## 4 Discussion  
Periodonal resident cells utilize various defense strategies to address challenges from environmental stimuli, such as periodontitis([PERSON] et al., 2018). Toll-like and NOD-like receptors expressing gingival resident cells could play key roles at the initial phase of innate immune responses. Many studies have investigated the expressions of these receptors in human cells and tissues ([PERSON] et al., 2018); however, limited studies are available reporting the expressions of such receptors in the periodontal cells and tissues in vivo ([PERSON] et al., 2018, 2019). To the best knowledge of this group, this is one of the more comprehensive reports characterizing expressions of TLRs and selected NLRs on periodontal tissue biopsies and resident cells. The current in vitro observations demonstrated that transcripts of TLR1-6, NOD1/2 and NLRP1/2 are observable in the latter. Similarly, the key proteins, i.e., TLR1/2/4/5/6, NOD1/2 and NLRP1/2, were also detected in periodontal biopsies, and HGK, and HGF with TLR2/4 and NOD1/2 appeared more readily detectable in the pocket vs. junctional epithelium in periodontal biopsies. All DAMP sensors of interest,  
Figure 3: Selected damage-associated molecular pattern (DAMP) sensors expression in human gingival keratinocytes (HGK) or human gingival fibroblasts (HGF) explants under 1% O2 and/or 2 ug/mL _Escherichia coli_ LPS, 24 h. (**A**) Detection of TLR1, TLR2, TLR4-6, NOD1/2, NLRP1/2 from HGK/HGF cell lysates after various treatments. Shown are representative blots from three independent experiments; (**B**) relative HGK TLR1, TLR2, TLR4-6, NOD1/2, NLRP1/2 protein levels after various treatments; (**C**) relative HGF TLR1, TLR2, TLR4-6, NOD1/2, NLRP1/2 protein levels after various treatments. *_p_ < 0.05, **_p_ < 0.01, one-way ANOVA with adjustments against multiple comparisons within same DAMP sensor group. Cells at passage three were used and the experiments were independently repeated three times. however, were significantly enhanced in periodontal pocket connective tissue, where heavy inflammatory cells infiltrate could be readily observed. Regarding the role of NOD-like receptors in periodontitis, the most thoroughly studied molecules are NOD1/2 [9], while not many reports are available for other NLRs. Contrary to NOD1, which is reported to be expressed almost in all cell types, NOD2 is found to be expressed in a rather restricted manner, mainly limited to macrophages, dendritic cells, epithelial cells in the intestine or oral cavity, etc. [31]. In the current study, the expressions of these four receptors were reported both in biopsies and resident cells in vitro. A moderate but enhanced stained area and the NOD1/2 intensity in the periodontitis pocket epithelium and subepithelial connective tissues, compared to that of the junctional epithelium and submuc in healthy biopsy sections indicated their potential roles in the innate immune defense at the periodontal front. This study confirmed the existence of NOD1/2 and NLRP1/2 expressions in periodontal biopsies for either healthy or periodontitis patients. Next, transcriptional signals for TLRs and NLRs under the influence of hypoxia or _E. coli_ LPS were investigated. Hypoxia appeared to be associated with the upregulation of TLR2/6 transcripts and proteins in human dendritic, monocytic, endothelial and intestinal epithelial cells [24]. In this study, hypoxia associated with enhanced TLR2, NOD2 and NLRP1 transcripts but reduced TLR1/3/6 NOD1, and NLRP2 transcripts detection in HGK were observable. Upon stimulation by _E. coli_ LPS, the upregulation of TLR2 and NOD2 only in the same cell was observed (Table 4). For HGF, hypoxia appeared to be associated more with transcript detection concerning TLR1/2/4, NOD1 and NLRP1, while that of TLR3/5 was reduced. Putting HGF under _E. coli_ LPS, only enhanced transcript detection of TLR1/2/6 and NOD2 was observed.