Free Radical Biology and Medicine 83 (2015) 149–158
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Original Contribution
Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9–p38 MAPK pathway Xiaoling Gu a, Guannan Wu a, Yanwen Yao a, Junli Zeng a, Donghong Shi b, Tangfeng Lv a, Liang Luo c, Yong Song a,n a
Department of Respiratory Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002, People’s Republic of China Department of Medical Imaging, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002, People’s Republic of China c Intensive Care Unit, Wuxi Second Affiliated Hospital, Nanjing Medical University, Wuxi, Jiangsu Province 210004, People’s Republic of China b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 November 2014 Received in revised form 14 February 2015 Accepted 25 February 2015 Available online 13 March 2015
An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1β, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1β, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9–p38 MAPK signal transduction pathway. & 2015 Elsevier Inc. All rights reserved.
Keywords: Mitochondrial DNA Lung Inflammation TLR9–p38 MAPK signal transduction pathway Free radicals
The etiological factors leading to pulmonary inflammation are often complicated. Although it has been well established that bacteria and specific environmental exposures are responsible for various lung diseases with profound inflammation, many cases of inflammatory lung disease are idiopathic and do not present any identifiable etiology. For instance, most cases of acute respiratory distress syndrome do not have a specific etiology and cannot be well explained by existing mechanisms. There has recently been increasing interest in clarifying the role of endogenous danger signals in the development of inflammatory lung injury and the systemic inflammatory response. The results of our previous study
Abbreviations: mtDNA, mitochondrial DNA; SIRS, systemic inflammatory response syndrome; nDNA, nuclear DNA; CpG, cytosine–phosphate–guanine; MAPK, mitogen-activated protein kinase; TLR9, Toll-like receptor 9; DAPI, 40 ,6diamidino-2-phenylindole; siRNA, small interfering RNA; CQ, chloroquine; SB, SB203580 n Corresponding author. Fax: þ 86 25 80863591. E-mail address: [email protected] (Y. Song). http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.034 0891-5849/& 2015 Elsevier Inc. All rights reserved.
showed that the plasma concentration of mitochondrial DNA (mtDNA)1, a newly identified damage-associated molecular pattern, is positively correlated with the risk of systemic inflammatory response syndrome (SIRS) in acute traumatic patients [1]. Additionally, a recently published study reported that acid aspiration results in a 120-fold increase in the mtDNA concentration in cell-free bronchoalveolar lavage fluid in an animal model of acute gastric aspiration lung injury [2]. However, the specific role of mtDNA in the development of inflammatory lung injury remains to be further determined. Mitochondria DNA is generally considered to be derived from the circular genomes of bacteria that evolved into the mitochondria of today’s eukaryotic cells [3–7]. Similar to bacterial DNA, mtDNA is circular and contains a higher frequency of unmethylated cytosine– phosphate–guanine (CpG) dinucleotides [8–10]. DNA that contains unmethylated CpG motifs can be recognized by the innate immune system and exerts powerful immunostimulatory effects [11–13]. Previous studies conducted by Oka et al. [14] have demonstrated that mtDNA that escapes from autophagy-mediated degradation can
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trigger local inflammation in the heart. Additionally, Zhang et al. [15] reported that mtDNA released into the circulation by shock can activate the neutrophil p38 mitogen-activated protein kinase (MAPK) signaling pathway via TLR9 and potentially contribute to the development of posttraumatic SIRS. The results of these recent studies indicate that mtDNA can not only participate in the activation of innate immunity in the original cell but also initiate systemic innate immunity. It is well known that CpG-containing DNA activates inflammatory responses via an endosomally located pattern recognition receptor, namely TLR9 [16–18], which is constitutively expressed in endothelial cells and macrophages in the lung [19,20]. It has been reported that the intravenous administration of mtDNA can cause SIRS and lung inflammation [21]. However, it is not clear whether the lung inflammation observed in this animal model is directly induced by mtDNA or is just a secondary result of SIRS. Even if this published study has confirmed the immunostimulatory potential of mtDNA in the lungs, the related molecular mechanisms remain unclear. Because DNA is readily endocytosed by macrophages [22], which act as the first line of host defense in the lung, it is logical to hypothesize that mtDNA is capable of causing lung inflammation via active alveolar macrophages and may be mediated by a signaling pathway similar to the TLR9–p38 MAPK pathway in neutrophils. To determine the possible role of mtDNA in the induction of lung inflammation, we intratracheally administered mtDNA to mice and simultaneously examined the in vitro effect of mtDNA on THP-1 macrophages, a human monocytic and macrophage cell line, to elucidate the specific signal transduction mechanism that mediates the inflammatory activation of THP-1 induced by mtDNA exposure.
Materials and methods Main reagents 40 ,6-Diamidino-2-phenylindole (DAPI), phorbol 12-myristate 13-acetate (PMA), chloroquine, and protease inhibitor cocktail were purchased from Sigma. Mouse anti-β-actin antibody and rabbit anti-TLR9 antibody were obtained from Abcam. Antibodies to p38 MAPK and phospho-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling. Alexa Fluor 594-conjugated goat anti-rabbit IgG was purchased from Invitrogen. Goat anti-rabbit IgG–horseradish peroxidase (HRP), goat anti-mouse IgG–HRP, and rabbit anti-CD68 antibody were obtained from Boster Biotechnology Co. Ltd. (Wuhan, China). The small interfering RNA (siRNA) targeting p38 MAPK, siRNA targeting TLR9, and negative control siRNA were purchased from GenePharma (Shanghai, China), and Lipofectamine 2000 and Opti-MEM I reduced-serum medium were obtained from Invitrogen Corp. (Carlsbad, CA, USA). Heattreated fetal bovine serum and RPMI 1640–Hepes medium were obtained from Wisent, Inc. Mice and ethics statement Healthy male C57BL/6 mice weighing 20–22 g and age 7–8 weeks were obtained from the Animal Feeding Center of Yangzhou University. The animal care and experimental procedures were performed in compliance with the Institutional Animal Care and User Guidelines and were approved by the Model Animal Research Center of Jinling Hospital. Cell line, culture, transfection, and differentiation THP-1 cells were obtained from the American Type Culture Collection and were cultured in complete medium (RPMI 1640–Hepes
containing 100 U/ml penicillin/streptomycin and 10% fetal bovine serum) according to the supplier’s guidelines. The transfection of THP-1 cells was performed according to the manufacturer’s recommended protocol (Invitrogen Corp.). Briefly, THP-1 cells were seeded at 5 105 cells/ml in 10-cm dishes in 12 ml of growth medium without antibiotics. The siRNA solution and Lipofectamine 2000 solution were prepared in 1.5 ml of Opti-MEM I reduced-serum medium. The siRNA and Lipofectamine solutions were then incubated together for 20 min at room temperature to form the siRNA–Lipofectamine complex, and this complex was then added to the cells to a final siRNA concentration of 50 nM. The cells were incubated at 37 1C in a CO2 incubator. The knockdown efficiency was verified 48 to 72 h after transfection (Supplementary Fig. s1). For differentiation into macrophages, THP-1 cells were plated at 1 10 6 cells/well in six-well plates and differentiated by exposure to 100 ng/ml PMA for 24 h. The differentiated, plastic-adherent cells were washed twice with fresh complete medium and allowed to rest for 6 h before exposure to various conditions. Preparation of mtDNA and nuclear DNA (nDNA) The mouse liver mitochondria were isolated using mitochondrial isolation kits for tissues (Pierce Scientific, Rockford, IL, USA) according to the protocol supplied by the manufacturer. Mitochondria isolation kits for cells (Pierce Scientific) were used to isolate the mitochondria from cultured THP-1 cell lines. The mitochondrial isolation was performed under sterile conditions at 4 1C. The isolated mitochondrial pellets were suspended in Hanks’ balanced salt solution buffer (Gibco Life Technologies, Gaithersburg, MD, USA). The nuclear fractions of THP-1 cells and hepatocytes were reserved for the subsequent preparation of nDNA. The mitochondrial DNA and nDNA were extracted from the isolated mitochondrial pellets and nuclear fractions, respectively, under sterile conditions with the DNeasy blood and tissue kits (Qiagen, Valencia, CA, USA), according to the protocol supplied by the manufacturer. The DNA concentrations and purity were examined by spectrophotometry. The A260/280 ratio of both the mtDNA and the nDNA samples was 1.8 to 2.0, confirming the lack of any significant protein contamination. The endotoxin levels in the DNA samples were assessed using the Limulus amoebocyte lysate assay, and no detectable levels were observed. To further exclude any significant nDNA contamination and ensure the purity of the mtDNA, we conducted real-time PCR to probe the samples for the presence of the nuclear genes GAPDH and 18S and for the presence of the mitochondrial genes MT-ND2 and mtCOI, and we found that the nDNA content was less than 0.1% in the isolated mtDNA samples. Primers for the nDNA markers GAPDH (forward 50 -CCATGTTCGTCATGGGTGTGAAC-30 and reverse 50 -GCCAGTAGAGGCAGGGATGATGTTC-30 ) and 18S (forward 50 -GTTCATCCTGTTCCTGCTCC-30 and reverse 50 -GTTCATCCTGTTCCTGCTCC-30 ) and for the mtDNA markers MT-ND2 (forward 50 -CACAGAAGCTGCCATCAAGTA-30 and reverse 50 -CCGGAGAGTATATTGTTGAAGAG-30 ) and mtCOI (forward 50 -GCCCCAGATATAGCATTCCC-30 and reverse 50 GTTCATCCTGTTCCTGCTCC-30 ) were all synthesized by Invitrogen. Animal procedures Seven- to eight-week-old mice were randomly divided into three groups (groups A, B, and C) with 25 animals per group. All of the mice were anesthetized with intraperitoneal pentobarbital sodium (3 mg/kg) and placed in the supine position with the head tilted back. The intratracheal administration of nDNA (3 mg/kg, group A) or mtDNA (3 mg/kg, group B and group C) in a volume of 60 ml was performed via a microsprayer (Penn-Century, Wyndmoor, PA, USA). The mice in group C were intraperitoneally pretreated
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with chloroquine (30 mg/kg) for 2 h before mtDNA exposure. All of the mice were sacrificed 0, 3, 6, 12, or 24 h after administration, and their lungs were subsequently harvested. The large left lobe was fixed in 4% paraformaldehyde for histological analysis, and the remaining lung lobes were stored at 80 1C before the subsequent experimental assays. Here, the 0-h time point means that the mice were untreated shams and were immediately sacrificed after tracheal intubation procedure.
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centrifugation at 12,000 rpm for 30 min. The concentrations of various cytokines in the mouse lung extracts and the supernatants (conditioned medium) of THP-1 macrophages were assessed using appropriate ELISA kits as recommended by the manufacturer (4A Biotech Co., Ltd, Beijing, China). The lowest limits of detection for mouse interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 were 7, 7, and 4 pg/ml, respectively, and the lowest detection limits for human IL-1β, IL-6, and TNF-α were 2, 1, and 4 pg/ml, respectively.
THP-1 macrophage activation assays Western blot analysis Differentiated THP-1 macrophages were exposed to mtDNA (10 mg/ml) or nDNA (10 mg/ml). For the inhibitor studies, THP-1 macrophages were pretreated with chloroquine (10 mg/ml, 30 min) or SB203580 (1 mM, 30 min), which is a specific inhibitor of the p38 MAPK signal transduction pathway, or were transfected with TLR9 siRNA or p38 MAPK siRNA before mtDNA exposure. The cellconditioned medium was collected by centrifugation 0, 3, 6, 12, or 24 h after exposure and then assayed for proinflammatory cytokines by ELISA as described below. THP-1 macrophages were lysed with extraction buffer containing a protease inhibitor cocktail (Roche) 0, 30, 60, or 120 min after exposure to determine the total and phosphorylated p38 MAPK levels by Western blot as described below. Histopathology and immunofluorescence of the lungs The left lungs were fixed overnight at 4 1C in 4% paraformaldehyde and processed by successive dehydration with an alcohol series and xylene. The tissues were then embedded in paraffin and cut into 5-mm-thick sections for hematoxylin–eosin staining or immunofluorescence assay. Hematoxylin–eosin (H&E) staining was carried out according to the instructions provided by the manufacturer to determine the severity of the lung inflammation. All of the tissue sections were evaluated by an experienced blinded pathologist and scored according to criteria described previously [23]. Briefly, according to three criteria, i.e., vascular congestion and interstitial edema, alveolar structural disturbance, and atelectasis and inflammatory cell infiltration, and using a semiquantitative scale, the histological score of the lung sections was calculated as the sum of the scores (0–3) given for each criterion. The cumulative histology score ranged from 0 to 9. To observe macrophages in the lung, we conducted an immunofluorescence assay as described below. Briefly, paraffinembedded lung tissue sections were dewaxed in xylene and rehydrated in graded ethanol washes (100, 90, 70, and 50%). The samples were then washed twice for 5 min with phosphatebuffered saline (PBS) and subsequently incubated with 0.3% Triton X-100 for 8 min at room temperature. The samples were washed in PBS for 10 min, blocked with 3% bovine serum albumin (BSA) for 1 h at 37 1C, and incubated overnight at 4 1C with a rabbit antiCD68 antibody at a 1:200 dilution in 2% BSA. The samples were then washed three times with PBS for 5 min each and subsequently incubated with a goat anti-rabbit Alexa Fluor 594conjugated secondary antibody (1:400) for 1 h at 37 1C in the dark. Finally, the nuclei were stained with DAPI (5 mg/ml). The samples were visualized with a laser scanning confocal microscope (FluoView FV10i, Japan) to detect the CD68 þ macrophages using red fluorescence. Enzyme-linked immunosorbent assay The middle lobe of the right lung was homogenized and lysed with extraction buffer containing a protease inhibitor cocktail (Roche), and we then collected the tissue supernatants via
The cell proteins were obtained from THP-1 macrophages, and the tissue proteins were obtained from the right lower lungs of the mice in each group. We performed a Western blot analysis of the cellular and tissue lysates as previously described [24]. Briefly, the cells or tissue homogenates were lysed in ice-cold protein extraction buffer containing a protease inhibitor cocktail (Roche) for 30 min. After the whole lysates were centrifuged at 12,000g for 30 min, the supernatants were collected. The protein concentrations in the supernatants were determined by the Bradford method. The proteins (20 mg) from the lung tissue and THP-1 macrophages were separated in 12% SDS–PAGE gels and electrophoretically transferred onto a polyvinylidene fluoride membrane using an electroblotting apparatus (Bio-Rad). The membranes were blocked in 5% BSA in PBS/Tween 20 (PBST) for 1 h at 37 1C, and the blocked membranes were then incubated overnight at 4 1C with anti-β-actin mouse monoclonal antibody (1:1000), antiphospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (1:800), or anti-p38 MAPK rabbit polyclonal antibody (1:800) in PBST containing 2% BSA. After three washes with PBST buffer, the membranes were incubated with secondary HRP-conjugated antibody for 1 h at 37 1C. The protein signals were enhanced by chemiluminescence detection kits (SuperSignal West Pico) and detected using an Odyssey Scanning System (Li-Cor Biosciences, Lincoln, NE, USA). Statistical analysis The study data are expressed as the means 7 standard deviation and assessed for statistical significance using Student’s t test. A two-tailed p value less than 0.05 was considered statistically significant. All of the calculations and statistical analyses were performed using SPSS software for Windows (version 17.0).
Results MtDNA induces histopathological changes in the lungs via TLR9 To examine the potential of mtDNA to induce lung inflammation, mtDNA was intratracheally instilled into the lungs of normal male C57BL/6 mice. Serial lung sections obtained from mice after mtDNA exposure were stained with H&E and examined by light microscopy. Representative microscopic images of these H&Estained lung specimens are shown in Fig. 1. The mtDNA administration caused marked inflammatory cell infiltration accompanied by thickening of the alveolar septa, which is suggestive of lung inflammation and acute lung injury. However, the mice in the nDNA control group showed no pathological changes in pulmonary inflammation. Additionally, as shown in Table 1, the histological score of the pulmonary tissues in the mtDNA exposure group was also markedly increased as early as 3 h after instillation, peaked at 6 h, and then gradually decreased, but the values after this decrease remained significantly higher compared with those
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Fig. 1. Representative photomicrographs of lung histopathology stained with H&E. (A) Control group treated with nDNA (3 mg/kg). (B) mtDNA challenge group treated with mtDNA (3 mg/kg). (C) Chloroquine inhibitory group, which was pretreated with CQ (30 mg/kg, ip) before mtDNA (3 mg/kg) challenge. Original magnification, 400. These observations are representative of results of experiments with five mice per time point per group.
Table 1 Histological scores of lung tissue. mtDNA
CQ þ mtDNA
Mean SD
Mean SD
Mean SD
0.3 0.5 0.5 0.3 0.3
0.3 3.6 4.9 4.8 3.9
0.3 2.5 3.5 3.0 3.0
Time point nDNA
0h 3h 6h 12 h 24 h a b
0.67495 0.70711 0.70711 0.48305 0.67495
0.67495 0.69921 1.19722 1.47573 0.99443
0.48305 1.08012 1.08012 0.66667 0.8165
pa
pb
1.0 o 0.001 o 0.001 o 0.001 o 0.001
1.0 0.015 0.013 0.002 0.04
mtDNA vs nDNA. CQ þ mtDNA vs mtDNA.
found for the nDNA control group (p o 0.001 for all four selected time points). Considering that TLR9 is the only known Toll-like pattern recognition receptor for mtDNA, to further determine whether TLR9 is a key upstream molecule that mediates the inflammatory response induced by mtDNA in the lung, mice were intraperitoneally pretreated with chloroquine for 2 h and then subjected to intratracheal mtDNA instillation. As shown in Fig. 1C and Table 1, pretreatment with chloroquine (CQ) significantly ameliorated the mtDNA-induced inflammatory pathological changes in the lungs and reduced the histological score of the pulmonary tissues after mtDNA exposure. These results suggested that TLR9 mediates the inflammatory histopathological changes in the lungs stimulated by mtDNA. Additionally, because alveolar macrophages act as the first line of host defense in the lung, we conducted an immunofluorescence assay with the paraffin-embedded lung tissue sections to determine whether macrophages had infiltrated into the mtDNA-treated pulmonary tissues. Representative
microscopic findings from these immunofluorescence-labeled lung specimens are shown in Fig. 2. The mice treated with control nDNA presented no obvious CD68 þ macrophage infiltration at all selected time points after the intratracheal instillation. However, exposure to mtDNA resulted in a profound increase in the amount of CD68 þ macrophages in the lung tissues at all selected time points, which suggested that the mtDNA-induced lung inflammation was accompanied by CD68 þ macrophage infiltration and that macrophages probably play an important role in the initiation and development of mtDNA-induced inflammation. MtDNA triggers cytokine responses in the lungs via TLR9 As further evidence of the mtDNA-induced inflammation, the concentrations of proinflammatory cytokines in the lung homogenate were assessed by ELISA. As shown in Fig. 3, in the mtDNA instillation group at all four time points (3, 6, 12, and 24 h), all of the three examined proinflammatory cytokines (IL-1β, IL-6, and TNF-α) exhibited statistically significant increases compared with the levels observed in the nDNA exposure control group. The concentrations of both IL-1β and TNF-α were highest 6 h after the mtDNA challenge and then gradually decreased, but these decreased levels remained significantly higher compared with those found in the respective nDNA controls. The levels of IL-6 in the lung homogenate reached a peak as early as 3 h after mtDNA instillation and then decreased, and the decreased levels remained significantly higher than the levels observed in the control group. These data indicated that mtDNA triggers a significant increase in IL-1β, IL-6, and TNF-α production in the lungs and provide evidence for the potential ability of mtDNA to induce lung inflammation.
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Fig. 2. Representative photomicrographs of immunofluorescence-labeled CD68 þ macrophages in lung specimens. The macrophages were labeled with antibodies to CD68 (red), and the nuclei were stained with DAPI (blue). Scale bar, 30 mm. These observations are representative of results from experiments with five mice per time point per group.
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Fig. 3. Cytokine responses in the lungs induced by control nDNA, mtDNA, and CQ þ mtDNA. (A) Concentration of IL-1β in the lung homogenate. (B) Concentration of IL-6 in the lung homogenate. (C) Concentration of TNF-α in the lung homogenate. *p o 0.05; **p o 0.001. The results are from experiments with five mice per time point per group.
The proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the mice of the CQ pretreatment group were also assessed and compared with those of the mtDNA-exposure group. As shown in Fig. 3, pretreatment with CQ resulted in a significant reduction in the IL-6 concentration in the lung homogenates at all four selected time points compared with the levels found in the mtDNA-exposure group (p o 0.05, Fig. 3B). Although pretreatment with CQ led only to a decreasing trend in the IL-1β level, with a limbic p value of 0.063 at 12 h, the suppression effect of CQ on IL-1β production assessed at the other three selected time points (3, 6, and 24 h) was statistically significant (p o 0.05, Fig. 3A). CQ pretreatment also significantly reduced the concentrations of TNF-α in the lung homogenates at 3 and 6 h after mtDNA instillation but did not significantly downregulate the levels of TNF-α at 12 and 24 h compared with the levels found in the mtDNA-exposure group (Fig. 3C). Taken together, these data suggested that TLR9 mediates the inflammatory cytokine response in the lungs after mtDNA exposure. Intratracheal mtDNA activates p38 MAPK in the lungs via TLR9 Because p38 MAPK is a key early intermediary in the inflammatory response and is one of the downstream targets of the TLR9 signaling pathway, which is the only known Toll-like receptor pathway for mtDNA, we performed a Western blot assay to determine the effect of mtDNA on the total and phosphorylated p38 MAPK levels in the lung. As shown in Fig. 4, mtDNA instillation induced a significant upregulation of phosphorylated p38 MAPK at 6 h, and this level returned to the baseline 12 h after
mtDNA stimulation. However, the expression of total p38 MAPK was not significantly changed within 24 h of mtDNA exposure. Conversely, nDNA administration had no effect on the expression of total or phosphorylated p38 MAPK (Fig. 4). These observations suggested that mtDNA induces p38 MAPK activation in vivo and that this activation is probably involved in the mtDNA-induced lung inflammation. Further more, pretreatment with CQ significantly suppressed p38 MAPK activation in lung tissue after mtDNA exposure (Fig. 4). It confirmed that mtDNA activates the p38MAPK pathway via TLR9 in the lung of model mice. MtDNA activates p38 MAPK in cultured THP-1 macrophages via TLR9 Given that the above-presented evidence indicated that mtDNA exposure can trigger significant pulmonary inflammation via the pattern recognition receptor TLR9 and that this inflammation was accompanied by CD68 þ macrophage infiltration, to clarify whether macrophages play an important role in the development of mtDNA-induced inflammation, we performed further experiments to determine whether mtDNA exposure can induce cytokine production and activate p38 MAPK in cultured THP-1 macrophages. Differentiated THP-1 macrophages were incubated with either nDNA or mtDNA (10 mg/ml), and a Western blot assay was performed to determine whether mtDNA stimulation activates the p38 MAPK pathway in THP-1 macrophages. As shown in Fig. 5, mtDNA exposure induced significant upregulation of phosphorylated p38 MAPK as early as 30 min after incubation, and the increased expression of phosphorylated p38 MAPK
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Fig. 4. Protein expression profiles of phosphorylated (p) p38 MAPK in the lungs induced by control nDNA, mtDNA, and CQ þ mtDNA. The results are representative of experiments with five mice per time point per group.
Fig. 5. Protein expression profiles of phosphorylated (p) p38 MAPK in THP-1 macrophages that were or were not pretreated with chloroquine, SB203580, TLR9 siRNA, or p38 MAPK siRNA and were then challenged with mtDNA. The results are representative of three individual experiments.
remained 1 h after exposure and then weakened to the baseline level at 2 h after mtDNA stimulation. However, the expression of total p38 MAPK was not significantly changed within 2 h of mtDNA administration. Conversely, nDNA exposure had no effect on the expression of total or phosphorylated p38 MAPK (Fig. 5). These observations suggested that mtDNA induces p38 MAPK activation in cultured THP-1 macrophages. Furthermore, we attempted to investigate whether TLR9 is a key upstream molecule that mediates the p38 MAPK activation induced by mtDNA in THP-1 macrophages. We pretreated THP-1 cells with TLR9 siRNA before mtDNA exposure. Additionally, because endosomal acidification is a prerequisite for several TLRs (including TLR9) [25–27], THP-1 macrophages were preincubated with CQ, an inhibitor of endosomal acidification, before mtDNA administration. As shown in Fig. 5, both TLR9 siRNA transfection and CQ preincubation resulted in no upregulation of the expression of phosphorylated p38 MAPK in THP-1 macrophages after mtDNA exposure. This means that mtDNA activates p38 MAPK in cultured THP-1 macrophages via TLR9. MtDNA induces cytokine production by THP-1 macrophages via the TLR9–p38 MAPK pathway The concentrations of proinflammatory cytokines in the supernatants of the THP-1 macrophages after mtDNA exposure were
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assessed by ELISA. As shown in Fig. 6, in the mtDNA stimulation group at all four time points (3, 6, 12, and 24 h), all three examined cytokines (IL-1β, IL-6, and TNF-α) exhibited significant increases compared with the levels found in the nDNA-exposure control group. The concentrations of both IL-1β and IL-6 were highest 12 h after the mtDNA challenge and then decreased at 24 h, but the decreased levels remained significantly elevated compared with those found in the nDNA controls. A persistent increase in the concentrations of TNF-α in the THP-1 macrophage culture medium treated with mtDNA was observed within 24 h after administration, and this increase was significantly higher than that found in the nDNA-exposure control group. These data indicated that mtDNA triggered a marked increase in IL-1β, IL-6, and TNF-α production in the cultured THP-1 macrophages. Because the above-presented evidence indicated that mtDNA exposure activates the p38 MAPK pathway in cultured THP-1 macrophages, we performed further experiments using the pharmacological probe SB203580 and p38 MAPK siRNA to clarify the causality between the p38 MAPK signal and the cytokine production induced by mtDNA. As shown in Fig. 5, both preincubation with SB203580 and pretreatment with p38 MAPK siRNA successfully suppressed the p38 MAPK activation triggered by mtDNA. In addition, pretreatment with SB203580 resulted in a significant reduction in IL-1β production by mtDNA-stimulated THP-1 macrophages at all four selected time points (p o 0.05, Fig. 6A). Although the suppression effect of SB203580 on TNF-α production was statistically significant only at 3 h after mtDNA administration, a trend toward TNF-α inhibition at 6, 12, and 24 h was also observed, with p values of 0.061, 0.06, and 0.062, respectively (Fig. 6C). Analogously, although preincubation with SB203580 led to a significant reduction in IL-6 production only at 3 and 24 h after mtDNA exposure, a trend toward IL-6 inhibition at the remaining two selected time points (6 and 12 h) was also observed, with p values of 0.093 and 0.066, respectively (Fig. 6B). Additionally, as shown in Fig. 6, pretreatment with p38 MAPK siRNA significantly reduced the production of all three inflammatory cytokines in mtDNA-stimulated THP-1 macrophages at all four selected time points. These data therefore suggested an important role for the p38 MAPK signal in mediating cytokine production in THP-1 macrophages stimulated with mtDNA. Furthermore, we attempted to investigate whether TLR9 is a key upstream molecule that mediates the inflammatory cytokine response induced by mtDNA in THP-1 macrophages. We pretreated THP-1 cells with TLR9 siRNA or with CQ before mtDNA exposure. As shown in Fig. 5, both TLR9 siRNA transfection and CQ preincubation suppressed the p38 MAPK activation induced by mtDNA. Additionally, the data shown in Fig. 6 demonstrate that pretreatment with CQ or TLR9 siRNA transfection resulted in a significant reduction in the production of all three examined cytokines (IL-1β, IL-6, and TNF-α) triggered by mtDNA exposure at all four selected time points (3, 6, 12, and 24 h). These observations suggested that TLR9 acts as a key upstream molecule in mtDNA-induced cytokine production and p38 MAPK activation in THP-1 macrophages.
Discussion Although it is well known that both bacteria and specific environmental exposures can lead to various lung diseases with profound inflammation, many cases of inflammatory lung disease are idiopathic, and the etiological factors leading to pulmonary inflammation are always unknown. Therefore, we investigated the potential of mtDNA, which is a newly identified danger-associated molecular pattern, for directly inducing lung inflammation in vivo. The present study showed that mtDNA instigated a series of
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Fig. 6. Effects of mtDNA on cytokine production by cultured THP-1 macrophages that were or were not pretreated with chloroquine, SB203580, TLR9 siRNA, or p38 MAPK siRNA. (A) Level of IL-1β in the cell-conditioned medium. (B) Level of IL-6 in the cell-conditioned medium. (C) Level of TNF-α in the cell-conditioned medium. *p o 0.05; **p o 0.001. The data are from three individual experiments. (Abbreviations: nDNA, nuclear DNA; mtDNA, mitochondrial DNA; CQ, chloroquine; SB, SB203580; NC-siRNA, negative control siRNA).
changes involving local inflammatory responses in the lung within a short period of time after instillation into the lung via the pattern recognition receptor TLR9. To further determine whether mtDNA activates innate immune cells in addition to polymorphonucleocytes (PMNs), we exposed THP-1 macrophages to mtDNA. The data presented clearly demonstrate that the exposure of THP-1 macrophages to mtDNA in vitro resulted in the upregulation of proinflammatory cytokines and that this upregulation was mediated by the activation of the TLR9–p38 MAPK signal pathway. These findings indicate that mtDNA may independently trigger lung inflammation and that it plays an important etiological role in certain inflammatory lung diseases in which the inflammatory
phenotype of macrophages induced by mtDNA plays an important role. To the best of our knowledge, the present study provides the first report of the direct proinflammatory effect of intratracheal administration of mtDNA into the local lung tissues. Specifically, mtDNA was found to trigger an inflammatory response in the lung that persisted for 24 h. This response was manifested by an increase in infiltrating inflammatory cells, a thickening of the alveolar septa, vascular congestion, the production of proinflammatory cytokines, and the activation of p38 MAPK in the lung. In line with these findings, previous studies conducted by Zhang et al. have shown that the
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intraperitoneal injection of mtDNA can induce systemic inflammation and acute lung injury [21]. However, that study did not clarify whether mtDNA itself can induce an inflammatory response in the lung because there are t least two possible mechanisms responsible for inflammation-associated lung injury in the context of a systemic inflammatory response: the lung injury may be due to inflammatory mediators that were extensively released into the bloodstream, and the phenomenon of lung injury may also be caused directly by mtDNA that traveled through the endothelial–epithelial barrier and reached the lung via the blood circulation. In addition, the time-course changes in the proinflammatory cytokines recorded in the present study first showed that the concentrations of both IL-1β and TNF-α in the lung were elevated as early as 3 h after mtDNA instillation, reached peaks at 6 h, and then decreased. Consistent with this finding, the immunofluorescence-labeled lung specimens also showed that the number of CD68 þ macrophages that had infiltrated into the lung was increasing at 3 h, reached maximal levels at 6 h, and then gradually decreased at 12 and 24 h after mtDNA challenge. This observation indirectly suggested that the macrophages were a possible source of the proinflammatory cytokines and that these cells were involved in the pulmonary inflammation induced by mtDNA administration. However, the time course for IL-6 in the lung homogenate was slightly different from that found for the infiltrated CD68 þ macrophages in the lung: the IL-6 levels reached a peak 3 h after mtDNA instillation and then gradually decreased within 24 h after mtDNA exposure. This finding indicates that the macrophages may not be the only source of IL-6 and that additional innate immune cells may also participate in the initiation and development of mtDNA-induced lung inflammation. Although the capacity of mtDNA to trigger innate immune responses through both the TLR9–p38 MAPK and the NLRP3– caspase-1 signaling pathways has been extensively investigated [15,28,29], the potential ability of cell-free mtDNA to induce an inflammatory phenotype in macrophages and the involved mechanisms are not fully understood. The data presented here clearly demonstrate that mtDNA exposure results in the upregulation of proinflammatory cytokines as well as the activation of p38 MAPK in cultured THP-1 macrophages. In agreement with our in vitro data, Zhang et al. have reported that mtDNA challenge induces a robust inflammatory response in primary cultured peritoneal macrophages, as manifested by the production of inflammatory mediators, including TNF-α, IL-6, and IL-10 [21]. However, it is interesting that the time course of the examined cytokines in the supernatants of THP-1 macrophages was not entirely consistent with that found in the in vivo experiments. This is probably because the cell lines do not fully reflect the responses of primary cells. Therefore, the reality in the lung may be slightly different from the in vitro results. The present finding that mtDNA induces pneumonitis in the murine model raises intriguing questions regarding the involvement of self-mtDNA in inflammation. It can be envisaged that certain pathological states resulting in tissue injury and necrosis could lead to the release of mtDNA from cells and that these endogenous mtDNA could subsequently promote destructive processes in the host through the initiation of autoinflammation and thereby create a vicious circle of cell destruction and inflammation. In fact, in a previous study [15], Zhang and co-workers showed that hemorrhagic shock-induced mtDNA release contributes to the development of posttraumatic SIRS and organ injury by activating PMNs through p38 MAPK. Additionally, in a recent report, we demonstrated that the plasma mtDNA concentration is positively correlated with the risk of SIRS in patients with acute traumatic injury [1]. Therefore, it seems likely that therapies including nuclease or suppressive oligodeoxynucleotides may
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minimize or counteract the impact of extracellular mtDNA, particularly in cases of mtDNA-induced sterile inflammation. Although the present study indicated that mtDNA is clearly involved in inflammation, there are several limitations that warrant further discussion. The major limitation of this study is that it still remains unclear which components of mtDNA contribute to its proinflammatory effect in the lung. Additionally, because the individual preparation of mtDNA is moderately different and the isolated mtDNA may contain scanty proteins or nDNA, each preparation of mtDNA probably has a slightly different ratio of these components. Thus, unlike using commercial reagent preparations, the application of these extracted mtDNA preparations at the same concentration does not always guarantee the same responses in different assays. Therefore, further studies applying commercial standardized mtDNA are required to determine the proinflammatory potential of mtDNA in the lung and to clarify the mechanisms involved in this process.
Conclusions To summarize, the present study demonstrated that the intratracheal administration of mtDNA induces a local inflammatory response in the mouse lung that depends on the interactions of mtDNA with TLR9 and may be correlated with infiltrated macrophages. Because the exposure of cultured THP-1 macrophages to mtDNA led to the upregulation of several proinflammatory cytokines via the TLR9–p38 MAPK signal pathway, it is likely that macrophage activation by mtDNA may play an important role in the mtDNA-induced lung inflammation. These events may participate in the genesis of certain idiopathic inflammatory lung diseases and provide novel therapeutic targets for these diseases.
Acknowledgments The present study was supported by the National Natural Scientific Foundation of China (Nos. 81170064 and 81370172). The National Natural Scientific Foundation of China grants did not directly participate in the literature search, determination of study eligibility criteria, data analysis or interpretation, or preparation, review, or approval of the manuscript for publication. Statements in this article should not be construed as an endorsement by the National Natural Scientific Foundation of China.
Appendix A.
Supplementary material
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Original Contribution
Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9–p38 MAPK pathway Xiaoling Gu a, Guannan Wu a, Yanwen Yao a, Junli Zeng a, Donghong Shi b, Tangfeng Lv a, Liang Luo c, Yong Song a,n a
Department of Respiratory Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002, People’s Republic of China Department of Medical Imaging, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002, People’s Republic of China c Intensive Care Unit, Wuxi Second Affiliated Hospital, Nanjing Medical University, Wuxi, Jiangsu Province 210004, People’s Republic of China b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 November 2014 Received in revised form 14 February 2015 Accepted 25 February 2015 Available online 13 March 2015
An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1β, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1β, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9–p38 MAPK signal transduction pathway. & 2015 Elsevier Inc. All rights reserved.
Keywords: Mitochondrial DNA Lung Inflammation TLR9–p38 MAPK signal transduction pathway Free radicals
The etiological factors leading to pulmonary inflammation are often complicated. Although it has been well established that bacteria and specific environmental exposures are responsible for various lung diseases with profound inflammation, many cases of inflammatory lung disease are idiopathic and do not present any identifiable etiology. For instance, most cases of acute respiratory distress syndrome do not have a specific etiology and cannot be well explained by existing mechanisms. There has recently been increasing interest in clarifying the role of endogenous danger signals in the development of inflammatory lung injury and the systemic inflammatory response. The results of our previous study
Abbreviations: mtDNA, mitochondrial DNA; SIRS, systemic inflammatory response syndrome; nDNA, nuclear DNA; CpG, cytosine–phosphate–guanine; MAPK, mitogen-activated protein kinase; TLR9, Toll-like receptor 9; DAPI, 40 ,6diamidino-2-phenylindole; siRNA, small interfering RNA; CQ, chloroquine; SB, SB203580 n Corresponding author. Fax: þ 86 25 80863591. E-mail address: [email protected] (Y. Song). http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.034 0891-5849/& 2015 Elsevier Inc. All rights reserved.
showed that the plasma concentration of mitochondrial DNA (mtDNA)1, a newly identified damage-associated molecular pattern, is positively correlated with the risk of systemic inflammatory response syndrome (SIRS) in acute traumatic patients [1]. Additionally, a recently published study reported that acid aspiration results in a 120-fold increase in the mtDNA concentration in cell-free bronchoalveolar lavage fluid in an animal model of acute gastric aspiration lung injury [2]. However, the specific role of mtDNA in the development of inflammatory lung injury remains to be further determined. Mitochondria DNA is generally considered to be derived from the circular genomes of bacteria that evolved into the mitochondria of today’s eukaryotic cells [3–7]. Similar to bacterial DNA, mtDNA is circular and contains a higher frequency of unmethylated cytosine– phosphate–guanine (CpG) dinucleotides [8–10]. DNA that contains unmethylated CpG motifs can be recognized by the innate immune system and exerts powerful immunostimulatory effects [11–13]. Previous studies conducted by Oka et al. [14] have demonstrated that mtDNA that escapes from autophagy-mediated degradation can
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trigger local inflammation in the heart. Additionally, Zhang et al. [15] reported that mtDNA released into the circulation by shock can activate the neutrophil p38 mitogen-activated protein kinase (MAPK) signaling pathway via TLR9 and potentially contribute to the development of posttraumatic SIRS. The results of these recent studies indicate that mtDNA can not only participate in the activation of innate immunity in the original cell but also initiate systemic innate immunity. It is well known that CpG-containing DNA activates inflammatory responses via an endosomally located pattern recognition receptor, namely TLR9 [16–18], which is constitutively expressed in endothelial cells and macrophages in the lung [19,20]. It has been reported that the intravenous administration of mtDNA can cause SIRS and lung inflammation [21]. However, it is not clear whether the lung inflammation observed in this animal model is directly induced by mtDNA or is just a secondary result of SIRS. Even if this published study has confirmed the immunostimulatory potential of mtDNA in the lungs, the related molecular mechanisms remain unclear. Because DNA is readily endocytosed by macrophages [22], which act as the first line of host defense in the lung, it is logical to hypothesize that mtDNA is capable of causing lung inflammation via active alveolar macrophages and may be mediated by a signaling pathway similar to the TLR9–p38 MAPK pathway in neutrophils. To determine the possible role of mtDNA in the induction of lung inflammation, we intratracheally administered mtDNA to mice and simultaneously examined the in vitro effect of mtDNA on THP-1 macrophages, a human monocytic and macrophage cell line, to elucidate the specific signal transduction mechanism that mediates the inflammatory activation of THP-1 induced by mtDNA exposure.
Materials and methods Main reagents 40 ,6-Diamidino-2-phenylindole (DAPI), phorbol 12-myristate 13-acetate (PMA), chloroquine, and protease inhibitor cocktail were purchased from Sigma. Mouse anti-β-actin antibody and rabbit anti-TLR9 antibody were obtained from Abcam. Antibodies to p38 MAPK and phospho-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling. Alexa Fluor 594-conjugated goat anti-rabbit IgG was purchased from Invitrogen. Goat anti-rabbit IgG–horseradish peroxidase (HRP), goat anti-mouse IgG–HRP, and rabbit anti-CD68 antibody were obtained from Boster Biotechnology Co. Ltd. (Wuhan, China). The small interfering RNA (siRNA) targeting p38 MAPK, siRNA targeting TLR9, and negative control siRNA were purchased from GenePharma (Shanghai, China), and Lipofectamine 2000 and Opti-MEM I reduced-serum medium were obtained from Invitrogen Corp. (Carlsbad, CA, USA). Heattreated fetal bovine serum and RPMI 1640–Hepes medium were obtained from Wisent, Inc. Mice and ethics statement Healthy male C57BL/6 mice weighing 20–22 g and age 7–8 weeks were obtained from the Animal Feeding Center of Yangzhou University. The animal care and experimental procedures were performed in compliance with the Institutional Animal Care and User Guidelines and were approved by the Model Animal Research Center of Jinling Hospital. Cell line, culture, transfection, and differentiation THP-1 cells were obtained from the American Type Culture Collection and were cultured in complete medium (RPMI 1640–Hepes
containing 100 U/ml penicillin/streptomycin and 10% fetal bovine serum) according to the supplier’s guidelines. The transfection of THP-1 cells was performed according to the manufacturer’s recommended protocol (Invitrogen Corp.). Briefly, THP-1 cells were seeded at 5 105 cells/ml in 10-cm dishes in 12 ml of growth medium without antibiotics. The siRNA solution and Lipofectamine 2000 solution were prepared in 1.5 ml of Opti-MEM I reduced-serum medium. The siRNA and Lipofectamine solutions were then incubated together for 20 min at room temperature to form the siRNA–Lipofectamine complex, and this complex was then added to the cells to a final siRNA concentration of 50 nM. The cells were incubated at 37 1C in a CO2 incubator. The knockdown efficiency was verified 48 to 72 h after transfection (Supplementary Fig. s1). For differentiation into macrophages, THP-1 cells were plated at 1 10 6 cells/well in six-well plates and differentiated by exposure to 100 ng/ml PMA for 24 h. The differentiated, plastic-adherent cells were washed twice with fresh complete medium and allowed to rest for 6 h before exposure to various conditions. Preparation of mtDNA and nuclear DNA (nDNA) The mouse liver mitochondria were isolated using mitochondrial isolation kits for tissues (Pierce Scientific, Rockford, IL, USA) according to the protocol supplied by the manufacturer. Mitochondria isolation kits for cells (Pierce Scientific) were used to isolate the mitochondria from cultured THP-1 cell lines. The mitochondrial isolation was performed under sterile conditions at 4 1C. The isolated mitochondrial pellets were suspended in Hanks’ balanced salt solution buffer (Gibco Life Technologies, Gaithersburg, MD, USA). The nuclear fractions of THP-1 cells and hepatocytes were reserved for the subsequent preparation of nDNA. The mitochondrial DNA and nDNA were extracted from the isolated mitochondrial pellets and nuclear fractions, respectively, under sterile conditions with the DNeasy blood and tissue kits (Qiagen, Valencia, CA, USA), according to the protocol supplied by the manufacturer. The DNA concentrations and purity were examined by spectrophotometry. The A260/280 ratio of both the mtDNA and the nDNA samples was 1.8 to 2.0, confirming the lack of any significant protein contamination. The endotoxin levels in the DNA samples were assessed using the Limulus amoebocyte lysate assay, and no detectable levels were observed. To further exclude any significant nDNA contamination and ensure the purity of the mtDNA, we conducted real-time PCR to probe the samples for the presence of the nuclear genes GAPDH and 18S and for the presence of the mitochondrial genes MT-ND2 and mtCOI, and we found that the nDNA content was less than 0.1% in the isolated mtDNA samples. Primers for the nDNA markers GAPDH (forward 50 -CCATGTTCGTCATGGGTGTGAAC-30 and reverse 50 -GCCAGTAGAGGCAGGGATGATGTTC-30 ) and 18S (forward 50 -GTTCATCCTGTTCCTGCTCC-30 and reverse 50 -GTTCATCCTGTTCCTGCTCC-30 ) and for the mtDNA markers MT-ND2 (forward 50 -CACAGAAGCTGCCATCAAGTA-30 and reverse 50 -CCGGAGAGTATATTGTTGAAGAG-30 ) and mtCOI (forward 50 -GCCCCAGATATAGCATTCCC-30 and reverse 50 GTTCATCCTGTTCCTGCTCC-30 ) were all synthesized by Invitrogen. Animal procedures Seven- to eight-week-old mice were randomly divided into three groups (groups A, B, and C) with 25 animals per group. All of the mice were anesthetized with intraperitoneal pentobarbital sodium (3 mg/kg) and placed in the supine position with the head tilted back. The intratracheal administration of nDNA (3 mg/kg, group A) or mtDNA (3 mg/kg, group B and group C) in a volume of 60 ml was performed via a microsprayer (Penn-Century, Wyndmoor, PA, USA). The mice in group C were intraperitoneally pretreated
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with chloroquine (30 mg/kg) for 2 h before mtDNA exposure. All of the mice were sacrificed 0, 3, 6, 12, or 24 h after administration, and their lungs were subsequently harvested. The large left lobe was fixed in 4% paraformaldehyde for histological analysis, and the remaining lung lobes were stored at 80 1C before the subsequent experimental assays. Here, the 0-h time point means that the mice were untreated shams and were immediately sacrificed after tracheal intubation procedure.
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centrifugation at 12,000 rpm for 30 min. The concentrations of various cytokines in the mouse lung extracts and the supernatants (conditioned medium) of THP-1 macrophages were assessed using appropriate ELISA kits as recommended by the manufacturer (4A Biotech Co., Ltd, Beijing, China). The lowest limits of detection for mouse interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 were 7, 7, and 4 pg/ml, respectively, and the lowest detection limits for human IL-1β, IL-6, and TNF-α were 2, 1, and 4 pg/ml, respectively.
THP-1 macrophage activation assays Western blot analysis Differentiated THP-1 macrophages were exposed to mtDNA (10 mg/ml) or nDNA (10 mg/ml). For the inhibitor studies, THP-1 macrophages were pretreated with chloroquine (10 mg/ml, 30 min) or SB203580 (1 mM, 30 min), which is a specific inhibitor of the p38 MAPK signal transduction pathway, or were transfected with TLR9 siRNA or p38 MAPK siRNA before mtDNA exposure. The cellconditioned medium was collected by centrifugation 0, 3, 6, 12, or 24 h after exposure and then assayed for proinflammatory cytokines by ELISA as described below. THP-1 macrophages were lysed with extraction buffer containing a protease inhibitor cocktail (Roche) 0, 30, 60, or 120 min after exposure to determine the total and phosphorylated p38 MAPK levels by Western blot as described below. Histopathology and immunofluorescence of the lungs The left lungs were fixed overnight at 4 1C in 4% paraformaldehyde and processed by successive dehydration with an alcohol series and xylene. The tissues were then embedded in paraffin and cut into 5-mm-thick sections for hematoxylin–eosin staining or immunofluorescence assay. Hematoxylin–eosin (H&E) staining was carried out according to the instructions provided by the manufacturer to determine the severity of the lung inflammation. All of the tissue sections were evaluated by an experienced blinded pathologist and scored according to criteria described previously [23]. Briefly, according to three criteria, i.e., vascular congestion and interstitial edema, alveolar structural disturbance, and atelectasis and inflammatory cell infiltration, and using a semiquantitative scale, the histological score of the lung sections was calculated as the sum of the scores (0–3) given for each criterion. The cumulative histology score ranged from 0 to 9. To observe macrophages in the lung, we conducted an immunofluorescence assay as described below. Briefly, paraffinembedded lung tissue sections were dewaxed in xylene and rehydrated in graded ethanol washes (100, 90, 70, and 50%). The samples were then washed twice for 5 min with phosphatebuffered saline (PBS) and subsequently incubated with 0.3% Triton X-100 for 8 min at room temperature. The samples were washed in PBS for 10 min, blocked with 3% bovine serum albumin (BSA) for 1 h at 37 1C, and incubated overnight at 4 1C with a rabbit antiCD68 antibody at a 1:200 dilution in 2% BSA. The samples were then washed three times with PBS for 5 min each and subsequently incubated with a goat anti-rabbit Alexa Fluor 594conjugated secondary antibody (1:400) for 1 h at 37 1C in the dark. Finally, the nuclei were stained with DAPI (5 mg/ml). The samples were visualized with a laser scanning confocal microscope (FluoView FV10i, Japan) to detect the CD68 þ macrophages using red fluorescence. Enzyme-linked immunosorbent assay The middle lobe of the right lung was homogenized and lysed with extraction buffer containing a protease inhibitor cocktail (Roche), and we then collected the tissue supernatants via
The cell proteins were obtained from THP-1 macrophages, and the tissue proteins were obtained from the right lower lungs of the mice in each group. We performed a Western blot analysis of the cellular and tissue lysates as previously described [24]. Briefly, the cells or tissue homogenates were lysed in ice-cold protein extraction buffer containing a protease inhibitor cocktail (Roche) for 30 min. After the whole lysates were centrifuged at 12,000g for 30 min, the supernatants were collected. The protein concentrations in the supernatants were determined by the Bradford method. The proteins (20 mg) from the lung tissue and THP-1 macrophages were separated in 12% SDS–PAGE gels and electrophoretically transferred onto a polyvinylidene fluoride membrane using an electroblotting apparatus (Bio-Rad). The membranes were blocked in 5% BSA in PBS/Tween 20 (PBST) for 1 h at 37 1C, and the blocked membranes were then incubated overnight at 4 1C with anti-β-actin mouse monoclonal antibody (1:1000), antiphospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (1:800), or anti-p38 MAPK rabbit polyclonal antibody (1:800) in PBST containing 2% BSA. After three washes with PBST buffer, the membranes were incubated with secondary HRP-conjugated antibody for 1 h at 37 1C. The protein signals were enhanced by chemiluminescence detection kits (SuperSignal West Pico) and detected using an Odyssey Scanning System (Li-Cor Biosciences, Lincoln, NE, USA). Statistical analysis The study data are expressed as the means 7 standard deviation and assessed for statistical significance using Student’s t test. A two-tailed p value less than 0.05 was considered statistically significant. All of the calculations and statistical analyses were performed using SPSS software for Windows (version 17.0).
Results MtDNA induces histopathological changes in the lungs via TLR9 To examine the potential of mtDNA to induce lung inflammation, mtDNA was intratracheally instilled into the lungs of normal male C57BL/6 mice. Serial lung sections obtained from mice after mtDNA exposure were stained with H&E and examined by light microscopy. Representative microscopic images of these H&Estained lung specimens are shown in Fig. 1. The mtDNA administration caused marked inflammatory cell infiltration accompanied by thickening of the alveolar septa, which is suggestive of lung inflammation and acute lung injury. However, the mice in the nDNA control group showed no pathological changes in pulmonary inflammation. Additionally, as shown in Table 1, the histological score of the pulmonary tissues in the mtDNA exposure group was also markedly increased as early as 3 h after instillation, peaked at 6 h, and then gradually decreased, but the values after this decrease remained significantly higher compared with those
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Fig. 1. Representative photomicrographs of lung histopathology stained with H&E. (A) Control group treated with nDNA (3 mg/kg). (B) mtDNA challenge group treated with mtDNA (3 mg/kg). (C) Chloroquine inhibitory group, which was pretreated with CQ (30 mg/kg, ip) before mtDNA (3 mg/kg) challenge. Original magnification, 400. These observations are representative of results of experiments with five mice per time point per group.
Table 1 Histological scores of lung tissue. mtDNA
CQ þ mtDNA
Mean SD
Mean SD
Mean SD
0.3 0.5 0.5 0.3 0.3
0.3 3.6 4.9 4.8 3.9
0.3 2.5 3.5 3.0 3.0
Time point nDNA
0h 3h 6h 12 h 24 h a b
0.67495 0.70711 0.70711 0.48305 0.67495
0.67495 0.69921 1.19722 1.47573 0.99443
0.48305 1.08012 1.08012 0.66667 0.8165
pa
pb
1.0 o 0.001 o 0.001 o 0.001 o 0.001
1.0 0.015 0.013 0.002 0.04
mtDNA vs nDNA. CQ þ mtDNA vs mtDNA.
found for the nDNA control group (p o 0.001 for all four selected time points). Considering that TLR9 is the only known Toll-like pattern recognition receptor for mtDNA, to further determine whether TLR9 is a key upstream molecule that mediates the inflammatory response induced by mtDNA in the lung, mice were intraperitoneally pretreated with chloroquine for 2 h and then subjected to intratracheal mtDNA instillation. As shown in Fig. 1C and Table 1, pretreatment with chloroquine (CQ) significantly ameliorated the mtDNA-induced inflammatory pathological changes in the lungs and reduced the histological score of the pulmonary tissues after mtDNA exposure. These results suggested that TLR9 mediates the inflammatory histopathological changes in the lungs stimulated by mtDNA. Additionally, because alveolar macrophages act as the first line of host defense in the lung, we conducted an immunofluorescence assay with the paraffin-embedded lung tissue sections to determine whether macrophages had infiltrated into the mtDNA-treated pulmonary tissues. Representative
microscopic findings from these immunofluorescence-labeled lung specimens are shown in Fig. 2. The mice treated with control nDNA presented no obvious CD68 þ macrophage infiltration at all selected time points after the intratracheal instillation. However, exposure to mtDNA resulted in a profound increase in the amount of CD68 þ macrophages in the lung tissues at all selected time points, which suggested that the mtDNA-induced lung inflammation was accompanied by CD68 þ macrophage infiltration and that macrophages probably play an important role in the initiation and development of mtDNA-induced inflammation. MtDNA triggers cytokine responses in the lungs via TLR9 As further evidence of the mtDNA-induced inflammation, the concentrations of proinflammatory cytokines in the lung homogenate were assessed by ELISA. As shown in Fig. 3, in the mtDNA instillation group at all four time points (3, 6, 12, and 24 h), all of the three examined proinflammatory cytokines (IL-1β, IL-6, and TNF-α) exhibited statistically significant increases compared with the levels observed in the nDNA exposure control group. The concentrations of both IL-1β and TNF-α were highest 6 h after the mtDNA challenge and then gradually decreased, but these decreased levels remained significantly higher compared with those found in the respective nDNA controls. The levels of IL-6 in the lung homogenate reached a peak as early as 3 h after mtDNA instillation and then decreased, and the decreased levels remained significantly higher than the levels observed in the control group. These data indicated that mtDNA triggers a significant increase in IL-1β, IL-6, and TNF-α production in the lungs and provide evidence for the potential ability of mtDNA to induce lung inflammation.
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Fig. 2. Representative photomicrographs of immunofluorescence-labeled CD68 þ macrophages in lung specimens. The macrophages were labeled with antibodies to CD68 (red), and the nuclei were stained with DAPI (blue). Scale bar, 30 mm. These observations are representative of results from experiments with five mice per time point per group.
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Fig. 3. Cytokine responses in the lungs induced by control nDNA, mtDNA, and CQ þ mtDNA. (A) Concentration of IL-1β in the lung homogenate. (B) Concentration of IL-6 in the lung homogenate. (C) Concentration of TNF-α in the lung homogenate. *p o 0.05; **p o 0.001. The results are from experiments with five mice per time point per group.
The proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the mice of the CQ pretreatment group were also assessed and compared with those of the mtDNA-exposure group. As shown in Fig. 3, pretreatment with CQ resulted in a significant reduction in the IL-6 concentration in the lung homogenates at all four selected time points compared with the levels found in the mtDNA-exposure group (p o 0.05, Fig. 3B). Although pretreatment with CQ led only to a decreasing trend in the IL-1β level, with a limbic p value of 0.063 at 12 h, the suppression effect of CQ on IL-1β production assessed at the other three selected time points (3, 6, and 24 h) was statistically significant (p o 0.05, Fig. 3A). CQ pretreatment also significantly reduced the concentrations of TNF-α in the lung homogenates at 3 and 6 h after mtDNA instillation but did not significantly downregulate the levels of TNF-α at 12 and 24 h compared with the levels found in the mtDNA-exposure group (Fig. 3C). Taken together, these data suggested that TLR9 mediates the inflammatory cytokine response in the lungs after mtDNA exposure. Intratracheal mtDNA activates p38 MAPK in the lungs via TLR9 Because p38 MAPK is a key early intermediary in the inflammatory response and is one of the downstream targets of the TLR9 signaling pathway, which is the only known Toll-like receptor pathway for mtDNA, we performed a Western blot assay to determine the effect of mtDNA on the total and phosphorylated p38 MAPK levels in the lung. As shown in Fig. 4, mtDNA instillation induced a significant upregulation of phosphorylated p38 MAPK at 6 h, and this level returned to the baseline 12 h after
mtDNA stimulation. However, the expression of total p38 MAPK was not significantly changed within 24 h of mtDNA exposure. Conversely, nDNA administration had no effect on the expression of total or phosphorylated p38 MAPK (Fig. 4). These observations suggested that mtDNA induces p38 MAPK activation in vivo and that this activation is probably involved in the mtDNA-induced lung inflammation. Further more, pretreatment with CQ significantly suppressed p38 MAPK activation in lung tissue after mtDNA exposure (Fig. 4). It confirmed that mtDNA activates the p38MAPK pathway via TLR9 in the lung of model mice. MtDNA activates p38 MAPK in cultured THP-1 macrophages via TLR9 Given that the above-presented evidence indicated that mtDNA exposure can trigger significant pulmonary inflammation via the pattern recognition receptor TLR9 and that this inflammation was accompanied by CD68 þ macrophage infiltration, to clarify whether macrophages play an important role in the development of mtDNA-induced inflammation, we performed further experiments to determine whether mtDNA exposure can induce cytokine production and activate p38 MAPK in cultured THP-1 macrophages. Differentiated THP-1 macrophages were incubated with either nDNA or mtDNA (10 mg/ml), and a Western blot assay was performed to determine whether mtDNA stimulation activates the p38 MAPK pathway in THP-1 macrophages. As shown in Fig. 5, mtDNA exposure induced significant upregulation of phosphorylated p38 MAPK as early as 30 min after incubation, and the increased expression of phosphorylated p38 MAPK
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Fig. 4. Protein expression profiles of phosphorylated (p) p38 MAPK in the lungs induced by control nDNA, mtDNA, and CQ þ mtDNA. The results are representative of experiments with five mice per time point per group.
Fig. 5. Protein expression profiles of phosphorylated (p) p38 MAPK in THP-1 macrophages that were or were not pretreated with chloroquine, SB203580, TLR9 siRNA, or p38 MAPK siRNA and were then challenged with mtDNA. The results are representative of three individual experiments.
remained 1 h after exposure and then weakened to the baseline level at 2 h after mtDNA stimulation. However, the expression of total p38 MAPK was not significantly changed within 2 h of mtDNA administration. Conversely, nDNA exposure had no effect on the expression of total or phosphorylated p38 MAPK (Fig. 5). These observations suggested that mtDNA induces p38 MAPK activation in cultured THP-1 macrophages. Furthermore, we attempted to investigate whether TLR9 is a key upstream molecule that mediates the p38 MAPK activation induced by mtDNA in THP-1 macrophages. We pretreated THP-1 cells with TLR9 siRNA before mtDNA exposure. Additionally, because endosomal acidification is a prerequisite for several TLRs (including TLR9) [25–27], THP-1 macrophages were preincubated with CQ, an inhibitor of endosomal acidification, before mtDNA administration. As shown in Fig. 5, both TLR9 siRNA transfection and CQ preincubation resulted in no upregulation of the expression of phosphorylated p38 MAPK in THP-1 macrophages after mtDNA exposure. This means that mtDNA activates p38 MAPK in cultured THP-1 macrophages via TLR9. MtDNA induces cytokine production by THP-1 macrophages via the TLR9–p38 MAPK pathway The concentrations of proinflammatory cytokines in the supernatants of the THP-1 macrophages after mtDNA exposure were
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assessed by ELISA. As shown in Fig. 6, in the mtDNA stimulation group at all four time points (3, 6, 12, and 24 h), all three examined cytokines (IL-1β, IL-6, and TNF-α) exhibited significant increases compared with the levels found in the nDNA-exposure control group. The concentrations of both IL-1β and IL-6 were highest 12 h after the mtDNA challenge and then decreased at 24 h, but the decreased levels remained significantly elevated compared with those found in the nDNA controls. A persistent increase in the concentrations of TNF-α in the THP-1 macrophage culture medium treated with mtDNA was observed within 24 h after administration, and this increase was significantly higher than that found in the nDNA-exposure control group. These data indicated that mtDNA triggered a marked increase in IL-1β, IL-6, and TNF-α production in the cultured THP-1 macrophages. Because the above-presented evidence indicated that mtDNA exposure activates the p38 MAPK pathway in cultured THP-1 macrophages, we performed further experiments using the pharmacological probe SB203580 and p38 MAPK siRNA to clarify the causality between the p38 MAPK signal and the cytokine production induced by mtDNA. As shown in Fig. 5, both preincubation with SB203580 and pretreatment with p38 MAPK siRNA successfully suppressed the p38 MAPK activation triggered by mtDNA. In addition, pretreatment with SB203580 resulted in a significant reduction in IL-1β production by mtDNA-stimulated THP-1 macrophages at all four selected time points (p o 0.05, Fig. 6A). Although the suppression effect of SB203580 on TNF-α production was statistically significant only at 3 h after mtDNA administration, a trend toward TNF-α inhibition at 6, 12, and 24 h was also observed, with p values of 0.061, 0.06, and 0.062, respectively (Fig. 6C). Analogously, although preincubation with SB203580 led to a significant reduction in IL-6 production only at 3 and 24 h after mtDNA exposure, a trend toward IL-6 inhibition at the remaining two selected time points (6 and 12 h) was also observed, with p values of 0.093 and 0.066, respectively (Fig. 6B). Additionally, as shown in Fig. 6, pretreatment with p38 MAPK siRNA significantly reduced the production of all three inflammatory cytokines in mtDNA-stimulated THP-1 macrophages at all four selected time points. These data therefore suggested an important role for the p38 MAPK signal in mediating cytokine production in THP-1 macrophages stimulated with mtDNA. Furthermore, we attempted to investigate whether TLR9 is a key upstream molecule that mediates the inflammatory cytokine response induced by mtDNA in THP-1 macrophages. We pretreated THP-1 cells with TLR9 siRNA or with CQ before mtDNA exposure. As shown in Fig. 5, both TLR9 siRNA transfection and CQ preincubation suppressed the p38 MAPK activation induced by mtDNA. Additionally, the data shown in Fig. 6 demonstrate that pretreatment with CQ or TLR9 siRNA transfection resulted in a significant reduction in the production of all three examined cytokines (IL-1β, IL-6, and TNF-α) triggered by mtDNA exposure at all four selected time points (3, 6, 12, and 24 h). These observations suggested that TLR9 acts as a key upstream molecule in mtDNA-induced cytokine production and p38 MAPK activation in THP-1 macrophages.
Discussion Although it is well known that both bacteria and specific environmental exposures can lead to various lung diseases with profound inflammation, many cases of inflammatory lung disease are idiopathic, and the etiological factors leading to pulmonary inflammation are always unknown. Therefore, we investigated the potential of mtDNA, which is a newly identified danger-associated molecular pattern, for directly inducing lung inflammation in vivo. The present study showed that mtDNA instigated a series of
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Fig. 6. Effects of mtDNA on cytokine production by cultured THP-1 macrophages that were or were not pretreated with chloroquine, SB203580, TLR9 siRNA, or p38 MAPK siRNA. (A) Level of IL-1β in the cell-conditioned medium. (B) Level of IL-6 in the cell-conditioned medium. (C) Level of TNF-α in the cell-conditioned medium. *p o 0.05; **p o 0.001. The data are from three individual experiments. (Abbreviations: nDNA, nuclear DNA; mtDNA, mitochondrial DNA; CQ, chloroquine; SB, SB203580; NC-siRNA, negative control siRNA).
changes involving local inflammatory responses in the lung within a short period of time after instillation into the lung via the pattern recognition receptor TLR9. To further determine whether mtDNA activates innate immune cells in addition to polymorphonucleocytes (PMNs), we exposed THP-1 macrophages to mtDNA. The data presented clearly demonstrate that the exposure of THP-1 macrophages to mtDNA in vitro resulted in the upregulation of proinflammatory cytokines and that this upregulation was mediated by the activation of the TLR9–p38 MAPK signal pathway. These findings indicate that mtDNA may independently trigger lung inflammation and that it plays an important etiological role in certain inflammatory lung diseases in which the inflammatory
phenotype of macrophages induced by mtDNA plays an important role. To the best of our knowledge, the present study provides the first report of the direct proinflammatory effect of intratracheal administration of mtDNA into the local lung tissues. Specifically, mtDNA was found to trigger an inflammatory response in the lung that persisted for 24 h. This response was manifested by an increase in infiltrating inflammatory cells, a thickening of the alveolar septa, vascular congestion, the production of proinflammatory cytokines, and the activation of p38 MAPK in the lung. In line with these findings, previous studies conducted by Zhang et al. have shown that the
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intraperitoneal injection of mtDNA can induce systemic inflammation and acute lung injury [21]. However, that study did not clarify whether mtDNA itself can induce an inflammatory response in the lung because there are t least two possible mechanisms responsible for inflammation-associated lung injury in the context of a systemic inflammatory response: the lung injury may be due to inflammatory mediators that were extensively released into the bloodstream, and the phenomenon of lung injury may also be caused directly by mtDNA that traveled through the endothelial–epithelial barrier and reached the lung via the blood circulation. In addition, the time-course changes in the proinflammatory cytokines recorded in the present study first showed that the concentrations of both IL-1β and TNF-α in the lung were elevated as early as 3 h after mtDNA instillation, reached peaks at 6 h, and then decreased. Consistent with this finding, the immunofluorescence-labeled lung specimens also showed that the number of CD68 þ macrophages that had infiltrated into the lung was increasing at 3 h, reached maximal levels at 6 h, and then gradually decreased at 12 and 24 h after mtDNA challenge. This observation indirectly suggested that the macrophages were a possible source of the proinflammatory cytokines and that these cells were involved in the pulmonary inflammation induced by mtDNA administration. However, the time course for IL-6 in the lung homogenate was slightly different from that found for the infiltrated CD68 þ macrophages in the lung: the IL-6 levels reached a peak 3 h after mtDNA instillation and then gradually decreased within 24 h after mtDNA exposure. This finding indicates that the macrophages may not be the only source of IL-6 and that additional innate immune cells may also participate in the initiation and development of mtDNA-induced lung inflammation. Although the capacity of mtDNA to trigger innate immune responses through both the TLR9–p38 MAPK and the NLRP3– caspase-1 signaling pathways has been extensively investigated [15,28,29], the potential ability of cell-free mtDNA to induce an inflammatory phenotype in macrophages and the involved mechanisms are not fully understood. The data presented here clearly demonstrate that mtDNA exposure results in the upregulation of proinflammatory cytokines as well as the activation of p38 MAPK in cultured THP-1 macrophages. In agreement with our in vitro data, Zhang et al. have reported that mtDNA challenge induces a robust inflammatory response in primary cultured peritoneal macrophages, as manifested by the production of inflammatory mediators, including TNF-α, IL-6, and IL-10 [21]. However, it is interesting that the time course of the examined cytokines in the supernatants of THP-1 macrophages was not entirely consistent with that found in the in vivo experiments. This is probably because the cell lines do not fully reflect the responses of primary cells. Therefore, the reality in the lung may be slightly different from the in vitro results. The present finding that mtDNA induces pneumonitis in the murine model raises intriguing questions regarding the involvement of self-mtDNA in inflammation. It can be envisaged that certain pathological states resulting in tissue injury and necrosis could lead to the release of mtDNA from cells and that these endogenous mtDNA could subsequently promote destructive processes in the host through the initiation of autoinflammation and thereby create a vicious circle of cell destruction and inflammation. In fact, in a previous study [15], Zhang and co-workers showed that hemorrhagic shock-induced mtDNA release contributes to the development of posttraumatic SIRS and organ injury by activating PMNs through p38 MAPK. Additionally, in a recent report, we demonstrated that the plasma mtDNA concentration is positively correlated with the risk of SIRS in patients with acute traumatic injury [1]. Therefore, it seems likely that therapies including nuclease or suppressive oligodeoxynucleotides may
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minimize or counteract the impact of extracellular mtDNA, particularly in cases of mtDNA-induced sterile inflammation. Although the present study indicated that mtDNA is clearly involved in inflammation, there are several limitations that warrant further discussion. The major limitation of this study is that it still remains unclear which components of mtDNA contribute to its proinflammatory effect in the lung. Additionally, because the individual preparation of mtDNA is moderately different and the isolated mtDNA may contain scanty proteins or nDNA, each preparation of mtDNA probably has a slightly different ratio of these components. Thus, unlike using commercial reagent preparations, the application of these extracted mtDNA preparations at the same concentration does not always guarantee the same responses in different assays. Therefore, further studies applying commercial standardized mtDNA are required to determine the proinflammatory potential of mtDNA in the lung and to clarify the mechanisms involved in this process.
Conclusions To summarize, the present study demonstrated that the intratracheal administration of mtDNA induces a local inflammatory response in the mouse lung that depends on the interactions of mtDNA with TLR9 and may be correlated with infiltrated macrophages. Because the exposure of cultured THP-1 macrophages to mtDNA led to the upregulation of several proinflammatory cytokines via the TLR9–p38 MAPK signal pathway, it is likely that macrophage activation by mtDNA may play an important role in the mtDNA-induced lung inflammation. These events may participate in the genesis of certain idiopathic inflammatory lung diseases and provide novel therapeutic targets for these diseases.
Acknowledgments The present study was supported by the National Natural Scientific Foundation of China (Nos. 81170064 and 81370172). The National Natural Scientific Foundation of China grants did not directly participate in the literature search, determination of study eligibility criteria, data analysis or interpretation, or preparation, review, or approval of the manuscript for publication. Statements in this article should not be construed as an endorsement by the National Natural Scientific Foundation of China.
Appendix A.
Supplementary material
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