The NALP3 inflammasome is required for collagen synthesis via the NF‑κB pathway

  • Authors:
    • Ju Kuang
    • Min Xie
    • Xiaolin Wei
  • View Affiliations

  • Published online on: January 18, 2018     https://doi.org/10.3892/ijmm.2018.3404
  • Pages: 2279-2287
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Abstract

The NALP3 inflammasome interacts with various immune and cell metabolic pathways and may participate in pulmonary fibrosis. However, little is known on its regulatory mechanism with respect to collagen synthesis. The objective of the present study was to investigate whether NALP3 inflammasome activation is involved in H2O2‑mediated collagen synthesis, in addition to examining the possible cell signaling mechanisms underlying this effect. It was demonstrated that the NF‑κB signaling pathway was activated under conditions of H2O2‑mediated oxidative stress in NIH‑3T3 mouse embryonic fibroblasts. H2O2‑exposed fibroblasts exhibited activated NALP3 inflammasomes via increased NALP3, apoptosis‑associated Speck‑like protein and caspase‑1 expression and the secretion of interleukin‑1β. H2O2 also elevated α‑SMA and type I collagen expression. In vitro silencing of NALP3 attenuated the degradation of IκBα and decreased the synthesis of type I collagen. Furthermore, the NALP3 inflammasome was found to be activated in bleomycin‑induced pulmonary fibrosis in mice, and this activation was relieved by a nuclear factor (NF)‑κB inhibitor. Taken together, these findings indicate that the NALP3 inflammasome is involved in H2O2‑induced type I collagen synthesis, which is mediated by the NF‑κB signaling pathway. Additionally, the NALP3 inflammasome contributes to the development of bleomycin‑induced pulmonary fibrosis.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease of unknown etiology, characterized by sequential episodes of acute lung injury with subsequent scarring. Pulmonary fibrosis may lead to respiratory failure due to damage to the lung structure and reduced gas exchange (1). No drugs are currently available that are able to control the accumulation of collagens in the lung once fibrosis has been established. Although several drugs have been applied to treat IPF, their clinical efficacy remains poor and the associated serious adverse effects pose problems during long-term treatment. Therefore, the development of novel therapeutic agents for this unmet medical need is required.

The NALP3 inflammasome represents the most extensively investigated inflammasome, and comprises a cytoplasmic multiprotein complex that consists of NACHT, LRR and PYD domains-containing protein 3 (NALP3), the adaptor protein apoptosis-associated Speck-like protein (ASC) containing a caspase recruitment domain (CARD), and pro-caspase-1, and interacts with various immune and cell metabolic pathways (2). Upon oligomerization, the NALP3 inflammasome activates the caspase-1 cascade, which produces the active pro-inflammatory cytokines interleukin (IL)-1β and IL-18 when triggered by a range of molecules, ranging from pathogen-associated molecular pattern molecules to damage-associated molecular pattern molecules, which are involved in infection, tissue injury and metabolic dysregulation. Evidence suggests that the inflammasome is involved in the development of fibrosis. Specifically, inorganic particulates associated with the development of pulmonary fibrosis, such as asbestos (3), silica (4) and nanoparticles (5,6), permeate lysosomal membranes, activating the NALP3 inflammasome, with subsequent IL-1β and IL-18 production. Furthermore, recent studies indicated that NF-κB mediates the reactive oxygen species (ROS)-induced NALP3 inflammasome by promoting the transcription of NALP3 and pro-IL-1β (7).

Bleomycin (BLM) is the chemotherapeutic agent most widely used to induce lung fibrosis in animal models and to identify the pathogenic mechanisms thereof, as the pathogenesis characteristics are very similar to those of IPF (8). Recently, NALP3 inflammasome activation in BLM-induced pulmonary fibrosis was reported by a series of studies (911). Specifically, BLM treatment has been shown to increase the production of ROS and uric acid (11), thereby inducing the development of lung fibrosis. In addition, these products have been shown in several cases to trigger NALP3 inflammasome activation. Furthermore, the purinergic receptor P2X7R is activated by ATP, which is released by injured lung cells following BLM treatment, leading to activation of the NALP3 inflammasome with subsequent cleavage and secretion of IL-1β and IL-18 (11,12). Notably, previous studies have demonstrated that a number of proinflammatory cytokines and profibrotic growth factors, including IL-1β, IL-6, IL-18, tumor necrosis factor-α and transforming growth factor (TGF)-β, are involved in pulmonary inflammation and fibrosis (13,14). Recent studies, primarily focusing on inflammasome/IL-1β secretion axis-mediated inflammatory actions, further suggested that the NALP3 inflammasome mediates the development of fibrosis in systemic sclerosis (15,16). NALP3 also appears to play a key role in promoting TGF-β signaling and Smad2/3 activation in renal epithelial cells (17). However, although TGF-β represents one of the most extensively investigated fibrogenic cytokines involved in the induction and development of pulmonary fibrosis, the mechanism underlying the role of NALP3 inflammasome in pulmonary fibrosis remains unclear.

Notably, an increasing volume of evidence indicates that NALP3 plays a key role in myofibroblast differentiation and collagen production in an IL-1β/Toll-like receptor 4 (TLR4)/MyD88/NF-κB-dependent manner, or an inflammasome-independent manner (18), as well as in liver fibrosis (19). Thus, in the present study, we hypothesized that the NALP3 inflammasome may participate in collagen production, and that NF-κB may serve as a link between NALP3 inflammasome activation and collagen synthesis.

Materials and methods

Cell culture, transfection and grouping

NIH-3T3 mouse embryonic fibroblasts were provided by the Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, Sichuan University (Chengdu, China). The cells were cultured in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare, Logan, UT, USA) supplemented with 10% fetal bovine serum (Sijiqing, Zhejiang, China) in a humidified atmosphere containing 5% CO2 at 37°C.

NALP3-siRNA (sense strand: 5′-CAGCCAGAGUGGAAUGACAdTdT-3′; antisense strand: 5′-UGUCAUUCCACUCUGGCUGdTdT-3′) and negative control (NControl) siRNA were synthesized by RiboBio, Guangzhou, China. Transient transfections were performed using the HiPerfect Transfection Reagent (Qiagen GmbH, Hilden, Germany) together with either an NControl siRNA (10 nM) or NALP3-siRNA (10 nM), according to the HiPerFect Transfection Reagent instruction manual. At 12 h after transfection, the cells were treated with H2O2 (200 μM). The silencing efficiency of the NALP3-siRNA was determined using reverse transcription-polymerase chain reaction (RT-PCR) after 12 h of treatment. Approximately 24 h after H2O2 stimulation, total RNA was extracted for RT-PCR and, 48 h later, total protein was extracted for western blot analysis, and the cell supernatants were frozen at -80°C for enzyme-linked immunosorbent assay (ELISA).

Additional groups of cells were treated with or without pyrrolidine dithiocarbamate (PDTC) (Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA) (50 μM) 1 h prior to H2O2 stimulation. The cells and cell supernatants were collected in the same manner as described above.

Animals

A total of 42 male C57BL/6 mice (age, 8 weeks; weight, 20–24 g) were purchased from the Laboratory Animal Center of Sichuan University (Chengdu, China). The mice were housed (n=5 per cage) in an air-conditioned animal facility under constant temperature and humidity, with a 12-h day-night cycle and free access to food and water. The mice were allowed to acclimatize for 2 weeks prior to the initiation of the experimental procedures. Animal experiments were performed according to protocols approved by the Laboratory Animal Welfare and Ethics Committee of the Sichuan University. Mice were randomly divided into three treatment groups as follows: i) BLM (Nippon Kayaku, Takasaki, Japan) + PDTC: On day 0, a single intratracheal injection of BLM (5 mg/kg in a final volume of 50 μl) was performed and PDTC (100 mg/kg) was intraperitoneally injected 2 h prior to the intratracheal injection. From day 1 onwards, PDTC (100 mg/kg) was intra-peritoneally injected once daily. ii) BLM + phosphate-buffered saline (PBS): On day 0, a single intratracheal injection of BLM (5 mg/kg in a final volume of 50 μl) was performed, and an equal volume of PBS was intraperitoneally injected 2 h prior to the surgery. From day 1 onwards, the same volume of PBS was intraperitoneally injected once daily. iii) Control group: A single intratracheal injection of PBS (50 μl) plus intraperi-toneal injections of PBS once daily. We utilized 10% chloral hydrate (3.5 ml/kg intraperitoneally) to anesthetize the mice. The mice (n=7/group) were sacrificed at 7 and 28 days after BLM intratracheal injection.

Western blotting

Whole protein from cells or lung tissue was lysed with RIPA lysis buffer (Beyotime, Shanghai, China) in the presence of protease inhibitor cocktail (Roche, Mannheim, Germany) for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. Protein concentrations were determined using the bicinchoninic acid protein assay (Beyotime, Shanghai, China) according to the manufacturer's instructions. Equal amounts of protein (60 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat dry milk in 0.1% Tween-20, 1X Tris-buffered saline (TBST; pH 7.4) for 1 h at room temperature, and then incubated with goat anti-NALP3 antibody (ab4207; Abcam, Cambridge, MA, USA), rabbit anti-IκBα antibody (cat. no. 4812, CST, Danvers, MA, USA), rabbit anti-pNF-κB antibody (cat. no. 3033, CST), rabbit anti-collagen I antibody (ab34710; Abcam), rabbit anti-α-SMA antibody (ab5694, Abcam), or rabbit anti-β-actin antibody (bs-0061R; Bioss, Beijing, China) in blocking solution overnight at 4°C, and washed three times with TBST at 10-min intervals. All above mentioned antibodies were diluted by 1:1,000. The membranes were then incubated with horseradish peroxidase-conjugated rabbit anti-goat (1:5,000; ZB-2306; ZSGB-Bio, Beijing, China) or mouse anti-rabbit IgG antibody (1:5,000; ZDR-5306; ZSGB-Bio) for 1 h at room temperature. After washing with TBST, antibody binding was detected by electro-chemiluminescence using fluorescence detection equipment (ChemiDoc MP; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were stripped using a buffer (10% sodium dodecyl sulfate, 25 mM glycine, pH 2.0) at room temperature for 30 min, followed by washing in TBST for 30 min. The membranes were blocked and reprobed for β-actin as a loading control.

Relative gene expression analysis

Total RNA was extracted from the lung tissue and from cells, using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA was reverse-transcribed into cDNA, using the ReverTra Ace qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan). PCR was performed in a final volume of 10 μl using a Thunderbird SYBR qPCR Mix (Toyobo Co., Ltd.). The cycling program involved initial denaturation at 95°C for 60 sec, followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec. The β-actin gene was used as an internal control. NALP3, ASC, caspase-1, type I collagen and α-smooth muscle actin (SMA) were detected. The sequences of the primers and products are listed in Table I. The relative expression of the genes was calculated by the 2−ΔΔq method.

Table I

Reverse transcription-polymerase chain reaction primers and products.

Table I

Reverse transcription-polymerase chain reaction primers and products.

Gene nameS/ASPrimer sequence (5′–3′)Product size (bp)
NALP3S ATTACCCGCCCGAGAAAGG83
AS TCGCAGCAAAGATCCACACAG
Caspase-1S AATACAACCACTCGTACACGTC78
AS AGCTCCAACCCTCGGAGAAA
ASCS GACAGTGCAACTGCGAGAAG106
AS CGACTCCAGATAGTAGCTGACAA
Collagen-1S GCTCCTCTTAGGGGCCACT91
AS ATTGGGGACCCTTAGGCCAT
α-SMAS CCCAGACATCAGGGAGTAATGG104
AS TCTATCGGATACTTCAGCGTCA
β-actinS GTGACGTTGACATCCGTAAAGA245
AS GCCGGACTCATCGTACTCC

[i] S, sense; AS, antisense; ASC, apoptosis-associated Speck-like protein; α-SMA, α-smooth muscle actin.

ELISA

The content of IL-1β in the lung tissue on day 28 after BLM instillation and in the cell supernatants was determined using an ELISA kit (ExCell Bio, Shanghai, China) according to the manufacturer's instructions.

Histopathological analysis

The tissue from the left lung was excised and immediately fixed with 4% paraformaldehyde for 48 h, and then embedded in paraffin. Serial 4-μm paraffin sections were prepared using a rotator microtome. Sirius Red staining is considered as the optimal method for identifying tissue collagen, as it can differentiate between collagen types I and III, whereas Masson's trichrome staining is the classical method for staining collagen used by numerous studies. Masson's trichrome and hematoxylineosin staining were selected in the present study to estimate the degree of fibrosis, rather than differentiate between collagen types. The tissues were visualized under a Zeiss AX10 imager A2 microscope and captured using a Zeiss AX10 cam HRC (Zeiss, Oberkochen, Germany). The criteria for grading lung fibrosis were based on the modified Ashcroft score (20). The grade of fibrotic changes in each lung section was assessed as a mean score of severity from 10 randomly selected high-power fields.

Hydroxyproline assay

The levels of lung collagen were determined by analysis of the hydroxyproline content on day 28 after BLM infusion using a hydroxyproline assay kit (Jiancheng Institute of Biotechnology, Nanjing, China) according to the manufacturer's instructions.

Statistical analysis

All the results are shown as mean ± standard error of the mean. Comparisons for multiple groups were performed by one-way analysis of variance followed by Tukey's multiple comparison test. For all analyses, a P-value of <0.05 was considered to indicate statistically significant differences.

Results

H2O2-induced collagen synthesis in mouse fibroblasts is mediated by the NF-κB signaling pathway

The IκBs is the most important inhibitor of NF-κB activation and the degradation of IκBs triggers NF-κB activation. Phosphorylation of NF-κB p65 at Ser536 is an indirect indicator of NF-κB activation, which results in translocation of the p65 subunit of NF-κB to the nucleus (21). In the present study, detection of the IκBα protein, which is the best studied IκB protein, and pNF-κB, were used to estimate the degree of NF-κB activation. Western blot analysis of cell lysates revealed degradation of the IκBα protein and increase in pNF-κB in the H2O2-treated group compared with controls. By contrast, the PDTC+H2O2 group exhibited preserved IκBα levels and decreased NF-κB p65 phosphorylation (Fig. 1A, C and D). H2O2 is known to be an important regulator of oxidative stress. The results confirmed that NF-κB was activated (Fig. 1A) via IκBα degradation during H2O2-mediated oxidative stress in fibroblasts. Stimulation with H2O2 significantly increased the expression of type I collagen at the mRNA and protein levels in NIH-3T3 cells (Figs. 1A and F, and 2A). Additionally, similar results for α-SMA at the mRNA and protein levels were obtained at 24 h after treatment in the H2O2 group (Figs. 1A and E, and 2A). These findings demonstrated that H2O2 serves as an important regulator of extracellular matrix (ECM) deposition for fibroblasts by increasing type I collagen expression, and may be involved in the transition from fibroblasts to myofibroblasts by promoting α-SMA expression. Furthermore, the upregulation of α-SMA and type I collagen was inhibited by the antioxidant and NF-κB inhibitor PDTC (Figs. 2A and 3A, E and F). Taken together, these data suggest that H2O2-induced oxidative stress stimulates type I collagen and α-SMA production in mouse fibroblasts, and that NF-κB signaling is required in this process.

The NALP3 inflammasome plays an important role in H2O2-induced collagen synthesis in mouse fibroblasts via the NF-κB signaling pathway

Cells treated with H2O2 exhibited overexpression of NALP3, ASC and caspase-1 at the mRNA level (Fig. 2A). In addition, H2O2 induced an increase in IL-1β content in cell supernatants, whereas cells treated with PDTC exhibited reduced expression of NALP3, ASC and caspase-1 mRNA and IL-1β content. The effect of PDTC on NALP3 protein levels was confirmed by western blot analysis. The results suggested that activation of the NF-κB signaling pathway promoted NALP3 inflammasome activation by increasing NALP3, ASC and caspase-1 expression during H2O2-mediated oxidative stress (Figs. 2B and 3A–D). Furthermore, we demonstrated that the expression of NALP3 could be effectively knocked down using siRNA-NALP3, and that the mRNA levels of ASC and caspase-1 were downregulated when exposed to H2O2 under conditions of NALP3 knockdown. In addition, the secretion of IL-1β was also decreased. Inhibition of NALP3 abolished H2O2-mediated type I collagen synthesis and increased the mRNA levels of α-SMA in fibroblasts. However, no effect on α-SMA protein was observed following administration of siRNA-NALP3 (Figs. 1E and 3E). The IκBα protein remained at a relatively high level and pNF-κB level was reduced compared with the siRNA-negative and H2O2 groups (Fig. 1A, C and D). Collectively, these findings suggest that activation of the NALP3 inflammasome is involved in H2O2-induced type I collagen production in fibroblasts via the NF-κB signaling pathway.

The NF-κB pathway is required for NALP3 inflammasome activation in BLM-induced pulmonary fibrosis

A single intratracheal injection of BLM constitutes a well-established animal model that results in airway epithelial cell damage, inflammation and formation of fibrotic lesions. In the present study, a well-alveolized normal histology was observed in the control group. By contrast, obvious alveolar wall thickening, massive infiltration of leukocytes and excessive deposition of mature collagen in the interstitium was observed in BLM-treated mice. Lung inflammation was contained on day 28, although the fibrotic changes became more severe. These histopathological changes were improved by PDTC pretreatment (Fig. 4A).

BLM administration induced a significant increase in Ashcroft scores compared with the control on day 7, and these scores were further increased by day 28 (Fig. 4C). Conversely, the scores of the mice administered PDTC were significantly lower. In addition, a similar trend was observed in the measurements of lung hydroxyproline content (Fig. 4B). Thus, these results indicate that the BLM-induced pulmonary fibrosis model was successfully established, and that the NF-κB signaling pathway played a key role in the process of fibrosis.

To examine the activation of NALP3 inflammasomes in BLM-induced pulmonary fibrosis, the mRNA levels of NALP3, ASC, caspase-1, α-SMA and type I collagen were measured and NALP3 protein levels were determined in the lungs of mice. BLM-treated mice exhibited significantly elevated levels of NALP3, ASC, caspase-1, α-SMA and type I collagen mRNA compared with the control group when analyzed on day 28 (Fig. 5E). The level of the NALP3 protein was markedly higher compared with that of the control on both days 7 and 28, whereas NALP3 protein expression was reduced on day 28 compared with day 7 after BLM injection (Fig. 5A and B). PDTC-pretreated mice exhibited a relatively lower expression of NALP3, ASC, caspase-1, α-SMA and type I collagen mRNA compared with the BLM group. Furthermore, PDTC reduced NALP3 protein levels to a statistically significant extent in the lungs of BLM-treated mice on days 7 and 28. In addition, ELISA analysis demonstrated that BLM administration resulted in a large increase in IL-1β production, and that PDTC pretreatment was able to attenuate the BLM-induced production of IL-1β in the lung tissues on day 28 (Fig. 5D). Western blot analysis revealed a decrease in IκBα levels in the BLM group as opposed to their preservation in the BLM+PDTC group (Fig. 5A and C). However, a more significant change in the level of IκBα was not observed over time. These results suggest that the NALP3 inflammasome is activated during the stages of early inflammation and fibrosis and, therefore, may play a role in fibrogenesis.

Discussion

NALP3 has been widely investigated with respect to immune response over the past several decades, and it is considered to act as a general sensor for cellular stress. In recent years, NALP3 has been found to play important roles in various pathological processes, including diabetes mellitus (22,23), non-alcoholic steatohepatitis (24,25), chronic kidney diseases (26) and IPF (27). Furthermore, an increasing volume of evidence indicates that NALP3 serves as an important factor in organ fibrosis. Pulmanary fibrosis develops as a consequence of abnormalities occurring in multiple biological pathways that affect inflammation and wound repair, which involve a series of cells and cytokines. Studies on the process of fibrogenesis have focused primarily on cell injury, macrophage activation, inflammation and ECM deposition. Tian et al (28) reported that siNALP3 may rescue A549 from BLM-induced pulmonary fibrosis. Accordingly, the biological characteristics of the NALP3 inflammasome during collagen metabolism in pulmonary fibrosis remain unclear. To further elucidate the role of the NALP3 inflammasome during type I collagen synthesis in fibroblasts, the effects of NALP3-siRNA on H2O2-treated mouse embryonic fibroblasts were examined.

In the present study, it was suggested that NALP3 plays an important role in H2O2-mediated type I collagen synthesis via the NF-κB signaling pathway. NF-κB activation and type I collagen production were shown to be significantly decreased by NALP3-siRNA. Additionally, the inhibition of NF-κB resulted in downregulation of type I collagen and the NALP3 inflammasome. In addition, consistent with the results from previous studies, we demonstrated that the NALP3 inflammasome was activated in BLM-induced pulmonary fibrosis and that this activation was attenuated by PDTC.

There are two essential criteria for triggering NALP3 inflammasome activation. First, NALP3 expression per se must be transcriptionally induced, which requires NF-κB. A second, post-transcriptional step then leads to the activation of NALP3, allowing for NALP3 inflammasome assembly. In the first step, NALP3 expression is considered as the limiting factor for inflammasome priming (29). The corresponding NF-κB binding sites (nt −1,303 to −1,292 and −1,238 to −1,228) are located in the NALP3 promoter in macrophages (30) and TLR2/MyD88/NF-κB and TLR4/MyD88/NF-κB signaling is required for pro-IL-1β and NALP3 gene expression (7,31). Our data further demonstrated that PDTC inhibits the expression of NALP3, ASC and caspase-1 to varying degrees. Taken together, this evidence suggests that NF-κB serves as a critical upstream mediator for NALP3 inflammasome priming. Accordingly, our study provides a new viewpoint regarding the NF-κB/inflammasome pathway. The partial protection of NF-κB activation by NALP3 silencing suggests that the inflammasome may function upstream of NF-κB. In previous studies, inflammasome activation leading to IL-1β maturation and release, IL-1β will also activate the IL-1R1/MyD88/NF-κB pathway (10). This may contribute to an autocrine/paracrine amplification loop of IL-1β and NF-κB during the process of collagen metabolism (19).

The second criterion for triggering NALP3 inflammasome activation involves intracellular ROS and potassium (K+) efflux due to the stimulation of ATP-sensitive ion channels, which promote inflammasome assembly leading to caspase-1 activation and subsequent IL-1β release. Oxidative stress is a strong NF-κB activator. We thus hypothesized that oxidative stress acts both up- and downstream of the NALP3 inflammasome, and it also plays a contributory role in the pathogenesis of NALP3 inflammasome activation and pulmonary fibrosis by inducing genetic overexpression of fibrogenic cytokines (32). Artlett et al indicated that the NALP3 inflammasome plays important roles in collagen synthesis via type IA and 3A1 collagen, and connective tissue growth factor production and myofibroblast differentiation (15). Inhibition of caspase-1 abrogated the expression of collagens, IL-1β and α-SMA in systemic sclerosis dermal and lung fibroblasts. As an NALP3 inflammasome effector, cytokine IL-1β exerts comprehensive biological effects associated with inflammation and fibrosis. The association between IL-1β and fibrosis has been widely investigated. IL-1 receptor antagonists or IL-1R deficiency may reduce liver or lung fibrosis (10,24). IL-1β mediates collagen expression mainly via the induction of TGF-β, the downstream cytokine of IL-1β, which is also known as the most essential cytokine in the biochemical processes of fibrosis. TGF-β directly increases the transcriptional activation of collagen genes and also stimulates the expression of a number of proinflamatory and fibrogenic cytokines, such as TNF-α, PDGF, IL-1β, or IL-13, thereby further enhancing the fibrotic response in macrophages, fibroblasts and myofibroblasts. TGF-β is also a direct mediator of epithelial-to-mesenchymal transition (EMT), which is one of the most important sources of myofibrosis. Another important TGF-β feature in increasing ECM deposition is the creation of a microenvironment that favors ECM deposition. Redox balance modulation may affect the NALP3 inflammasome/Smad pathway in terms of collagen synthesis. Smads are the most crucial intracellular proteins that transduce extracellular signals from TGF-β ligands to activate downstream gene transcription. Thus, the mechanism underlying collagen synthesis may be the NALP3/IL-1β/TGF-β pathway. Of note, when NALP3−/− mouse primary cardiac fibroblasts (CFs) were treated with TGF-β, which plays a direct role in fibroblast differentiation into myofibroblasts and EMC deposition, they displayed impaired and delayed myofibroblast differentiation with reduced α-SMA expression (18). Furthermore, the expression of α-SMA and MMP-9 was significantly decreased in mouse NALP3−/− renal tubular epithelial cells that had been stimulated by TGF-β. Taken together, these findings may suggest an upstream role for TGF-β to NALP3 in myofibroblast differentiation. Cai et al demonstrated that angiotensin 2 (Ang-2) increased NALP3 and pro-IL-1β levels by activating the TLR4/MyD88/NF-κB pathway, and first demonstrated that Ang-2-induced collagen synthesis in hepatic stellate cells could be inhibited by NALP3 depletion. NF-κB may therefore affect inflammasome activation and downstream IL-1β-mediated collagen metabolism.

Consistent with prior findings, our data suggest that NALP3 may act as a new mediator in the pathomechanism of fibrosis via regulating type I collagen and α-SMA expression. NALP3 also affects the collagen synthesis rate-limiting enzyme (P4Hα1) and collagen breakdown enzymes (MMP-2 and MMP-9) (28). However, our data demonstrated decreased mRNA expression with inhibition at the protein level of α-SMA after transfection. A possible explanation for this apparent discrepancy is that H2O2-mediated myofibroblast differentiation may involve multiple pathways in which NALP3 acts upon only a subset. Another possible explanation may be associated with limitations of cell culture studies including the short duration of transfection and the transfection efficiency. To resolve this issue, we must design a more durable transfection system in our future experiment.

H2O2 generates excessive amounts of ROS, and both factors represent the primary mediators of the effects of TGF-β in various cells (3335). In particular, H2O2-mediated collagen synthesis is associated with TGF-β (36). The association between NALP3 inflammasome and TGF-β is mentioned above. Notably, the TGF-β effects involving NALP3 have been identified as occurring in another inflammasome-independent manner. The involvement of NALP3 in TGF-β-induced R-Smad phosphorylation, nuclear accumulation and myofibroblast differentiation was demonstrated; however, TGF-β did not induce the upregulation or secretion of activated IL-1, IL-18, or caspase-1 in CFs. By contrast, the protective effect of NALP3 deficiency was consistently reported; simultaneously, IL-1β, IL-18, ASC, and caspase-1 were shown to be less important under certain conditions (37,38). It was suggested that ASC can induce MAPK phosphorylation independently of cytokine production in macrophages (39) to regulate mRNA stability; this, in turn, affected DOCK-2 protein expression and phagocytosis in leukocytes (40). These findings demonstrated that the physiological roles of NALP3 and ASC are not limited to the caspase-1/IL-1β axis. As TGF-β represents one of the most critical cytokines in the EMT and in ECM deposition, whether the associated regulatory mechanisms involving both NALP3 and TGF-β act via an inflammasome-dependent or -independent manner remains unclear. The independent biological effects of these components were mainly reported in non-monocytes/macrophages, such as renal tubular epithelial cells or CFs; thus, we may infer that the inflammasome-independent pathway may be a mechanism distinguishing epithelial cell lines from monocytes/macrophages, but this hypothesis requires further investigation.

As TGF-β represents one of the most critical cytokines in EMT and in ECM deposition, whether the associated regulatory mechanisms involving both NALP3 and TGF-β act in an inflammasome-dependent or -independent manner remains unclear.

In the present study, it was first demonstrated that NALP3 deficiency alleviates H2O2-induced type I collagen synthesis via the NF-κB signaling pathway. However, further research is required to identify the exact mechanism through which the NALP3 inflammasome plays a role in collagen production. As fibroblast activation represents the main checkpoint for ECM deposition, these results suggest that modulation of the effects of the NALP3 inflammasome on fibroblasts may have important therapeutic implications in pulmonary fibrosis.

Abbreviations:

NF-κB

nuclear factor κB

IL-1β

interleukin 1β

IL-18

interleukin 18

TGF-β

transforming growth factor-β

TNF-α

tumor necrosis factor-α

PDTC

pyrrolidine dithiocarbamate

ASC

apoptosis-associated Speck-like protein

IκBα

nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α

pNF-κB

phosphorylated NF-κB

α-SMA

α-smooth muscle actin

BLM

bleomycin

IPF

idiopathic pulmonary fibrosis

ROS

reactive oxygen species

ECM

extracellular matrix

EMT

epithelial-to-mesenchymal transition

TLR

toll-like receptor 4

Ang-2

angiotensin 2

MMP-9

matrix metalloproteinase 9

CFs

cardiac fibroblasts

TECs

renal tubular epithelial cells

Acknowledgments

The present was supported by a grant from the Sichuan Provincial Department of Science and Technology (no. 0040205301A53).

Notes

[1] Competing interests

The authors declare that they have no competing interests.

References

1 

Harari S and Caminati A: IPF: New insight on pathogenesis and treatment. Allergy. 65:537–553. 2010. View Article : Google Scholar : PubMed/NCBI

2 

Latz E, Xiao TS and Stutz A: Activation and regulation of the inflammasomes. Nat Rev Immunol. 13:397–411. 2013. View Article : Google Scholar : PubMed/NCBI

3 

Hillegass JM, Miller JM, MacPherson MB, Westbom CM, Sayan M, Thompson JK, Macura SL, Perkins TN, Beuschel SL, Alexeeva V, et al: Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells. Part Fibre Toxicol. 10:392013. View Article : Google Scholar : PubMed/NCBI

4 

Peeters PM, Eurlings IM, Perkins TN, Wouters EF, Schins RP, Borm PJ, Drommer W, Reynaert L and Albrecht C: Silica-induced NLRP3 inflammasome activation in vitro and in rat lungs. Part Fibre Toxicol. 11:582014. View Article : Google Scholar : PubMed/NCBI

5 

Naji A, Muzembo BA, Yagyu K, Baba N, Deschaseaux F, Sensebé L and Suganuma N: Endocytosis of indiumtin-oxide nanoparticles by macrophages provokes pyroptosis requiring NLRP3-ASC-Caspase1 axis that can be prevented by mesenchymal stem cells. Sci Rep. 6:261622016. View Article : Google Scholar

6 

Sun B, Wang X, Ji Z, Wang M, Liao YP, Chang CH, Li R, Zhang H, Nel AE and Xia T: NADPH oxidase-dependent NLRP3 inflammasome activation and its important role in lung fibrosis by multiwalled carbon nanotubes. Small. 11:2087–2097. 2015. View Article : Google Scholar : PubMed/NCBI

7 

Segovia J, Sabbah A, Mgbemena V, Tsai SY, Chang TH, Berton MT, Morris IR, Allen IC, Ting JP and Bose S: TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection. PLoS One. 7:e296952012. View Article : Google Scholar

8 

Mouratis MA and Aidinis V: Modeling pulmonary fibrosis with bleomycin. Curr Opin Pulm Med. 17:355–361. 2011. View Article : Google Scholar : PubMed/NCBI

9 

dos Santos G, Rogel MR, Baker MA, Troken JR, Urich D, Morales-Nebreda L, Sennello JA, Kutuzov MA, Sitikov A, Davis JM, et al: Vimentin regulates activation of the NLRP3 inflammasome. Nat Commun. 6:65742015. View Article : Google Scholar : PubMed/NCBI

10 

Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S, Schnyder B, Akira S, Quesniaux VF, Lagente V, et al: IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice. J Clin Invest. 117:3786–3799. 2007.PubMed/NCBI

11 

Gasse P, Riteau N, Charron S, Girre S, Fick L, Pétrilli V, Tschopp J, Lagente V, Quesniaux VF, Ryffel B and Couillin I: Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis. Am J Respir Crit Care Med. 179:903–913. 2009. View Article : Google Scholar : PubMed/NCBI

12 

Gicquel T, Victoni T, Fautrel A, Robert S, Gleonnec F, Guezingar M, Couillin I, Catros V, Boichot E and Lagente V: Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages. Clin Exp Pharmacol Physiol. 41:279–286. 2014. View Article : Google Scholar : PubMed/NCBI

13 

Hoshino T, Okamoto M, Sakazaki Y, Kato S, Young HA and Aizawa H: Role of proinflammatory cytokines IL-18 and IL-1beta in bleomycin-induced lung injury in humans and mice. Am J Respir Cell Mol Biol. 41:661–670. 2009. View Article : Google Scholar : PubMed/NCBI

14 

Phan SH and Kunkel SL: Lung cytokine production in bleomycin-induced pulmonary fibrosis. Exp Lung Res. 18:29–43. 1992. View Article : Google Scholar : PubMed/NCBI

15 

Artlett CM, Sassi-Gaha S, Rieger JL, Boesteanu AC, Feghali-Bostwick CA and Katsikis PD: The inflammasome activating caspase 1 mediates fibrosis and myofibroblast differentiation in systemic sclerosis. Arthritis Rheum. 63:3563–3574. 2011. View Article : Google Scholar : PubMed/NCBI

16 

Martínez-Godínez MA, Cruz-Domínguez MP, Jara LJ, Domínguez-López A, Jarillo-Luna RA, Vera-Lastra O, Montes-Cortes DH, Campos-Rodríguez R, López-Sánchez DM, Mejía-Barradas CM, et al: Expression of NLRP3 inflammasome, cytokines, and cascular mediators in the skin of systemic sclerosis patients. Isr Med Assoc J. 17:5–10. 2015.

17 

Wang W, Wang X, Chun J, Vilaysane A, Clark S, French G, Bracey NA, Trpkov K, Bonni S, Duff HJ, et al: Inflammasome-independent NLRP3 augments TGF-β signaling in kidney epithelium. J Immunol. 190:1239–1249. 2013. View Article : Google Scholar

18 

Bracey NA, Gershkovich B, Chun J, Vilaysane A, Meijndert HC, Wright JR Jr, Fedak PW, Beck PL, Muruve DA and Duff HJ: Mitochondrial NLRP3 protein induces reactive oxygen species to promote smad protein signaling and fibrosis independent from the inflammasome. J Biol Chem. 289:19571–19584. 2014. View Article : Google Scholar : PubMed/NCBI

19 

Cai S, Yang R, Li Y, Ning ZW, Zhang LL, Zhou GS, Luo W, Li DH, Chen Y, Pan MX and Li X: Angiotensin-(1–7) improves liver fibrosis by regulating the NLRP3 inflammasome via redox balance modulation. Antioxid Redox Sign. 24:795–812. 2016. View Article : Google Scholar

20 

Hübner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, Freitag-Wolf S and Bewig B: Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques. 44:507–511. 514–517. 2008. View Article : Google Scholar : PubMed/NCBI

21 

Zhang Q, Lenardo MJ and Baltimore D: 30 Years of NF-κB: A blossoming of relevance to human pathobiology. Cell. 168:37–57. 2017. View Article : Google Scholar : PubMed/NCBI

22 

Henriksbo BD, Lau TC, Cavallari JF, Denou E, Chi W, Lally JS, Crane JD, Duggan BM, Foley KP, Fullerton MD, et al: Fluvastatin causes NLRP3 inflammasome-mediated adipose insulin resistance. Diabetes. 63:3742–3747. 2014. View Article : Google Scholar : PubMed/NCBI

23 

Luo B, Li B, Wang W, Liu X, Xia Y, Zhang C, Zhang M, Zhang Y and An F: NLRP3 gene silencing ameliorates diabetic cardio-myopathy in a type 2 diabetes rat model. PLoS One. 9:e1047712014. View Article : Google Scholar

24 

Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay A, Hoffman HM and Feldstein AE: NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology. 59:898–910. 2014. View Article : Google Scholar :

25 

Wree A, McGeough MD, Peña CA, Schlattjan M, Li H, Inzaugarat ME, Messer K, Canbay A, Hoffman HM and Feldstein AE: NLRP3 inflammasome activation is required for fibrosis development in NAFLD. J Mol Med. 92:1069–1082. 2014. View Article : Google Scholar : PubMed/NCBI

26 

Vasileiou E, Montero RM, Turner CM and Vergoulas G: P2X7 receptor at the heart of disease. Hippokratia. 14:155–163. 2010.PubMed/NCBI

27 

Lasithiotaki I, Giannarakis I, Tsitoura E, Samara KD, Margaritopoulos GA, Choulaki C, Vasarmidi E, Tzanakis N, Voloudaki A, Sidiropoulos P, et al: NLRP3 inflammasome expression in idiopathic pulmonary fibrosis and rheumatoid lung. Eur Res J. 47:910–918. 2016. View Article : Google Scholar

28 

Tian R, Zhu Y, Yao J, Meng X, Wang J, Xie H and Wang R: NLRP3 participates in the regulation of EMT in bleomycin-induced pulmonary fibrosis. Exp Cell Res. 357:328–334. 2017. View Article : Google Scholar : PubMed/NCBI

29 

Simard J, Cesaro A, Chapeton-Montes J, Tardif M, Antoine F, Girard D and Tessier PA: S100A8 and S100A9 induce cytokine expression and regulate the NLRP3 inflammasome via ROS-dependent activation of NF-κB1. PLoS One. 8:e721382013. View Article : Google Scholar

30 

Qiao Y, Wang P, Qi J, Zhang L and Gao C: TLR-induced NF-κB activation regulates NLRP3 expression in murine macrophages. FEBS Lett. 586:1022–1026. 2012. View Article : Google Scholar : PubMed/NCBI

31 

Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, Fernandes-Alnemri T, Wu J, Monks BG, Fitzgerald KA, et al: Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol. 183:787–791. 2009. View Article : Google Scholar : PubMed/NCBI

32 

Poli G and Parola M: Oxidative damage and fibrogenesis. Free Radic Biol Med. 22:287–305. 1997. View Article : Google Scholar : PubMed/NCBI

33 

Hong YH, Peng HB, La Fata V and Liao JK: Hydrogen peroxide-mediated transcriptional induction of macrophage colony-stimulating factor by TGF-beta1. J Immunol. 159:2418–2423. 1997.PubMed/NCBI

34 

Junn E, Lee KN, Ju HR, Han SH, Im JY, Kang HS, Lee TH, Bae YS, Ha KS, Lee ZW, et al: Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: Involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression. J Immunol. 165:2190–2197. 2000. View Article : Google Scholar : PubMed/NCBI

35 

Koo HY, Shin I, Lee ZW, Lee SH, Kim SH, Lee CH, Kang HS and Ha KS: Roles of RhoA and phospholipase A2 in the elevation of intracellular H2O2 by transforming growth factor-beta in Swiss 3T3 fibroblasts. Cell Signal. 11:677–683. 1999. View Article : Google Scholar

36 

Park SK, Kim J, Seomun Y, Choi J, Kim DH, Han IO, Lee EH, Chung SK and Joo CK: Hydrogen peroxide is a novel inducer of connective tissue growth factor. Biochem Biophys Res Commun. 284:966–971. 2001. View Article : Google Scholar : PubMed/NCBI

37 

Mizushina Y, Shirasuna K, Usui F, Karasawa T, Kawashima A, Kimura H, Kobayashi M, Komada T, Inoue Y, Mato N, et al: NLRP3 protein deficiency exacerbates hyperoxia-induced lethality through Stat3 protein signaling independent of interleukin-1β. J Biol Chem. 290:5065–5077. 2015. View Article : Google Scholar

38 

Shigeoka AA, Mueller JL, Kambo A, Mathison JC, King AJ, Hall WF, Correia Jda S, Ulevitch RJ, Hoffman HM and McKay DB: An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury. J Immunol. 185:6277–6285. 2010. View Article : Google Scholar : PubMed/NCBI

39 

Taxman DJ, Holley-Guthrie EA, Huang MT, Moore CB, Bergstralh DT, Allen IC, Lei Y, Gris D and Ting JP: The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome. J Biol Chem. 286:19605–19616. 2011. View Article : Google Scholar : PubMed/NCBI

40 

Ippagunta SK, Malireddi RK, Shaw PJ, Neale GA, Vande Walle L, Green DR, Fukui Y, Lamkanfi M and Kanneganti TD: The inflammasome adaptor ASC regulates the function of adaptive immune cells by controlling Dock2-mediated Rac activation and actin polymerization. Nat Immunol. 12:1010–1016. 2011. View Article : Google Scholar : PubMed/NCBI

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April-2018
Volume 41 Issue 4

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Spandidos Publications style
Kuang J, Xie M and Wei X: The NALP3 inflammasome is required for collagen synthesis via the NF‑κB pathway. Int J Mol Med 41: 2279-2287, 2018
APA
Kuang, J., Xie, M., & Wei, X. (2018). The NALP3 inflammasome is required for collagen synthesis via the NF‑κB pathway. International Journal of Molecular Medicine, 41, 2279-2287. https://doi.org/10.3892/ijmm.2018.3404
MLA
Kuang, J., Xie, M., Wei, X."The NALP3 inflammasome is required for collagen synthesis via the NF‑κB pathway". International Journal of Molecular Medicine 41.4 (2018): 2279-2287.
Chicago
Kuang, J., Xie, M., Wei, X."The NALP3 inflammasome is required for collagen synthesis via the NF‑κB pathway". International Journal of Molecular Medicine 41, no. 4 (2018): 2279-2287. https://doi.org/10.3892/ijmm.2018.3404