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

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Abstract

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 E2 (PGE2), 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, PGE2, tumor necrosis factor-α (TNF-α), 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-1β. 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, PGE2, TNF-α, 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.

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Acknowledgments

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (no. 2012R1A5A2051384) and by a grant of the Korea Healthcare Technology R & D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A111412).

Conflict of interest

The authors declare no conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Dental Hygiene, School of Health Sciences, Dankook University, Cheonan, Republic of Korea

    Sang-Im Lee

  2. Department of Oral and Maxillofacial Pathology & Research Center for Tooth and Periodontal Regeneration (MRC), School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 130-701, Republic of Korea

    Ha-Jin Jung & Eun-Cheol Kim

  3. Department of Oral Medicine, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea

    Soo-Kyung Kang & Yang-Hyun Chun

  4. Department of Oral and Maxillofacial Surgery, School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 130-701, Republic of Korea

    Young-Dae Kwon

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  1. Sang-Im Lee
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Correspondence to Young-Dae Kwon or Eun-Cheol Kim.

Additional information

Young-Dae Kwon and Eun-Cheol Kim contributed equally to this work as corresponding authors.

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Lee, SI., Kang, SK., Jung, HJ. et al. Muramyl dipeptide activates human beta defensin 2 and pro-inflammatory mediators through Toll-like receptors and NLRP3 inflammasomes in human dental pulp cells. Clin Oral Invest 19, 1419–1428 (2015). https://doi.org/10.1007/s00784-014-1361-8

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