Hypoxia and E. coli LPS-induced periodontal resident cells PAMP expressions : implication for periodontitis pathogenesis

Abstract

Toll-like (TLRs) and NOD-like receptors (NLRs) are families of transmembrane or intracellular pattern recognition receptors that play parts in innate immunity. They mainly recognize the nonself molecular patterns outside or inside host cells triggering defense mechanisms and cellular changes. At the periodontal front, pathogen related molecular patterns (PAMP) e.g. lipopolysaccharides (LPS), lipopeptides or glycolipids were recognized by corresponding receptors of the lining cells. Microbial anaerobic PAMP accumulation would eventually lead to local chronic inflammation under low redox environment in the periodontitis loci. Through recognition of these patterns, the lining cells receptors activate intracellular signaling for instance via the NF-κB pathway to trigger various cell reactions.

The aim of this project is to characterize the role of periodontal resident cells PAMP recognition, in particular under the influence of low oxygen levels. This project hypothesized that hypoxia in combination with LPS may influence and modulate the expressions of TLRs and NLRs in periodontal resident cells, which might be related to intracellular signaling involving nuclear factor kappa B (NF-κB).

Human gingival biopsies were collected from periodontitis or healthy sites from donors without systemic diseases. Immunohistochemistry were carried out to detect TLRs and selected NLRs (NOD1/2 and NLRP1/2) peptides expression. Biopsies of healthy gingivae were collected to cultivate human gingival resident cells, i.e. human gingival keratinocytes (HGKs) and human gingival fibroblasts (HGFs). TLRs, NLRs and NF-κBs transcripts and peptides expressions on HGK/HGF were screened under hypoxia (1% O2), normoxia (18% O2) with/without treatment of 2μg/ml Escherichia coli LPS over various time periods. Cellular levels of ROS and ATP were also followed. We attempted immortalization of keratinocyte explants from healthy donors.

TLR1/2/4/5/6, NOD1/2 and NLRP1/2 peptides were detected in both healthy and periodontitis biopsies. TLR2/4 and NOD1/2 immunostaining appeared stronger in pocket compared to junctional epithelium, while TLR1/2/5 and NOD1 immunostaining seemed stronger in periodontitis than healthy oral epithelium. In the periodontitis sub-epithelial connective tissue, immunostaining of TLR1/2/4/5/6, NOD1/2 and NLRP1/2 were enhanced.

Transcripts of TLR1/2/3/4/5/6/7/8/10, NOD1/2 and NLRP1/2 were detected in periodontitis tissue. In HGK and HGF explants, transcripts of TLR1-6, NOD1/2 and NLRP1/2 were detected. Hypoxia and/or E. coli LPS induced HGK/HGF-TLR1/2/6 and NOD2 signals and/or peptides accumulation.

Regarding intracellular signals followed, hypoxia and/or LPS induced NF-κB2 and/or RelB signal and peptide accumulation in HGK and HGF, while no significant influence was observed for other NF-κBs. The increased expression of RelB and NF-κB2 suggested that the noncanonical NF-κB pathway may be involved in the corresponding cellular responses towards hypoxia and LPS.

Our preliminary data showed one immortalized HGK line was successfully maintained till passages 33 with normal cellular contours, suggesting the attempt was successful.

In conclusions, with the limitation of this project, TLR2 and NOD2 in HGKs and HGFs appeared reacting to hypoxia and LPS, during which NF-κB2 and RelB may contribute in the subsequent intracellular signaling. Further studies, are required to characterize and confirm the biology of hypoxia and E.coli LPS mediated periodontal residential cells pattern recognition in human periodontitis.