Recombinant overexpression of camel hepcidin cDNA in Pichia pastoris: purification and characterization of the polyHis‐tagged peptide HepcD‐His

Hepcidin, a liver‐expressed antimicrobial peptide, has been demonstrated to act as an iron regulatory hormone as well as to exert a wide spectrum of antimicrobial activity. The aim of this work was the expression, as secreted peptide, purification, and characterization of a new recombinant polyHis‐tagged camel hepcidin (HepcD‐His) in yeast Pichia pastoris . The use of this eukaryotic expression system, for the production of HepcD‐His, having 6 histidine residues at its C terminus, was simpler and more efficient compared with the use of the prokaryotic system Escherichia  coli . Indeed, a single purification step was required to isolate the soluble hepcidin with purity estimated more that 94% and a yield of 2.8 against 0.2 mg/L for the E coli system. Matrix‐assisted laser desorption/ionization time‐of‐flight (TOF)/TOF mass spectrometry of the purified HepcD‐His showed 2 major peaks at m /z 4524.64 and 4634.56 corresponding to camel hepcidin with 39 and 40 amino acids. Evaluation of disulfide bond connectivity with the Ellman method showed an absence of free thiol groups, testifying that the 8 cysteine residues in the peptide are displayed, forming 4 disulfide bridges. Circular dichroism spectroscopy showed that camel hepcidin structure was significantly modified at high temperature of 90°C and returns to its original structure when incubation temperature drops back to 20°C. Interestingly, this peptide showed also a greater bactericidal activity, at low concentration of 9.5μM, against E coli , than the synthetic analog DH3. Thus, the production, at a large scale, of the recombinant camel hepcidin, HepcD‐His, may be helpful for future therapeutic applications including bacterial infection diseases.     

Structural studies of the tethered N‐terminus of the Alzheimer's disease amyloid‐β peptide

Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid‐β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal‐binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1–16 fused to the N‐terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti‐Aβ N‐terminal antibody WO2. The structure demonstrates that Aβ residues 10–16, which are not in complex with the antibody, adopt a mixture of local polyproline II‐helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10–16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13‐metal‐His14 coordination in the Aβ_1–16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758. © 2013 Wiley Periodicals, Inc.     

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma

The β‐subunit of the human eukaryotic elongation factor 1 complex (heEF1β) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α‐ and γ‐subunits (eEF1γ). To biophysically characterize heEF1β, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL‐heEF1β), N‐terminus (NT‐heEF1β), and the C‐terminus (CT‐heEF1β) regions of the protein. Our results suggest that heEF1β is predominantly alpha‐helical and possesses an accessible hydrophobic cavity in the CT‐heEF1β. Both FL‐heEF1β and NT‐heEF1β form dimers of size 62 and 30 kDa, respectively, but the CT‐heEF1β is monomeric. FL‐heEF1β interacts with the N‐terminus glutathione transferase‐like domain of heEF1γ (NT‐heEF1γ) to form a 195‐kDa complex or a 230‐kDa complex in the presence of oxidized glutathione. On the other hand, NT‐heEF1β forms a 170‐kDa complex with NT‐heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL‐heEF1β associated with monomeric or dimeric NT‐heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (K_D = 9.6 nM for monomeric or 11.3 nM for dimeric NT‐heEF1γ). We postulate that the N‐terminus region of heEF1β may be responsible for its dimerization and the C‐terminus region of heEF1β modulates the formation of an ordered heEF1β‐γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.     

XOMA 052, an anti‐IL‐1β monoclonal antibody,prevents IL‐1β‐mediated insulin resistance in 3T3‐L1 adipocytes

Objective:

Interleukin‐1β (IL‐1β) has recently been implicated as a major cytokine that is involved in the pancreatic islet inflammation of type 2 diabetes mellitus. This inflammation impairs insulin secretion by inducing beta‐cell apoptosis. Recent evidence has suggested that in obesity‐induced inflammation, IL‐1β plays a key role in causing insulin resistance in peripheral tissues.

Design and Methods:

To further investigate the pathophysiological role of IL‐1β in causing insulin resistance, the inhibitory effects of IL‐1β on several insulin‐dependent metabolic processes in vitro has been neutralized by XOMA 052. The role IL‐1β plays in insulin resistance in adipose tissue was assessed using differentiated 3T3‐L1 adipocytes and several parameters involved in insulin signaling and lipid metabolism were examined.

Results and Conclusion:

IL‐1β inhibited insulin‐induced activation of Akt phosphorylation, glucose transport, and fatty acid uptake. IL‐1β also blocked insulin‐mediated downregulation of suppressor of cytokine signaling‐3 expression. Co‐preincubation of IL‐1β with XOMA 052 neutralized nearly all of these inhibitory effects in 3T3‐L1 adipocytes. These studies provide evidence, therefore, that IL‐1β is a key proinflammatory cytokine that is involved in inducing insulin resistance. These studies also suggest that the monoclonal antibody XOMA 052 may be a possible therapeutic to effectively neutralize cytokine‐mediated insulin resistance in adipose tissue.     

Biochemical characterization and molecular modeling of a unique lipase from Staphylococcus arlettae JPBW‐1

A three‐step purification of a unique lipase with halo‐, solvent‐, detergent‐, and thermo‐tolerance from Staphylococcus arlettae JPBW‐1 gave raise to a 27‐fold purification with a specific activity of 32.5 U/mg. The molecular weight of the purified lipase was estimated to be 45 kDa using SDS–PAGE, and its amino acid sequence was characterized using MALDI‐TOF‐MS analysis. The sequence obtained from MALDI‐TOF‐MS showed significant similarity with the capsular polysaccharide biosynthesis protein (CapD) of Staphylococcus aureus through comparative modeling approach using ROBETTA server. Identification of responsible fragments for homodimer formation was performed using comparative modeling and substrate binding domain through C‐terminus matching of this new lipase with the CapD of Staphylococcus aureus was executed. Thus, the experimental coupled molecular modeling postulated a structure–activity relationship of lipase from S. arlettae JPBW‐1, a potential candidate for detergent, leather, pulp, and paper industries.     

Characterization and high‐yield production of non‐N‐glycosylated recombinant human BCMA‐Fc in Pichia pastoris

B‐cell maturation antigen (BCMA) fused at the C‐terminus to the Fc portion of human IgG1 (BCMA‐Fc) blocks B‐cell activating factor (BAFF) and proliferation‐inducing ligand (APRIL)‐mediated B‐cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA‐Fc (rhBCMA‐Fc) in Pichia pastoris under the control of the AOX1 methanol‐inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N‐linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed‐batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA‐Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA‐Fc possessed high‐binding affinity to APRIL and dose‐dependent inhibition of APRIL‐induced proliferative activity in vitro through three‐step purification. Thus, this yeast‐derived expression method could be a low‐cost and effective alternative to the production of rhBCMA‐Fc in mammalian cell lines.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

ATP‐dependent activation of an inflammasome in primary gingival epithelial cells infected by Porphyromonas gingivalis

Production of IL‐1β typically requires two‐separate signals. The first signal, from a pathogen‐associated molecular pattern, promotes intracellular production of immature cytokine. The second signal, derived from a danger signal such as extracellular ATP, results in assembly of an inflammasome, activation of caspase‐1 and secretion of mature cytokine. The inflammasome component, Nalp3, plays a non‐redundant role in caspase‐1 activation in response to ATP binding to P2X₇ in macrophages. Gingival epithelial cells (GECs) are an important component of the innate‐immune response to periodontal bacteria. We had shown that GECs express a functional P2X₇ receptor, but the ability of GECs to secrete IL‐1β during infection remained unknown. We find that GECs express a functional Nalp3 inflammasome. Treatment of GECs with LPS or infection with the periodontal pathogen, Porphyromonas gingivalis, induced expression of the il‐1β gene and intracellular accumulation of IL‐1β protein. However, IL‐1β was not secreted unless LPS‐treated or infected cells were subsequently stimulated with ATP. Conversely, caspase‐1 is activated in GECs following ATP treatment but not P. gingivalis infection. Furthermore, depletion of Nalp3 by siRNA abrogated the ability of ATP to induce IL‐1β secretion in infected cells. The Nalp3 inflammasome is therefore likely to be an important mediator of the inflammatory response in gingival epithelium.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β). New in vitro testing systems are needed to evaluate efficacies of new anti‐inflammatory biological drugs, ideally in a patient‐specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti‐inflammatory drugs, spheroids were exposed to TNF‐α, IL‐1β, or to supernatant containing secretome from activated macrophages (MCM). The anti‐inflammatory efficacies of anti‐TNF‐α biologicals adalimumab, infliximab, and etanercept, and the anti‐IL‐1β agent anakinra were assessed in short‐term microspheroid and long‐term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF‐α or IL‐1β. The differences in potency of anti‐TNF‐α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short‐term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti‐TNF‐α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045–1058, 2018     

Human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSC) inhibit the proliferation of K562 (human erythromyeloblastoid leukaemic cell line)

hUCB‐MSC (human umbilical cord blood‐derived mesenchymal stem cells) offer an attractive alternative to bone marrow‐derived MSC for cell‐based therapy by being less invasive a source of biological material. We have evaluated the effect of hUCB‐MSC on the proliferation of K562 (an erythromyeloblastoid cell line) and the cytokine secretion pattern of hUCB‐MSC. Co‐culturing of hUCB‐MSC and K562 resulted in inhibition of proliferation of K562 in a dose‐dependent manner. However, the anti‐proliferative effect was reduced in transwells, suggesting the importance of direct cell‐to‐cell contact. hUCB‐MSC inhibited proliferation of K562, arresting them in the G₀/G₁ phase. NO (nitric oxide) was not involved in the hUCB‐MSC‐mediated tumour suppression. The presence of IL‐6 (interleukin 6) and IL‐8 were obvious in the hUCB‐MSC conditioned media, but no significant increase was found in 29 other cytokines. Th1 cytokines, IFNα (interferon α), Th2 cytokine IL‐4 and Th17 cytokine, IL‐17 were not secreted by hUCB‐MSC. There was an increase in the number of hUCB‐MSC expressing the latent membrane‐bound form of TGFβ1 co‐cultured with K562. The anti‐proliferative effect of hUCB‐MSC was due to arrest of the growth of K562 in the G₀/G₁ phase. The mechanisms underlying increased IL‐6 and IL‐8 secretion and LAP (latency‐associated peptide; TGFβ1) by hUCB‐MSC remains unknown.     

MiR‐149 sensitizes esophageal cancer cell lines to cisplatin by targeting DNA polymerase β

Human DNA polymerase β (polβ) is a small, monomeric protein essential for short‐patch base excision repair (BER). polβ plays an important role in the regulation of chemotherapy sensitivity in tumour cells. In this study, we determined that the expression levels of polβ mRNA and miR‐149 in tumour tissues were significantly higher than in adjacent non‐tumour tissues. We also found that the expression level of miR‐149 in EC tumour tissues was inverse to that of polβ expression. Bioinformatics analysis and dual‐luciferase reporter assay predicted that miR‐149 negatively regulates polβ expression by directly binding to its 3′UTR. CCK‐8 assay indicated that miR‐149 could enhance the anti‐proliferative effects of cisplatin in EC1 and EC9706 cell lines. Flow cytometry, caspase 3/7 activity, and immunofluorescence microscopy results indicated that miR‐149 could enhance the apoptotic effects of cisplatin in EC1 and EC9706 cell lines. We also showed that the expression of polβ lacking the 3′UTR sequence could override the proliferative and apoptotic functions of miR‐149, suggesting that miR‐149 negatively regulates polβ expression by binding to its 3′UTR. Surface plasmon resonance results also showed that miR‐149 could bind with wild‐type polβ. In addition, we identified a new variant of polβ (C1134G). In conclusion, this study confirms that miR‐149 may enhance the sensitivity of EC cell lines to cisplatin by targeting polβ, and that miR‐149 may be unable to regulate the C1134G variant of polβ. Based on these findings, potential drugs could be developed with a focus on enhanced sensitivity of EC patients to chemotherapy.     

His‐tag binding by antibody C706 mimics β‐amyloid recognition

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ₄₂ peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2₁ and C2. Although the Fab was crystallized in the presence of Aβ₁₆, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.     

MXRA5 is a TGF‐β1‐regulated human protein with anti‐inflammatory and anti‐fibrotic properties

Current therapy for chronic kidney disease (CKD) is unsatisfactory because of an insufficient understanding of its pathogenesis. Matrix remodelling‐associated protein 5 (MXRA5, adlican) is a human protein of unknown function with high kidney tissue expression, not present in rodents. Given the increased expression of MXRA5 in injured tissues, including the kidneys, we have suggested that MXRA5 may modulate kidney injury. MXRA5 immunoreactivity was observed in tubular cells in human renal biopsies and in urine from CKD patients. We then explored factors regulating MXRA5 expression and MXRA5 function in cultured human proximal tubular epithelial cells and explored MXRA5 expression in kidney cancer cells and kidney tissue. The fibrogenic cytokine transforming growth factor‐β1 (TGFβ1) up‐regulated MXRA5 mRNA and protein expression. TGFβ1‐induced MXRA5 up‐regulation was prevented by either interference with TGFβ1 activation of the TGFβ receptor 1 (TGFBR1, ALK5) or by the vitamin D receptor agonist paricalcitol. By contrast, the pro‐inflammatory cytokine TWEAK did not modulate MXRA5 expression. MXRA5 siRNA‐induced down‐regulation of constitutive MXRA5 expression resulted in higher TWEAK‐induced expression of chemokines. In addition, MXRA5 down‐regulation resulted in a magnified expression of genes encoding extracellular matrix proteins in response to TGFβ1. Furthermore, in clear cell renal cancer, von Hippel–Lindau (VHL) regulated MXRA5 expression. In conclusion, MXRA5 is a TGFβ1‐ and VHL‐regulated protein and, for the first time, we identify MXRA5 functions as an anti‐inflammatory and anti‐fibrotic molecule. This information may yield clues to design novel therapeutic strategies in diseases characterized by inflammation and fibrosis.     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Th17 can regulate silica‐induced lung inflammation through an IL‐1β‐dependent mechanism

Silicosis is an occupational lung disease caused by the inhalation of silica dust and characterized by lung inflammation and fibrosis. Interleukin (IL)‐1β is induced by silica and functions as the key pro‐inflammatory cytokine in this process. The Th17 response, which is induced by IL‐1β, has been reported very important in chronic human lung inflammatory diseases. To elucidate the underlying mechanisms of IL‐1β and IL‐17 in silicosis, we used anakinra and an anti‐IL‐17 monoclonal antibody (mAb) to block the receptor of IL‐1β (IL‐RI) and IL‐17, respectively, in a mouse model of silicosis. We observed increased IL‐1β expression and an enhanced Th17 response after silica instillation. Treatment with an IL‐1 type I receptor (IL‐1RI) antagonist anakinra substantially decreased silica‐induced lung inflammation and the Th17 response. Lung inflammation and the accumulation of inflammatory cells were attenuated in the IL‐17‐neutralized silicosis group. IL‐17 may promote lung inflammation by modulating the differentiation of Th1 and regulatory T cells (Tregs) and by regulating the production of IL‐22 and IL‐1β during the lung inflammation of silicosis. Silica may induce IL‐1β production from alveolar macrophages and promote inflammation by initiating a Th17 response via an IL‐1β/IL‐1RI‐dependent mechanism. The Th17 response could induce lung inflammation during the pathogenesis of silicosis by regulating the homoeostasis of the Th immune responses and affecting the production of IL‐22 and IL‐1β. This study describes a potentially important inflammatory mechanism of silicosis that may bring about novel therapies for this inflammatory and fibrotic disease.     

Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL‐1β/IL‐1R1/MyD88/NF‐κB pathway

Interleukin (IL)‐1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL‐1β is dependent upon caspase‐1‐containing multiprotein complexes called inflammasomes and IL‐1R1/MyD88/NF‐κB pathway. In this study, we explored whether a potential anti‐fibrotic agent fluorofenidone (FD) exerts its anti‐inflammatory and anti‐fibrotic effects through suppressing activation of NACHT, LRR and PYD domains‐containing protein 3 (NALP3) inflammasome and the IL‐1β/IL‐1R1/MyD88/NF‐κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α‐smooth muscle actin (α‐SMA), fibronectin, collagen I, caspase‐1, IL‐1R1, MyD88 were measured by Western blot and/or RT‐PCR. The human actue monocytic leukaemia cell line (THP‐1) were incubated with monosodium urate (MSU), with or without FD pre‐treatment. The expression of caspase‐1, IL‐1β, NALP3, apoptosis‐associated speck‐like protein containing (ASC) and pro‐caspase‐1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome‐associated molecules were measured by Co‐immunoprecipitation. RLE‐6TN (rat lung epithelial‐T‐antigen negative) cells were incubated with IL‐1β, with or without FD pre‐treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL‐1β, IL‐6, monocyte chemotactic protein‐1 (MCP‐1), myeloperoxidase (MPO), α‐SMA, fibronectin, collagen I, caspase‐1, IL‐1R1 and MyD88 in mice lung tissues. And FD inhibited MSU‐induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome‐associated molecules, decreased the level of caspase‐1 and IL‐1β in THP‐1 cells. Besides, FD inhibited IL‐1β‐induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM‐induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL‐1β/IL‐1R1/MyD88/ NF‐κB pathway.