## 4 Discussion
Such observations lined up nicely with the in vitro DAMP sensor protein expression of HGF when increased TLR1/6 and NOD2 protein were reported under hypoxia and/or _E. coli_ LPS challenge (Figure 3C). However, this investigation also observed the unstable expression of TLR4 in HGK and NLRP2 in HGF both at normoxia and hypoxia; the authors postulated that TLR4 expression remains at a low level in healthy epithelium [26], and so it is the same with NLRP2 expression in HGF. Since it is the first report concerning NLRP2 expression in gingival cells [32], further investigation is needed. The current study results perhaps implied the potential roles of TLR1/2/6 and NOD2 in periodontal resident cells immune responses against periodontal infection. According to the previous research studies, TLR1/2/6 is known to be activated by various ligands, such as bacteria, fungi, virus and certain endogenous substances [11], while NOD2 is reported to be a universal sensor for peptidoglycans from Gram-positive and muramyl dipeptide moiety from Gram-negative bacteria. Taking the above together, the authors postulate that TLR1/2/6 and NOD2 are potential potent receptors at the dento-gingival front responsible for periodontal innate immunity. It is regularly believed that specific receptors might be preferentially expressed upon stimulation of corresponding target ligands, e.g., LPS for TLR4 [6]. However, in the current study, HGK or HGF TLR4 transcript and protein expression did not appear to be significantly influenced/changed under LPS stimulation. Only in 46%/38% HGK explants under normoxia/hypoxia could TLR4 transcript be detected, implying either an inherited low cellular transcription level in periodontal resident cells or perhaps that a specific yet tightly regulated mechanism is in effect [26; 33]. Considering the fact that previous reports regarding the expression of TLR4 upon LPS stimulation remained ambiguous [34], the present group postulated that the diverse observation concerning TLR4 expression under LPS/hypoxia stimulation in this study was actually in line with earlier reports. There are reports describing the downregulated expression or cellular tolerance via reduced cell surface TLR4 expression under LPS stimulation [35]. However, the molecular mechanisms of endotoxin tolerance remained yet unclear. During tolerance, TLR4 is transiently suppressed or unchanged, with proximal post-receptor signaling proteins also altered, such as interleukin-1 receptor-associated kinase (IRAK), TLR4-myeloid differentiation factor 88 (MyD88) and IRAK-MyD88 association [36]. Therefore, it is hypothesized that gingival tissue under repeated exposure to Gram-negative periodontitis that carries LPS may lead to host cell tolerance; therefore, low expressions of TLR4 were observed in the experiment reported. This may also aid in explaining the rare distribution of TLR4 in the junctional/pocket epithelium and even the lack of expression in certain donors' gingival primary cell culture. Previous studies have shown that TLR4 is an important receptor involved in periodontitis immune defense. However, these studies relied heavily on the use of human oral keratinocyte from oral mucosa instead of that from gingival tissue origin [37]. It is hypothesized that perhaps the expressions of TLR4 could also be location-specific or cellular type- and physiological status-dependent. Taking all information in consideration, this research team postulated that DAMP sensor regulation, such as TLR4, may not operate merely under a direct feedback mechanism, implying that various factors, such as cell types, environmental stimuli, and duration/persistence of ligand exposure, may also be involved [34; 38; 39]. The mechanism modulating the expression of TLR4 at resident cells at the dento-gingival junction, therefore, warrants further investigations. On the other hand, it is indicated _E.