Study On Correlation Of Inflammasome With Lung Cancer And Pulmonary Fibrosis

Lung cancer is the leading cause of cancer death worldwide. Base on the report of CA: a cancer journal for clinicians, the incidence of lung cancer accounted for 14% in new cancer cases in 2014, and it is the second most common cancer among both men and women in the United States. Moreover, more than one-quarter of all cancer deaths due to lung cancer(27%), this is far more that the second cause of cancer death both in man(prostate cancer, 10%) and women(breast cancer, 15%). In China, data from “annual report on status of cancer in China” published in 2015 by National Central Cancer Registry(NCCR) indicated that lung cancer accounted for 19.31% in new cancer cases, and accounted for 25.04% of all cancer deaths in 2011. Both of these two data of lung cancer ranked first among various cancers. During these years, due to the decreasing of air quality, the numbers of patients suffering from respiratory disease are increasing. Though no epidemiological research focus on the relationship between smog and lung cancer has been founded in China, a multicentre European Study of Cohorts for Air Pollution Effects(ESCAPE) reported that increments of particulate matter 10(PM 10) and PM2.5 in air were associated with high hazard ratio for adenocarcinomas of the lung. Thus, it is worried about that China will have the biggest population of lung cancer patients in the world in 2025. Till now, more and more molecules involved in the development of lung cancer have been discovered. Moreover, new drugs targeting at the cellular signaling transduction have been wildly used in the treatment of lung caner. Although novel therapeutic methods such as targeted molecular therapy, gene therapy, as well as immunotherapy have improved the out come of lung cancer, the 5-year survival rate for all stages of lung cancer patients is less than 16%. Therefore, exploring the pivotal molecules participating the development and progression of lung cancer will make great contributions to the prevention, diagnosis and therapeutics of lung cancers.Chronic and repeated inflammations have long been observed to be associated with various types of cancer. Indeed, today, aberrant inflammation has been identified as the seventh hallmark of cancer besides limitless replicative potential, tissue invasion and metastasis, insensitivity to growth inhibitors, self-sufficiency in growth signals, evasion of apoptosis, and sustained angiogenesis. In some types of cancer, inflammatory conditions precede development of malignancy; in others, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumors. Regardless of its origin, persistent inflammation in the tumor microenvironment promotes proliferation and survival of malignant cells, stimulates angiogenesis and metastasis, subverts adaptive immune responses, and alters responses to chemotherapeutic agents. So, exploring the key molecular switch linking inflammation to cancer will be helpful to inhibit cancer-related inflammation and provide novel target for the treatment of lung cancer.Pulmonary fibrosis is a common respiratory disease. It includes a heterogeneous group of lung disorders characterized by the progressive and irreversible destruction of lung architecture caused by scar formation that ultimately leads to organ malfunction, disruption of gas exchange, and death from respiratory failure. Chronic inflammation has been implicated in the development of pulmonary fibrosis. Many forms of the disease are believed to be induced initially by a strong inflammatory response. Consequently, the proliferation, activation, and differentiation of collagen-secreting myofibroblasts initiated the preogression of fibrosis in lung. There are various known etiologies of pulmonar fibrosis, sucn as silicon dust and asbestos, chemical toxins, pathogens, radiation, and some special drugs(bleomycin and amiodarone). Moreove, there are also unknown etiologies of pulmonar fibrosis. Due to the air pollution, the incidence of pulmonar fibrosis is increasing. Besides injury or antigen-induced chronic inflammstion, it has been comfirmed that damage and regeneration impairment of epithelial cells and/or endothelial cells and dysregulation of transformation growth factor-β(TGF-β) signaling are involved in the development of pulmonar fibrosis. Notably, till now, it has been well recognized that endothelial-to-mesenchymal transition(EMT) is a key cellular mechanism for pulmonary fibrosis. Since pulmonary fibrosis is a lethal pathological process with limited therapeutic options, a more detailed and integrated understanding of the cellular and molecular mechanisms of pulmonary fibrosis could help pave the way for effective therapeutics for this devastating and complex disease.Inflammasomes are large cytosolic multi-protein complexes that assemble in response to endogenous and exogenous dangers and result in the activation of caspase-1-mediated inflammatory responses, including cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18. The concept of inflammasome was firstly introduced by Tschopp et al. in 2002. A typical inflammasome consists of a sensor/scaffolding protein [in general a nucleotide-binding oligomerization domain(NOD)-like receptors(NLR)], an adaptor protein termed apoptosis-associated speck-like protein containing a Card domain(ASC), and pro-caspase-1. Within the NLR family, NALP1, NLRP3, and NLRC4, as well as the PYHIN family member absent in melanoma 2(AIM2), have been shown to form large multi-protein functional inflammasomes. It is confirmed that persistent activation of inflammasomes by infection or injury is the vital biological basis for chronic inflammation. Till now, the roles of inflammasome are well characterized in the development and progression of gastric cancer, colon cancer, melanoma, and prostate cancer. However, the roles of different inflammasome subtypes in the development of lung cancer have not been elucidated. In addition, it is comfirmed that silica crystal particulates could activate NLRP3 inflammasome. As one of the major componet of air pollution, silica particulate is one of the major etiologies of occupational pulmonary fibrosis. In mice model, NLPR3 knockout inhibites the silica-induced granuloma formation, decreases collagen deposition and pulmonary fibrosis. However, as the most important cellular mechanism in pulmonary fibrosis, whether endothelial-to-mesenchymal transition(EMT) is regulated by NLRP3 inflammasome is unclear.This study aimed to determine the expression and function of inflammasomes in various lung cancer cells, as well as the role of NLRP3 inflammasome in pulmonary fibrosis. In the present study, we first investigated expression and function of inflammasome in various lung cancer cell lines with different histological, invasion ability, and chemoresistance. Then, resected lung cancer tissues were used to study the expression of inflammasome components in stage Ⅰ non-small cell lung cancer patients and limited stage of small cell lung caner patients. Next, we investigated the role of NLRP3 inflammasome in silica PM2.5-induced epithelial to mesenchymal transition(EMT). Finally, by using lung tissues form video-assisted thoracoscopic surgery(VATS) we investigated the role of NLRP3 inflammasome in a case of rituximab-induced interstitial lung disease(R-ILD). The results revealed in this paper will be beneficial for extending the pathophysisological effects of inflammasome in respiratory diseases, and provide a new target for developing therapeutic options for respiratory disease, especially for lung cancer and fibrosis. Part I. Expression of inflammasome in human lung cancercell lines AIM: To investigate the subtype and level of inflammasome expressed in human lung cancer cell lines with different histological classification, invasive ability, chemoresistance, and the pro-inflammatory factors(IL-1β and IL-18) releasing activity of inflammasome in different lung cancer cell lines. METHODS: Eight cell lines including human pulmonary alveolar epithelial cells(HPAEpiCs) and bronchi epithelial cell line(16HBE), alveolar epithelial adenocarcinoma cell line(A549) and its cisplatin-resistant variant A549/DDP, lung squamous cell cancer line(SK-MES-1), human large-cell lung carcinoma cell lines with low(95C) or high(95D) metastasis potential, and human small cell lung cancer(SCLC) cell line(NCI-H446) were cultured for this study. Quantitative real-time polymerase chain reaction(qRT-PCR) and Western blotting were used to determine the mRNA and protein expressions of NOD-like receptors(NALP1, AIM2, NLRP3, and NLRC4), ASC, caspase-1, IL-1β, and IL-18. The inflammasome activators including anthrax lethal factor(LF), poly(dA:dT), lipopolysaccharide combined with adenosine-triphosphate(LPS+ATP), and flagellin were used to stimulate the NALP1, AIM2, NLRP3, and NLRC4 inflammasome, respectively. After stimulation, cell supernatants were collected for detection of IL-1β by enzyme-linked immunosorbent assay(ELISA). Results: The components of inflammasome were expressed in normal and lung cancer cell lines. NOD-like receptor NALP1 is highly expressed in lung cancer cell line A549, NCI-446, as well as normal bronchi epithelial cell line 16 HBE, moderately expressed in 549/DDP、SK-MES-1, and 95 D cell lines, and lowly expressed in HPAEpiC. Compared to both HPAEpiC and 16 HBE, the mRNA expressions of AIM2 were significantly higher in NSCLC cell line, rather than SCLC cell line. The expression of NLRP3 mRNA was low in both of the two normal lung cell lines, whereas it was significantly higher in A549 and NCI-446 lung cancer cell lines. For the transcripts and protein expressions of NLRC4, no significant difference was found among the eight lung cell lines. The mRNA expression of ASC was significantly higher in A549 than that in other lung cell lines, while the protein expressions of ASC were significantly higher in A549, 95 D, and NCI-446 lung cancer cell lines than that in two normal lung cell lines. As to the mRNA expression of pro-caspase-1, pro-IL-1β, and pro-IL-18, one-way ANOVA found no statistical difference among these eight lung cell lines. However, the cleaved caspase-1, mature IL-1β and IL-18 protein levels were significantly higher in lung cancer cell lines. Additionally, the expression levels of IL-1β and IL-18 in A549/DDP and 95 C cells were significantly lower than that in their counterpart A549 and 95 D cells respectively. ELISA data showed that the releases of IL-1β by the activation of various inflammasomes were different among individual lung cancer cell lines. After the activation of inflammasomes, IL-1β levels were high in the culture supernatants of adenocarcinoma, giant-cell carcinoma, and SCLC, but relative low in squamous cell carcinoma. Moreover, among these four types of inflammasome, NLRP3 inflammasome displayed more potent activity in releasing IL-1β than others. CONCLUSION: Inflammasomes are differentially expressed in various lung cancers. The phenotypes and levels of inflammasome in lung cancer cell lines are depended on the histological type, invasion ability, and chemoresistance. Moreover, inflammasomes in lung cancer cell can be stimulated by activators, and elicited the release of IL-1β.Part II. Expressions of inflammasome components inresected stage I lung cancer tissues AIM: To investigate the expressions of inflammasomes and their products in resected stage I non-small cell lung cancer(NSCLC) tissues, including squamous cell carcinoma(SCC), adenocarcinoma(ADC), and cancer tissues of limited stage of small cell lung cancer(SCLC). METHODS: The lung cancer and cancer-adjacent normal tissues and paraffin-embedded sections were obtained from the Tumor Tissue Samples Library and Pathology Department of the First affiliated Hospital of Nanjing Medical University(Nanjing, Jiangsu, China) from January 2011 to December 2012. The liquid nitrogen-frozen tissue specimens for quantitative real-time polymerase chain reaction(qRT-PCR) included 17 cases of SCC, 20 cases of low-grade ADC(grade I–II), 16 cases of high-grade ADC(grade III–IV), 8 cases of SCLC, and 20 cases of lung cancer-adjacent normal tissues(composed of corresponding adjacent normal tissues to 5 random lung cases out of SCC, low-grade ADC, high-grade ADC, and SCLC groups, respectively). For immunohistochemical study, paraffin-embedded sections of another 4 cases of SCC, low-grade ADC, high-grade ADC, and SCLC respectively were used respectively. The expressions of NOD-like receptors(NALP1, AIM2, NLRP3, and NLRC4), ASC, caspase-1, IL-1β, and IL-18 were determined by immunohistochemistry, qRT-PCR, and Western blotting. RESULTS: NOD-like receptors, ASC, caspase-1, IL-1β, and IL-18 were barely expressed in cancer-adjacent normal tissues. No statistical differences were found in both mRNA and protein expressions of NALP 1 and NLRC4 between normal and tumorous tissues by qRT-PCR. Moreover, the immunostaining signals of NALP1 and NLRC4 were undetectable in lung cancer tissue sections. Compared to the normal tissues, the AIM2, NLRP3 and ASC were significantly up-regulated in lung cancer tissues. AIM2 was most highly expressed in ADC followed by SCC, but lowly expressed in SCLC and normal tissues. Relative to normal controls, the mRNA and protein levels of ASC were significantly higher in high-grade ADC followed by low-grade ADC, SCLC, and SCC tissues. The levels of cleaved caspase-1 and mature IL-1β and IL-18 in lung cancer tissues were significantly higher than that in normal tissues, with the highest level in high-grade ADC tissues. CONCLUSION: Inflammasomes and their products are highly expressed in lung cancer tissues than normal tissues. In lung cancer tissues, the expression levels of inflammasomes and their products were higher in ADC than that in SCC and SCLC. Moreover, NLRP3 inflammasome is predominantly expressed in ADC, and it is over-expressed in high-grade ADC than low-grade ADC.Part III. The effects of NLRP3 inflammasome in silica PM2.5-induced epithelial to mesenchymal transition(EMT)of 16 HBE cell line AIM: To investigate whether silica PM2.5(SiO2 PM2.5) can induce epithelial to mesenchymal transition(EMT) as well as NLRP3 inflammasome activation in 16 HBE cell, and further investigate the role and potential mechanism of NLRP3 inflammasome in this process. METHODS: The effects of different concentrations of SiO2 PM2.5 on the cell viability of 16 HBE cell line were determined by cell counting Kit-8 assay(CCK8). The markers of EMT including EMT rate, E-cadherin, and α-smooth muscle actin(α-SMA) as well as the markers of activated NLRP3 inflammasome in 16 HBE cell were determined by Western blotting and immunofluorescence followed by the treatment of SiO2 PM2.5. A proper concentration of SiO2 PM2.5 and a proper time for cell culture was determined by the above researches. Down-regulation of NLRP3 in 16 HBE cells was achieved by lentivirus shRNA transfection. The effects of NLRP3 on EMT were further investigated by using NLRP3 knockdown 16 HBE cells. Finally, the expression and phosphorylation of intracellular signal transduction molecules including Smad2/3, GSK3β, β-catenin, as well as Snail were determined by Western blotting. RESULTS: SiO2 PM2.5 inhibited the cell viability of 16 HBE cell in dose- and timedependent ways. It induced EMT and meanwhile activated the NLRP3 inflammasome in 16 HBE cells. SiO2 PM2.5 induced EMT of 16 HBE cell also in dose- and timedependent ways. The protein expression of NLRP3 was downregulated in 16 HBE cells by lentivirus transfection of shRNA. In NLRP3 knockdown 16 HBE cell, the SiO2 PM2.5 induced EMT and NLRP3 inflammasome activation was inhibited. However, NLRP3 knockdown had no effects on the release of IL-1βand IL-18 in 16 HBE cells after 72h-treatment of Si O2 PM2.5. Moreover, molecular biological studies indicated that NLRP3 knockdown could alleviate SiO2 PM2.5 induced EMT by inhibiting Smad2/3-Snail signal transduction. CONCLUSION: SiO2 PM2.5 could induce EMT and activate NLRP3 inflammasome in 16 HBE cells simultaneously. Inhibiting the expression and activation of NLRP3 inflammasome could alleviate SiO2 PM2.5-induced EMT of 16 HBE cells by suppressing the Smad2/3-Snail signaling pathway. Therefore, NLRP3 inflammasome could be a potential target for preventing against air pollution particles related chronic lung injury, fibrosis, or even lung cancer.Part IV. Involvement of NLRP3 inflammasome inrituximab-induced interstitial lung disease(R-ILD) AIM: To investigate the expression of NLRP3 inflammasome in a clinical case of R-ILD, and determine the relationship between serum IL-1β and IL-18 levels and radiological change of the lung during the period of therapy. METHODS: A 30-year-old male patient diagnosed with respiratory failure and R-ILD due to the usage of rituximab for idiopathic thrombocytopenic purpura(ITP) was treated with glucocorticoid and antibiotics. Serum IL-1β and IL-18 levels were detected by ELISA. Repeat chest high resolution CT imaging was used to evaluate the resolution of the infiltrates in the lung. After the consent, a video assisted thoracoscopic surgery(VATS) lung biopsy of posterobasal segment of left lower lobe was performed to establish the pathological diagnosis. Collagen in lesion area of lung tissues was observed by Sirius red staining. The expressions of NLRP3, ASC, and caspase-1 were evaluated by immunohistochemistry. RESULTS: Respiratory failure was relieved by the treatment of glucocorticoid and antibiotics. Repeat chest CT imaging showed pulmonary infiltrates resolved continuingly 5 days after the treatment, and part of infiltrates had resolved two weeks later. Systemic steroids were tapered over 4 weeks after discharge, and follow-up examination was scheduled for 1 month later. At that time, chest CT imaging showed complete resolution of the pulmonary infiltrates. The pathological diagnosis of lesion in lung was non-specific interstitial pneumonitis(NSIP). The majority of the infiltrating cells expressed CD19, a marker of B lymphocytes and plasma cells. Sirius red staining indicated a significant accumulation of collagen in thickened alveolar septa in lesion areas. Immunohistochemical studies of NLRP3 inflammasome components showed high expressions of NLPR3, ACS and caspase-1 in lesion areas. These pathological changes were disappeared in the recovery area of the lung followed by the treatment. Moreover, patient demonstrated higher levels of IL-1β and IL-18 at the early time of hospitalization. However, they began to decline after 4-day steroid therapy and kept falling to the end of the therapy period. CONCLUSION: NLRP3 inflammasome is involved in R-ILD. Glucocorticoid can effectively inhibit the activation of NLRP3 inflammasome and reverse the pathological changes in lung. Serum IL-1β and IL-18 levels are correlated to the interstitial lesion in lung, and they could be potential molecular markers for the interstitial lung disease induced by rituximab. In summary, our work firstly investigated expression and function of inflammasome in different lung cancer cell lines and tissues. Our findings show that inflammasomes are functionally and differentially expressed in various lung cancer cells, depending on the histological type and grading, invasion ability, and chemosensitivity. These data suggests that inhibiting the activation of inflammasomes could be a novel therapeutic strategy for the treatment of lung cancer. Moreover, inhibiting the expression of NLRP3 inflammasome could reduce silica PM2.5 induced EMT, indicating that NLRP3 inflammasome could be a potential switch linking inflammation to EMT in pulmonary fibrosis. Furthermore, activation of NLRP3 inflammasome also participates in the development of R-ILD, suggesting inflammasome related pro-inflammatory factors may be useful biomarkers for interstitial lung diseases.