Muramyl dipeptide activates human beta defensin 2 and pro-inflammatory mediators through Toll-like receptors and NLRP3 inflammasomes in human dental pulp cells

作者：Lee Sang Im; Kang Soo Kyung; Jung Ha Jin; Chun Yang Hyun; Kwon Young Dae; Kim Eun Cheol^*

来源：Clinical Oral Investigations, 2015, 19(6): 1419-1428.

DOI：10.1007/s00784-014-1361-8

• NOD2
• Human beta defensin-2
• Cytokines
• TLR
• NLRP3
• Defense

Purpose The expression levels of intracellular pyrin domain-containing 3 (NLRP3) and microbial pattern-recognition receptors, such as nucleotide-binding oligomerization domain 2 (NOD2), have been reported in human dental pulp cells (HDPCs) and inflamed dental pulp tissue, but the role of NLRP3 and Toll-like receptors (TLRs) in the production of human beta defensin 2 (hBD2) and inflammatory cytokines against invading pathogens remains poorly defined. The aim of this study was to determine whether the NOD2 ligand muramyl dipeptide (MDP) upregulates hBD2 and inflammatory cytokines and whether this response is dependent on TLRs and NLRP inflammasomes in HDPCs.
Methodology The effects of MDP on the expression of hBD2, TLRs, inflammasomes, and pro-inflammatory mediators in HDPCs were examined using Western blotting and reverse transcription-polymerase chain reaction. Levels of pro-inflammatory cytokines, such as nitric oxide (NO) and prostaglandin E-2 (PGE(2)), were determined by enzyme-linked immunosorbent assay.
Results MDP upregulated hBD2, TLR2, and TLR4 mRNAs and protein levels in a dose-and time-dependent manner. TLR2 and TLR4 neutralizing blocking antibodies and NOD2-and hBD2-specific small interfering RNAs (siRNAs) attenuated the MDP-induced production of NO, PGE(2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8 and upregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) in HDPCs. Additionally, MDP activated inflammasome-related genes, such as NLRP3, caspase 1, apoptotic speck protein containing a caspase recruitment domain, and IL-1 beta. Furthermore, silencing of the NLRP3 gene using a siRNA significantly decreased the MDP-induced expression of hBD2 and cytokines, such as iNOS-derived NO, COX2, PGE(2), TNF-alpha, IL-6, and IL-8.
Conclusion These results suggest that NOD2 activates the TLR2, TLR4, and NLRP3 inflammasome-signaling pathways in HDPCs to induce the production of multiple inflammatory mediators and antimicrobial peptides, which in turn promote pulp immune defense against microbial challenge.
Clinical relevance The TLR and NLRP3 inflammasome pathways may represent an important modulatory mechanism of immune defense responses during the progression of pulpitis. Our results suggest that local inhibition of NLRP3 and TLRs may reduce the impact of cytokine-mediated host destructive processes in pulpitis.

• 出版日期2015-7