50 kD) corresponds to Mlkl protein, which was also based on the absence of staining in lung tissue from Mlkl−/− mice (Figures 2L and 2M). Mlkl protein was increased in the lungs of mice exposed to CS for 8 weeks. In results similar to those for human COPD, immunofluorescence staining revealed increased MLKL intensity in the airway epithelial cells and macrophages of CS-exposed mice (8 wk), which was significantly elevated in macrophages (Figure 2N; see also Figures E2G and E2H). Thus, core necrosome components, particularly Mlkl, were increased at the mRNA and protein levels in lung tissue and alveolar macrophages in experimental COPD, which is similar to what is observed in the airways and alveolar macrophages from human samples.
Figure 2. Necroptosis-related mRNA and protein levels are increased in the lungs in experimental chronic obstructive pulmonary disease. (A) TUNEL staining in the lungs of normal air–exposed and nose-only cigarette smoke (CS)-exposed mice (4, 8, and 12 wk of nose-only CS exposure; n = 3–4/group). (B–D) Mlkl (mixed lineage kinase domain-like) mRNA expression in whole-lung tissues relative to the Hprt (hypoxanthine guanine phosphoribosyl transferase) housekeeping control after 4 weeks (B), 8 weeks (C), and 12 weeks (D) (n = 6/group). (E) Expression of Mlkl mRNA after 8 weeks of CS exposure in airway and parenchymal tissue (n = 6/group). (F–H) Ripk1 (receptor-interacting protein kinase 1) (F), Ripk3 (G), and Mlkl (H) mRNA expression in alveolar macrophages sorted by using flow cytometry from the BAL fluid of mice exposed to whole-body CS for 4 weeks (n = 5/group). (I) Immunoblot images for Ripk1, Ripk3, and the β-actin loading control in whole-lung homogenates after 8 weeks of nose-only CS exposure; the approximate molecular weights are indicated. (J and K) Ripk1 (J) and Ripk3 (K) changes determined by using densitometry normalized to β-actin (n = 3/group). (L) Immunoblot images for Mlkl and the vinculin loading control; the approximate molecular weights are indicated. (M) Mlkl levels determined by using densitometry normalized to vinculin (n = 5/group). (N) Representative images of immunofluorescence staining for MLKL in the airway epithelium and macrophages after 8 weeks of CS exposure. Arrows point to MLKL-positive macrophages. Scale bars, 50 μm. Data are the mean ± SEM. *P < 0.05 and ***P < 0.001 compared with other groups indicated. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with wild-type air-exposed control animals. For data with four groups, one-way ANOVA with Bonferroni correction for multiple comparisons was used, and for data with two groups, the Student’s t test was used. NS = not significant; TUNEL = terminal deoxynucleotide transferase–mediated dUTP nick end label.
[More] [Minimize]Ripk3−/−, Mlkl−/−, and wild-type (WT) mice were exposed to nose-only CS or normal air for 4 days. On Day 5, BALF was collected and total and differential leukocytes were enumerated (Figure 3). The numbers of total leukocytes, macrophages, neutrophils, and lymphocytes in the BALF significantly increased after CS exposure in the Ripk3−/−, Mlkl−/−, and WT groups (Figures 3A–3H). However, the numbers of total leukocytes and neutrophils were significantly reduced in CS-exposed Ripk3−/− and Mlkl−/− mice compared with WT control animals (Figures 3A, 3C, 3E, and 3G). Airway macrophage numbers tended to be lower in Ripk3−/− and Mlkl−/− mice (Figure 3B), and lymphocytes were significantly reduced in CS-exposed Ripk3−/− compared with WT mice, with a similar trend being observed in Mlkl−/− mice (Figure 3D). To further evaluate whether RIPK3 and MLKL deficiency affected acute CS-induced pulmonary inflammation, qPCR analysis was used to assess the mRNA expression of proinflammatory factors in whole-lung tissues of mice after 1 week of nose-only CS exposure. The transcript levels of Cxcl1, Mmp8, Mmp12, Ym1, Marco, and Mip1a all increased after CS exposure compared with air exposure in WT mice (see Figures E3A–E3M). Mlkl deficiency prevented the increase in Cxcl1, Mmp12, Ym1, and Marco, with some similar trends being observed in Ripk3−/− mice. Mlkl mRNA was significantly increased in WT mice (see Figure E3N). There was no detectable expression of Ripk3 or Mlkl mRNA in the lung tissue from the respective deficient mice, confirming their deletion (see Figures E3G and E3N).
Figure 3. Ripk3 (receptor-interacting protein kinase 3) and Mlkl (mixed lineage kinase domain-like) drive airway inflammation in response to acute cigarette smoke (CS) exposure (1-wk exposure, nose-only). (A–D) Numbers of total leukocytes (A), macrophages (B), neutrophils (C), and lymphocytes (D) in the BALF of normal air–exposed and CS-exposed wild-type (WT) and Ripk3−/− mice (n = 6/group). (E–H) Numbers of total leukocytes (E), macrophages (F), neutrophils (G), and lymphocytes (H) in air- and CS-exposed WT and Mlkl−/− mice (n = 6/group). Data are the mean ± SEM. *P < 0.05 and ****P < 0.0001 compared with other groups indicated. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with WT air-exposed control animals. One-way ANOVA with Bonferroni correction for multiple comparisons was used. BALF = BAL fluid; NS = not significant.
[More] [Minimize]To examine the role of Ripk3 in inflammation and pathology in experimental COPD, we exposed Ripk3−/− and WT mice to nose-only CS for 8 weeks. The numbers of total leukocytes, macrophages, neutrophils, and lymphocytes in the BALF were significantly increased after CS exposure in WT mice (Figures 4A–4D). Ripk3 deficiency prevented CS-induced increases in the total number of BALF leukocytes, macrophages, and lymphocytes, with a trend toward decreased neutrophils compared with WT mice being shown. mRNA expression of Cxcl1, Mmp8, Mmp12, Ym1, Marco, and Mip1a all increased after CS exposure in the WT groups (see Figure E4). Ripk3 deficiency suppressed the increases in Ym1 and Marco mRNA in deficient CS-exposed mice compared with WT CS-exposed control animals but did not affect the expression of other factors. CS exposure significantly increased the histopathologic scores in both the airway and large peribronchial blood vessels in both genotypes; however, Ripk3−/− mice had significantly less airway inflammation than their WT counterparts, with a similar trend toward decreased perivascular inflammation being shown (Figures 4E and 4F). Alveolar macrophage numbers were significantly increased with CS exposure in WT mice and were significantly reduced in Ripk3−/− mice (Figure 4G). The CS exposure of WT mice significantly increased collagen deposition around the airways, which was attenuated in the absence of Ripk3 (Figures 4H and 4I). CS-induced epithelial thickening was also significantly reduced in Ripk3−/− mice compared with WT mice (Figure 4J). CS exposure increased the mean linear intercept in WT mice, which was significantly reduced in Ripk3−/− mice, indicating the attenuation of emphysematous pathology (Figures 4K and 4L).
Figure 4. Ripk3 (receptor-interacting protein kinase 3) deficiency reduces airway inflammation, airway remodeling, and emphysema in experimental chronic obstructive pulmonary disease (8-wk exposure, nose-only). (A–D) Numbers of total leukocytes (A), macrophages (B), neutrophils (C), and lymphocytes (D) in the BALF of normal air–exposed and cigarette smoke–exposed wild-type and Ripk3−/− mice (n = 5–6/group). (E–G) Airway (E) and vascular (F) inflammatory score and average macrophage numbers (G) in 15 random fields in lung histologic analysis (n = 5–6/group). (H) Lung sections stained with Sirius red. Scale bar, 60 μm. (I and J) Collagen deposition (I) and epithelial thickness (J) (n = 4–6/group). (K) Lung sections stained with hematoxylin and eosin. Scale bar, 60 μm. (L) Mean linear intercept. Data are the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.01 compared with other groups indicated. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with wild-type air-exposed control animals. One-way ANOVA with Bonferroni correction for multiple comparisons was used. BALF = BAL fluid; NS = not significant.
[More] [Minimize]RIPK3 has additional functions in inflammatory cytokine production and apoptosis, whereas MLKL has only been reliably attributed to functions in necroptosis (45, 46). Thus, to examine whether the effects of RIPK3 loss could be attributed exclusively to necroptosis deficiency, we examined the disease features of Mlkl−/− mice relative to WT mice in experimental COPD. In addition, to assess the role of apoptosis and investigate the potential of combined inhibition of the apoptotic and necroptotic cell death pathways in COPD, some WT groups were treated with the pan-caspase inhibitor qVD-OPh or vehicle. The numbers of total leukocytes in the BALF significantly increased after CS exposure in vehicle-treated WT mice but did not significantly increase in vehicle-treated Mlkl−/− mice or qVD-OPh–treated WT or Mlkl−/− mice (Figure 5A). This reduction in total leukocytes appeared to be specifically driven by significant inhibition of airway macrophage and neutrophil accumulation, whereas lymphocytes accumulated in high numbers in CS-exposed Mlkl−/− qVD-OPh–treated mice versus air-exposed Mlkl−/− qVD-OPh–treated mice, albeit at lower numbers than macrophages or neutrophils (Figures 5B–5D). Mlkl−/− mice that underwent treatment with qVD-OPh showed decreases in CS-induced cellular inflammation in the BALF that were similar to those of Mlkl−/− mice that did not undergo qVD-OPh treatment or WT mice that underwent qVD-OPh treatment. Cxcl1, Mmp8, Mmp12, and Ym1 mRNA expression significantly increased after CS exposure in vehicle-treated WT mice, and the levels of expression were significantly reduced in vehicle-treated Mlkl−/− mice and qVD-OPh–treated WT or Mlkl−/− mice (Figure E5). Marco mRNA expression was significantly increased in all groups after CS exposure but was reduced in qVD-OPh–treated WT mice (Figure E5E). Mip1a mRNA expression was also significantly increased after CS exposure, but this only occurred in vehicle-treated WT mice, and the levels of expression were significantly lower in qVD-OPh–treated WT and Mlkl−/− mice (Figure E5F). Mlkl was significantly induced in CS-exposed, vehicle-treated WT treated mice. qVD-OPh treatment partially reduced CS-induced Mlkl mRNA expression in WT mice (see Figure E5G).
Figure 5. Mlkl (mixed lineage kinase domain-like) deficiency or caspase inhibition by qVD-OPh treatment reduces chronic cigarette smoke (CS)-induced airway inflammation (8-wk exposure, nose-only). (A–D) Numbers of total leukocytes (A), macrophages (B), neutrophils (C), and lymphocytes (D) in the BALF of normal air–exposed and CS-exposed untreated or qVD-OPh–treated wild-type or Mlkl−/− mice (n = 5–6/group). Mice were treated with qVD-OPh or vehicle three times per week throughout the CS exposure period. Data are the mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with other groups indicated. #P < 0.05, ###P < 0.001, and ####P < 0.0001 compared with wild-type air-exposed control animals. One-way ANOVA with Bonferroni correction for multiple comparisons was used. BALF = BAL fluid; qVD-OPh = quinoline-Val-Asp-difluorophenoxymethylketone.
[More] [Minimize]To examine the impact of inhibiting apoptosis and/or necroptosis on lung pathology in experimental COPD, we performed histopathologic analysis (Figures 6A and 6B). Inflammation was induced in the airways all CS-exposed groups compared with normal air–exposed control animals (Figure 6C). However, the histopathologic score was significantly reduced in both CS-exposed, vehicle-treated Mlkl−/− mice and qVD-OPh–treated WT or Mlkl−/− mice compared with vehicle-treated WT control animals. Similarly, perivascular inflammation also increased after CS exposure in all groups but was significantly reduced in qVD-OPh–treated WT and Mlkl−/− mice compared with vehicle-treated WT mice (Figure 6D). There were significantly fewer parenchymal macrophages in CS-exposed Mlkl−/− mice than in their WT counterparts with or without qVD-OPh treatment (Figure 6E). CS exposure induced collagen deposition in vehicle-treated WT mice, but this was prevented in Mlkl−/− mice, irrespective of qVD-OPh treatment (Figure 6F). Similarly, epithelial thickness was increased in CS-exposed WT mice but was not increased in CS-exposed Mlkl−/− mice, irrespective of qVD-OPh treatment (Figure 6G). The mean linear intercept significantly increased in CS-exposed WT mice, irrespective of vehicle or qVD-OPh treatment, whereas Mlkl−/− mice did not develop emphysema in any experimental condition (Figure 6H).
Figure 6. Mlkl (mixed lineage kinase domain-like) deficiency or caspase inhibition by qVD-OPh treatment reduces chronic cigarette smoke (CS)-induced lung inflammation, but only Mlkl deficiency reduces airway remodeling and emphysema (8-wk exposure, nose only). (A and B) Lung sections stained with Sirius red (A) or hematoxylin and eosin (B). Scale bars, 60 μm. (C–H) The airway (C) and vascular (D) histopathologic scores, average macrophage numbers in 15 random fields (E), collagen deposition (F), airway epithelial thickness (G), and mean linear intercept (H) in 15 random fields in lung histologic analysis of normal air–exposed or CS-exposed untreated or qVD-OPh–treated wild-type or Mlkl−/− mice (n = 6/group). Mice were treated with qVD-OPh or vehicle three times per week throughout the CS exposure period. Data are the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 compared with other groups indicated. ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared with wild-type air-exposed control animals. One-way ANOVA with Bonferroni correction for multiple comparisons was used. qVD-OPh = quinoline-Val-Asp-difluorophenoxymethylketone.
[More] [Minimize]TUNEL staining for total apoptotic and necrotic cell death and active caspase-3 staining for apoptosis were performed on sequential, formalin-fixed lung sections of mice exposed to CS or normal air for 8 weeks. The number of both TUNEL+ and active (cleaved) caspase-3–positive cells increased in CS-exposed groups compared with air-exposed groups (Figures 7A–7D). In Ripk3−/− mice, the CS-induced increases in the number of TUNEL+ and active caspase-3–positive cells were attenuated compared with those of WT mice (Figures 7A and 7B). Similarly, Mlkl deficiency and/or qVD-OPh treatment reduced CS-induced increases in TUNEL+ events (Figure 7C) and prevented CS-induced increases in active caspase-3 staining, which was only observed in the WT vehicle-treated group (Figure 7D).
Figure 7. Ripk3 (receptor-interacting protein kinase 3) and Mlkl (mixed lineage kinase domain-like) deficiency suppresses cell death and caspase-3 activation (8-wk exposure, nose-only). (A and B) TUNEL+ (A) and active (cleaved) caspase-3–positive (B) cells in the lungs of normal air–exposed and cigarette smoke–exposed wild-type (WT) or Ripk3−/− mice (n = 5–6/group). (C and D) TUNEL+ (C) and active caspase-3–positive (D) cells in the lungs of normal air–exposed and cigarette smoke–exposed untreated or qVD-OPh–treated WT or Mlkl−/− mice (n = 6/group). Results are shown as the mean ± SEM. *P < 0.05 , **P < 0.01, and ****P < 0.0001 compared with other groups indicated. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared with WT air-exposed control animals. One-way ANOVA with Bonferroni correction for multiple comparisons was used. qVD-OPh = quinoline-Val-Asp-difluorophenoxymethylketone; TUNEL = terminal deoxynucleotide transferase–mediated dUTP nick end label.
[More] [Minimize]Here, we describe the dysregulation and activation of the core necrosome components RIPK3 and MLKL in clinical COPD lung tissue samples and demonstrate similar upregulation of necroptosis-related signaling in CS-induced experimental COPD. Through a combination of genetic deletion of core necrosome components (RIPK3 and MLKL) and pan-caspase inhibition to prevent apoptosis, we define for the first time the differential contributions of necroptosis signaling versus apoptosis signaling to key pathologies in experimental COPD. This highlights potential roles for regulated necrosis, particularly necroptosis, in COPD pathogenesis and suggests that regulation of necroptosis represents a novel therapeutic target in COPD.
We profiled the necroptosis signaling components RIPK3 and MLKL in lung tissue samples from patients with COPD and control subjects without COPD. We found that MLKL, the critical membrane-disrupting component that executes necroptotic cell death, was increased in the bronchial epithelium and alveolar macrophages in patients with severe COPD compared with control subjects. During necroptosis initiation, MLKL becomes phosphorylated by RIPK3, which is required to trigger a conformational change allowing plasma membrane translocation, oligomerization, and membrane disruption, resulting in membrane leakage, rupture, and necrotic cell death (6). Critically, pRIPK3 and pMLKL were elevated in the lung tissues of patients with severe COPD, which remained significant after adjustment for age, sex, and current smoking. Moreover, we found a strong positive correlation between pRIPK3 and pMLKL protein levels and found negative associations between pRIPK3 and pMLKL levels and gas exchange (DlCO). These data indicate activation of the necroptosis pathway, RIPK3, and the key terminal effector molecule MLKL in clinical COPD. Importantly, we observed similar dysregulation of necrosome components in murine experimental COPD, including increased mRNA and protein levels of Mlkl and the upstream regulators Ripk1 and Ripk3. Our human and mouse data indicate that this dysregulation occurs in multiple cellular compartments, including the airways, airway epithelium, and pulmonary macrophages. Further delineating the key cellular locations where necroptosis may promote features of COPD will be a future goal.
Having established that necroptosis-related signaling was increased in human and experimental COPD, we tested its role in promoting pathologic responses to acute and chronic CS exposure by using Ripk3−/− and Mlkl−/− mice. Deletion of Ripk3 or Mlkl protected against acute and chronic CS-induced cellular and molecular airway inflammation. Reduced recruitment of neutrophils and macrophages to the airways was observed. Notably, the molecular markers of inflammation Cxcl1 (in Mlkl−/− mice), Mmp12, Ym1, and Marco were suppressed in the lung tissue of Ripk3−/− and/or Mlkl−/− mice in the setting of both acute and chronic CS exposure. Suppression of other chemoattractants, such as CXCL2, IFNγ, complement, etc., are likely responsible for the reduced number of neutrophils in Ripk3−/− mice. These molecules are all involved in promoting macrophage-related airway inflammation, and RNA-sequencing analysis of isolated alveolar macrophages from mice that underwent CS exposure for 4 weeks demonstrated increased mRNA expression of all three factors as well as Mmp12 and Marco compared with mice that were exposed to air. Marco is a scavenger receptor on the surface of macrophages (47). It promotes inflammation by binding and inducing the phagocytosis of low-density lipoprotein and pathogen-associated molecular patterns (48) but is also important for removal of these same ligands. The chitinase-like protein YM1 is a marker of alternatively activated macrophages but is also able to induce neutrophil recruitment through expansion of IL-17–producing γδT cells (49). There have been no studies of YM1+ macrophages in COPD. The decrease in Marco and Ym1 expression (putative M2 markers) in Ripk3−/− and Mlkl−/− mice corresponded with reduced macrophage numbers and may represent the suppression of inflammatory activation of these cells or could reflect an overall modulation of the macrophage phenotypic subsets in response to CS exposure. Previous studies in patients with COPD suggest that M1 and M2 macrophages dominate in the airway wall and lumen, respectively (50). Reductions in CS-induced expression of Mmp12 were more pronounced with chronic CS exposure than with acute CS exposure in both deficient mouse strains. Mmp12 is an enzyme that breaks down the extracellular matrix that can be generated through macrophage activation. It is highly upregulated in smokers, is associated with the development of emphysema (51), and also contributes to experimental COPD (52). These data suggest that the protective effects of Ripk3 or Mlkl deletion may occur through the suppression of the inflammatory activation of macrophages and the reduced macrophage production of emphysema-inducing factors. The role of necrosome components in macrophage activation is poorly understood; however, genetic deletion of necroptosis-related genes in CS exposure models may alter the macrophage activation status indirectly through blockade of proinflammatory DAMP release or other responses (53).
Prior studies have linked acute CS exposure to the induction of necroptosis and release of DAMPs that promote airway inflammation. The exposure of lung epithelial cells to CSE induced MLKL phosphorylation (54). Administration of the RIPK1 kinase inhibitor necrostatin-1 can inhibit CSE-induced death of lung epithelial cells in vitro and reduces DAMP release and neutrophilic airway inflammation during acute CS exposure in mice (54, 55). However, necrostatin-1 has off-target effects, and RIPK1 has roles in proinflammatory cytokine signaling and apoptosis. Our data extend these findings by demonstrating roles for the core necrosome components RIPK3 and MLKL in CS-induced airway inflammation and show that both the airway epithelium and alveolar macrophages are important sites of dysfunction of this pathway. Increased expression of necrosome components could sensitize cells to undergo necroptosis (54, 56–61), and further mechanisms that inhibit necroptosis under physiologic circumstances may be suppressed by CS exposure. A recent study demonstrated that the transcript levels of CFLAR, an apoptosis and necroptosis modulator, were reduced in lung biopsy specimens from smokers versus nonsmokers and in epithelial cells after CS exposure in vitro (62). CFLAR encodes c-FLIP proteins, which heterodimerize with caspase-8 to modulate their cell death–related activities, including suppression of necroptosis (62). Knockdown of CFLAR sensitized epithelial cells to CS-induced cell death and the related DAMP release, demonstrating a further potential mechanism whereby CS exposure induces necroptosis in the lung. Further studies are required to understand the mechanisms of CS-mediated modulation of signaling upstream of necrosome formation and activation in vivo.
Apoptosis has long been considered to have roles in lung remodeling and tissue destruction in COPD, particularly emphysema. Early studies reported increased cell death (TUNEL positivity) in the emphysematous regions of COPD lungs (63); however, TUNEL staining is not a bona fide marker of apoptosis and also detects cells undergoing necrosis, including necroptosis (62). Furthermore, there is a lack of studies demonstrating the effect of or lack of protection afforded by inhibiting apoptosis (or other types of cell death) in experimental COPD. Thus, the pathogenic roles of apoptosis and the broader mechanisms of cell death were unclear. CSE is reported to induce the apoptotic cell death of epithelial cells, resulting in TUNEL+ staining (64). Consistent with this observation, by using TUNEL staining, we found that CS exposure significantly increased cell death in the lung parenchyma from 8 weeks onward. Positive cells occurred in the airways, in the parenchyma, and among immune cells but were not more prominent in a particular cell type. We attempted unsuccessfully to perform costaining for TUNEL and active caspase-3. Importantly, this increased incidence of cell death was not present at 4 weeks and coincided with the development of emphysema, which is observed from 8 weeks onward in our model (17, 23–30). Thus, the induction of alveolar epithelial-cell death may be an important pathophysiologic event that contributes to the destruction of alveolar structures in emphysema. Importantly, our data suggest that necroptosis, but not apoptosis, promotes the destruction of alveoli in CS-induced emphysema. Both genetic deletion of necroptosis-related genes and pharmacologic inhibition of apoptosis reduced airway inflammation. Our approach to inhibit apoptosis was to employ a pan-caspase inhibitor. Although this was sufficient to suppress TUNEL positivity and the levels of cleaved caspase-3 in the lungs in experimental COPD, it is likely that pharmacologic inhibition was not completely efficient and did not reduce airway remodeling or emphysema. Previous studies showed that these chronic disease features can occur independently of chronic inflammation, such as in the absence of Ccr5 (65). Moreover, we cannot exclude that some other inflammatory processes not affected by caspase inhibition contributed to the induction of pathologic remodeling and emphysema. In addition, this approach may have blocked processes additional to apoptosis, such as inflammasome-associated caspase-1 activity and IL-1β production (66). The observation that BALF lymphocytes were increased by dual blockade of apoptosis and necroptosis after CS exposure is intriguing and merits further investigation in terms of the role of cell death in regulating lymphocyte survival during ongoing inflammatory processes. Additional inhibitory approaches targeting other components of the apoptosis machinery are required to fully delineate the role of apoptosis in the pathogenesis of experimental COPD. That apoptosis is usually a noninflammatory form of cell death, whereas necroptosis is proinflammatory, may explain why blocking of either apoptosis or necroptosis reduces cell death but only blocking necroptosis reduces remodeling and emphysema. However, this does not mean that our findings conflict with previous work indicting that apoptosis contributes to lung remodeling and emphysema. This is because most previous studies used nonspecific measurements to evaluate the level of apoptosis, and these measurements could not be used to separate it from necroptosis. Furthermore, most evidence supporting a role of apoptosis in emphysema is based on clinical studies without causative data.
Our study has some other limitations. Some of the experimental studies would benefit from an increase in the sample size. It is currently not possible to confirm activation of the necrosome components through coimmunoprecipitation or detection of the phosphorylated necrosome components in experimental COPD, as there are no specific antibody tools for mice. However, we did detect increased phosphorylation of RIPK3 and MLKL, which reflects necroptosis activation, in human lung resections. In addition, deletion of two separate necrosome components (RIPK3 and MLKL) strongly support roles for necroptosis-related signaling in promoting experimental COPD. This shows that the effects are caused by necroptosis rather than by inflammatory signaling. It is possible that using pharmacologic inhibition of caspases may not have completely inhibited apoptosis at all times or in all tissues and cells; however, we confirmed reduction of total lung-cell death in pan-caspase inhibitor–treated, CS-exposed mice. The use of caspase-3–specific inhibitors or genetic models of apoptosis inhibition may be useful in future studies for separating the apoptotic roles from the nonapoptotic roles of caspases in experimental COPD. Future studies should employ novel inhibitors of necroptosis, which will also be necessary to further establish the therapeutic potential of targeting necroptosis in COPD. Our study is unique in that we used defined genetic or pharmacologic inhibition to specifically delineate the contribution of cell death pathways to experimental COPD pathology; we used these methods together with measurements of bona fide markers of necroptotic cell death in the lungs of patients with COPD.
In summary, we provide compelling evidence that the activity of the necroptosis pathway is increased in the lungs in human COPD and in experimental COPD. Genetic and pharmacologic inhibition shows that RIPK3 and MLKL promote cell death and contribute to the pathogenesis of airway inflammation, airway remodeling, and emphysema. Inhibiting necroptosis may be a novel therapeutic approach in COPD.
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* These authors contributed equally to this manuscript.
Supported by the National Health and Medical Research Council of Australia (1175134, 1120252, 1099095, and 1159582), the Australian Research Council (150102153), the University of Newcastle, the Hunter Medical Research Institute, and the University of Technology Sydney (P.M.H.); the Rebecca Cooper Foundation (M.F.); the Victorian Government Operational Independent Research Institutes Infrastructure Support Scheme (J.M.M.); the European Union Horizon 2020 Marie Skłodowska-Curie grant agreement number 796450 (C.M.G.); Flemish grants (Molecular Mechanisms of Cellular Death and Life Decisions in Inflammation, Degeneration and Infection Research Project, Excellence of Science Program, 30826052), Research Foundation–Flanders research grants (G.0E04.16N, G.0C76.18N, G.0B71.18N, and G.0B96.20N), the Methusalem Program (BOF16/MET_V/007), iBOF20/IBF/039 ATLANTIS, the Foundation against Cancer (FAF-F/2016/865 and F/2020/1505), the Cancer Research Institute Ghent and Ghent Gut Inflammation Group consortia, and the Vlaams Instituut voor Biotechnologie (P.V.); and the Consortium of Excellence Focusing on Inflammation (INFLA-MED) (T.V.B.).
Author Contributions: Z.L., H.P.V.E., C.M.G., K.R.B., M.F., and P.M.H. designed and performed experiments and analyzed the data, and G.L., B.C.N., B.J, P.M.N., J.C.H., J.M.M., and P.A.W. contributed. Z.L., H.P.V.E., K.R.B., M.F., and P.M.H. conceptualized the studies and wrote the manuscript. H.P.V.E., C.M.G., F.V., T.B.-H., P.V., T.V.B., G.G.B., J.C.H., and K.R.B. analyzed clinical data, assisted in performing experiments, helped analyze data, helped interpret data, and edited and revised the manuscript. K.R.B., M.F., and P.M.H. supervised the studies and edited and revised the manuscript.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1164/rccm.202009-3442OC on June 16, 2021
Author disclosures are available with the text of this article at www.atsjournals.org.
