title: Mechanisms that lead to the regulation of NLRP3 inflammasome expression and activation in human dental pulp fibroblasts.
abstract: BACKGROUND
  The NLRP3 inflammasome plays an important role in the cellular defense against invading pathogens and is reported to be expressed in human dental pulp fibroblasts (HDPFs). However, the role of the NLRP3 inflammasome in HDPFs during pulpal infection and inflammation remains unclear.
  
  
  OBJECTIVE
  To elucidate the function of the NLRP3 inflammasome and the mechanisms that lead to its expression and activation in HDPFs.
  
  
  METHODS
  The test model used lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to simulate an inflammatory environment. Lentiviral vectors encoding short hairpin RNAs were used to knock down NLRP3 and caspase-1 in HDPFs. Specific inhibitors were used to determine whether the toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), or nuclear factor-kappa B (NF-κB) pathways were involved in the regulation of NLRP3 expression. Reactive oxygen species (ROS) production was measured by fluorescent microscopy and flow cytometry using the total ROS/superoxide detection kit. Gene and protein expression were quantified by real-time polymerase chain reaction and Western blot, while cytokine release was measured by an enzyme-linked immunosorbent assay.
  
  
  RESULTS
  LPS up-regulated NLRP3 and IL-1β expression while ATP induced the activation of caspase-1 and the release of IL-1β in LPS-primed HDPFs. The knockdown of NLRP3 or caspase-1 expression significantly inhibited IL-1β secretion. Pretreatment with a TLR4 inhibitor, a MyD88 inhibitory peptide, or an I Kappa B alpha (IκBα) phosphorylation inhibitor significantly inhibited LPS-induced NLRP3 and IL-1β expression. ATP potently promoted ROS generation in HDPFs; N-acetyl cysteine inhibited ROS production, caspase-1 activation and IL-1β secretion induced by ATP.
  
  
  CONCLUSIONS
  Our results demonstrated that the NLRP3 inflammasome in HDPFs is crucial for IL-1β secretion in response to LPS plus ATP. LPS engaged the TLR4/MyD88/NF-κB pathway to enhance NLRP3 and pro-IL-1β expression in HDPFs. ATP promoted the generation of ROS and activated the NLRP3 inflammasome in a ROS-dependent manner.
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China.
authors: Ansheng
authors: A
authors: Zhang
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China.
authors: Peina
authors: P
authors: Wang
authors: Out-patient Department, The 323 Hospital of PLA, Xi'an 710054, Shaanxi, China.
authors: Xiaoying
authors: X
authors: Ma
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China.
authors: Xiao
authors: X
authors: Yin
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China.
authors: Jiguo
authors: J
authors: Li
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China.
authors: Haijing
authors: H
authors: Wang
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China.
authors: Wenkai
authors: W
authors: Jiang
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China.
authors: Qian
authors: Q
authors: Jia
authors: State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, 145 Changle West Road, Xi'an 710032, Shaanxi, China. Electronic address: nilongx@fmmu.edu.cn.
authors: Longxing
authors: L
authors: Ni
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001641:Bicuspid / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D020170:Caspase 1 / Q000235:genetics / Q000276:immunology; D003782:Dental Pulp / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D005786:Gene Expression Regulation; D006801:Humans; D020672:I-kappa B Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D058847:Inflammasomes / Q000187:drug effects* / Q000276:immunology; D053583:Interleukin-1beta / Q000235:genetics / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D053594:Myeloid Differentiation Factor 88 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D000072000:NF-KappaB Inhibitor alpha; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology*; D014081:Tooth Extraction
chemical_list: D002352:Carrier Proteins; D020672:I-kappa B Proteins; D058847:Inflammasomes; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; C000606204:NFKBIA protein, human; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D010455:Peptides; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D000072000:NF-KappaB Inhibitor alpha; D000255:Adenosine Triphosphate; D020170:Caspase 1; D000111:Acetylcysteine
keywords: Human dental pulp fibroblasts; Innate immunity; NLRP3 inflammasome; Reactive oxygen species; TLR