| Objective:Silicosis is the most serious occupational lung disease.Over the past decades,many preventive measures have been set up to counter this disease in countries all around the world,however,the incidence and prevalence of silicosis are still increasing.The main pathogenic factor of silicosis is caused by the accumulation of crystalline silica（CS）in the lung tissue.The deposition of CS in the lungs will continue to act on the lung tissue,resulting in chronic lung inflammation and progressive pulmonary fibrosis.Due to the irreversibility of pulmonary fibrosis,progression of the disease is accompanied with aggravated damage to lung function,eventually resulting in disability and death.There is no effective treatment for silicosis,so it is critical important to explore the pathogenesis of silicosis.We have previously shown that blocking 4-1BB signaling attenuated CS-induced inflammation and pulmonary fibrosis.However,the cells that express 4-1BB,which plays a vital role in promoting fibrosis,are still unknown.Therefore,we need to explore which cell express 4-1BB.Activation and inhibition interventions were used to study the regulatory mechanism of 4-1BB signaling in this cell and the effect of the cell on pulmonary fibrosis in experimental silicosis.Materials:In this study,C57BL/6 mice and mouse alveolar macrophages cell line,MH-S,were selected to study the effect of 4-1BB signaling in macrophages mediated pro-fibrotic responses after exposure to CS,and the effect of blocking 4-1BB signaling on pro-fibrotic mediators in the lung of CS-injured mice.The specific methods as follows:1.Flow cytometryLung tissue was minced into a slurry in digestion solution containing collagenase,DNase I,and complete media.The suspension was incubated on a rocker at 37°C for 30min.Cells were dispersed by repetitive suction and passed through a 70-μm cell strainer.Lung cells were washed,resuspended in PBS and blocked with Fc receptor antibodies.Cells were stained with antibodies to CD137,F4/80,CD45,CD11c,CD137L,CD4,CD44and CD62L for 30 min at room temperature in the dark place,and then washed and resuspended in staining buffer.A FACS Canto II flow cytometer and FlowJo V10 software were used to analyze the expression of 4-1BB and 4-1BBL on lung cells.2.Extraction of Alveolar MacrophagesMice were anesthetized with 10%chloral hydrate.After draining blood,the thoracic cavity was partially dissected,and then lungs were harvested.The trachea was cannulated with an angiocatheter,infused with 1 mL of ice-cold sterile saline and withdrawn for ten times.Cell suspensions were centrifuged at 4°C,and the pellet was resuspended and cultured in RPMI 1640 medium with 10%FBS,100 U/m L penicillin and 100μg/m L streptomycin.Cells firmly adhered to a cover glass in 24-well plates（1×105 cells per well）and were incubated for 2 h;the attached cells were considered as alveolar macrophages.3.Western BlotMH-S macrophages and lungs of different group mice were lysed by RIPA buffer containing protease inhibitors and phosphatase inhibitors.The BCA Protein Assay Kit was used to evaluate the concentration of the protein of cell lysates and lungs.Lysates from MH-S macrophages and from lungs of different group mice were subjected to 8%SDS-PAGE（30μg per sample）,and the removed proteins were electrophoretically transferred to PVDF membranes.After blocking in 5%skim milk for 1h at room temperature,the membranes were incubated with rat anti-CD137,rabbit anti-ASK1,rabbit anti-phospho-ASK1,rabbit anti-p38,rabbit anti-phospho-p38,rabbit anti-JNK,rabbit anti-phospho-JNK,rabbit anti-MMP9,rabbit anti-MMP12,and rabbit anti-β-Actin overnight at 4°C.Membranes were then incubated with HRP-conjugated antibody for 2h at room temperature.The bands were visualized with a high-performance luminol substrate solution.Expression was respectively normalized with the results fromβ-Actin.4.Realtime-PCR AnalysisThe cells were scratched and collected the cell suspension in a centrifuge tube.After centrifugation.Total RNA was extracted from cultured cells with TRIzol following the manufacturer’s instructions.The RNA pellet was dried for 2 min and dissolved in 20-40μL RNase—free dH20.cDNA was synthesized using a Prime Script RT-PCR Kit.Quantitative real-time PCR assays with a SYBR Premix ExTaq Kit were performed by a7500-sequence detector.GAPDH was used as the normalizing gene for determiningΔCT values.Fold changes in gene expression were compared with 2-ΔΔCT values.5.ELISA AnalysisCulture supernatants were collected and centrifuged at 2000 rpm for 10 mins after 12 h culture.The supernatants were stored at-80°C and used for subsequent experiments.Lung lysates of different mice groups were diluted to a concentrate of 1μg/μL.The cytokines,interleukin（IL）-1β,IL-6,tumor necrosis factor（TNF）-αand monocyte chemoattractant protein-1,were measured by ELISA according to the manufacturer’s instructions.6.ImmunohistochemistryAfter deparaffinization in xylene and subsequent rehydration in graded alcohol series,tissue sections were blocked by exposure to 3%H2O2 and boiled in a citrate buffer（p H5.9-6.2）at 95°C for 20 mins.After blocking in 5%BSA for 1 h at room temperature,tissue sections were incubated at 4°C overnight with antibodies to MMP9,MMP12,or collagenⅠ.The sections were washed with phosphate buffered saline（PBS）,and then incubated with horse radish peroxidase（HRP）secondary antibodies at room temperature for 2 h.A DAB kit was used to perform the positive staining.Nuclei were stained by hematoxylin.7.Statistical AnalysisResults were expressed as mean±standard error of the mean.Two data sets were compared by Student’s t-test.Differences among groups were assessed using one way-ANOVA followed by a Student-Newman-Keuls test.Significance was defined as P<0.05or smaller.The data were presented from at least three independent experiments.Results:1.4-1BB is highly expressed in alveolar macrophages from CS-injured miceFlow cytometry was used to detect the expression of 4-1BB in the lung of CS-injured mice.The proportion of alveolar macrophages from CS-injured mice was increased in contrast to control at days 7.The percentage of 4-1BB and 4-1BBL in alveolar macrophages from CS-injured mice were also added（P<0.05）.However,the proportion of pulmonary interstitial macrophages was decreased in CS-injured mice.The percentage of4-1BB and 4-1BBL in pulmonary interstitial macrophages from CS-injured mice did not differ from control（P<0.05）.The proportion of effector Th cells from CS-injured mice was augmented relative to control at days 7（P<0.05）;however,the percentage of 4-1BB did not change between CS-injured mice and control mice.The proportion of na?ve Th cells from CS-injured mice was declined（P<0.05）;the percentage of 4-1BB did not have a difference between CS-injured mice and control mice.2.MH-S cells express 4-1BB after exposure to CSFlow cytometry,western blot and Realtime-PCR were used to verify the expression of4-1BB protein and mRNA levels in MH-S cells exposed to CS.As flow cytometry analysis shown,the percentage of 4-1BB added after exposure to CS（P<0.05）.As Western blot analysis shown,the protein level of 4-1BB significantly increased after exposure to CS（P<0.05）.As Realtime-PCR analysis shown,the relative expression of 4-1BB mRNA in MH-S cells cultured with CS was dramatically elevated（P<0.05）.As immunofluorescence analysis shown,significantly greater numbers of 4-1BB positive MH-S cells were obvious after exposure of cells to CS than saline（P<0.05）.3.Activated 4-1BB signaling exaggerate the pro-fibrotic responses of macrophagesAs Western blot analysis shown,the phosphorylation of ASK-1 was markedly elevated in macrophages that were stimulated with CS in the presence of agonist 4-1BB mAb（10μg/mL）,however the total level of ASK-1 was not affected（P<0.05）.Agonist 4-1BB mAb（10μg/mL）could increase the level of the phosphorylation of ASK-1 downstream mitogen-activated protein kinase（MAPK）proteins（p38 and c-Jun N-terminal kinase[JNK]/stress activated protein kinase[SAPK]）in macrophages that were stimulated with CS（P<0.05）,however the total levels of p38 and JNK were not affected.As Realtime-PCR analysis shown,CS accompanied with agonist 4-1BB mAb（10μg/mL）stimulated MH-S cells to express significantly more MMP9 and MMP12 than CS alone（P<0.05）.The relative expression of MCP-1 was markedly upregulated in macrophages treated with CS and agonist 4-1BB mAb（10μg/mL）compared to CS alone（P<0.05）.As ELISA analysis shown,macrophages that exposed to CS,secreted notably more IL-1β,IL-6 and TNF-αafter activation of 4-1BB signaling by agonist 4-1BB mAb（10μg/m L）（P<0.05）.4.Blocking 4-1BB signaling alleviates pro-fibrotic responses of macrophagesAs Western blot analysis shown,compared to macrophages exposed to CS alone,the phosphorylation of ASK-1 was downregulated in macrophages treated with NQDI 1,4-1BBIg and 4-1BB silencing after exposure to CS,however the total level of ASK-1 was not affected（P<0.05）.The protein levels of phosphorylated p38 and JNK were decreased in macrophages treated with NQDI 1,4-1BBIg and 4-1BB silencing after exposure to CS,relative to macrophages exposed to CS alone（P<0.05）.As Realtime-PCR analysis shown,compared with exposure to CS alone,the expression of MMP12 was significantly attenuated in macrophages in which 4-1BB was silenced and in macrophages treated with NQDI 1 or 4-1BBIg（P<0.05）.In contrast,we found that NQDI 1 markedly inhibited the expression of MMP9 in macrophages（P<0.05）,while 4-1BBIg and silenced 4-1BB did not.The relative expression of MCP-1 was notably alleviated in macrophages blocked by NQDI 1 and silenced 4-1BB（P<0.05）,while it was prominently increased in macrophages treated with 4-1BBIg（P<0.05）.As ELISA analysis shown,the use of NQDI 1 and silenced4-1BB suppressed the CS-induced secretion of pro-inflammatory and pro-fibrotic cytokines,including IL-1βand TNF-α,in macrophages（P<0.05）.In contrast,4-1BBIg stimulated macrophages to secrete much more IL-1β,IL-6 and TNF-αrelative to CS treatment alone（P<0.05）.5.Blockade of 4-1BB signaling reduce the secretion of pro-fibrotic mediators in the lung of CS-injured miceCompared to mice exposed to CS alone,the phosphorylation of p38 and JNK were decreased in mice treated with 100?g 4-1BBIg after exposure to CS,however the total levels of p38 and JNK were not affected.While the phosphorylation of p38 and JNK were differently influenced in mice treated with 50?g 4-1BBIg after exposure to CS.Western blot analysis indicated that CS-injured mice treated with 100?g 4-1BBIg exhibited a dramatic reduction in protein levels of MMP9 and MMP12（P<0.05）,while CS-injured mice treated with 50?g 4-1BBIg.As ELISA analysis shown,relative to mice exposed to CS alone,CS-injured mice treated with 100?g 4-1BBIg had markedly lower levels of IL-1β,IL-6 and TNF-αin their lungs（P<0.05）.Nevertheless,the level of MCP-1 did not differ among the groups of mice.Immunohistochemistry analysis illustrated that NQDI 1treatment could reduce MMP9 and MMP12 expressions in the lungs at 7 and 56 days（P<0.05）.As Western blot analysis shown,compared to mice exposed to CS alone,MMP9and MMP12 protein levels were noticeably downregulated in NQDI 1 treated CS-injured mice at different time points（P<0.05）.As immunohistochemistry analysis shown,the deposition of type 1 collagen was markedly reduced in the lungs of mice treated with NQDI1 after exposed to CS 56 days.Conclusion:1.Macrophages could be induced to express 4-1BB by CS2.Activated 4-1BB signaling in macrophages promoted the release of pro-inflammatory and pro-fibrotic cytokines,chemokines and MMPs.3.Blocking 4-1BB signaling reduced the secretion of pro-fibrotic mediators in the lung of CS-injured mice,and then alleviated pulmonary fibrosis. |
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