## 4 Discussion
coli_ LPS under hypoxia-stimulated HGF, HGK TLR1/6 and NOD2 protein expression (to a certain extent), and TLR2 and NOD2 transcripts' upregulation were readily observed under LPS challenge alone in this in vitro study. It is postulated that for HGK/HGF, TLR1/2/6 and NOD2 instead of TLR4 could be the more important/relevant DAMP sensors against _E. coli_ LPS. TLR1/2 and TLR2/6 are not considered typical oral/periodonal LPS receptors compared to TLR4, reasons being that the former could bind to various ligands, including lipoproteins, glycolipids, fungi, virus and certain endogenous substances [40]. However, in reports concerning intestinal epithelium, TLR2 expression was reported to be relatively upregulated in intestinal epithelial cell lines upon LPS stimulation, and the same was reported in LPS-treated adipocytes [41]. One point worth noticing, however, is that LPS with relatively low endotoxic activity, such as those from _Bacteroides fragilis_, _Chlamydia trachomatis_ and _Pseudomonas aeruginosa_, commonly transduces signals via TLR2 instead of TLR4 [4]. Highly purified _Helicobacter pylori_ LPS also induce weak inflammatory reactions and utilize the TLR2, but not the TLR4, pathway [42]. In this research, it was noticed that TLR4/5, NOD1 and NLRP1/2 appeared constitutively expressed, especially in immunohistochemistry staining, and remained more or less unaffected by low oxygen tension and/or _E. coli_ LPS, both at transcriptional and peptide levels. Therefore, the authors postulated that these receptors may be persistently expressed by periodontal resident cells or they are regulated by other DAMPs, such as those from _Eubacterium sapheum_, _Eubacterium nodatum_ and _Filitator alocis_ other than low oxygen tension and/or _E. coli_ LPS [43; 44]. Interestingly, previous reports held different opinions on the regulation of _Porphyomonas gingivalis_ on the expression of inflammosomes, such as NLRP1, and some papers described the activation of inflammasome members via _P. gingivalis_ stimulation [32], while others published the opposite result that _P. gingivalis_ inhibited inflammasome-involved immune responses [45]. However, both reports indicated that IL-1 plays an important role in the arena of inflammasome complex [32; 45]. The current study utilized _E. coli_ instead of _P. gingivalis_ LPS. _E. coli_ LPS was reported to be more readily recognized by TLR4 and TLR2 compared to _P. gingivalis_ LPS [46], and the former could pose further influence on downstream gene expression, such as CXC chemokine ligand 5 [47]. More importantly, _E. coli_ LPS could induce stronger cellular expressions compared to that of _P. gingivalis_[47]. Obviously, _E. coli_ are less often detected in human mouths, unless associated with recurrent aphthous stomatitis [48], while _P. gingivalis_ is an established keystone periodontopathogen [49]. Therefore, in theory, _E. coli_ LPS could influence periodontal resident cells differently compared to _P. gingivalis_ LPS, and the latter could facilitate experiments more accurately, mimicking the pathogenesis of periodontitis. This group hypothesized that perhaps HGF/HGK TLR1/2 or TLR2/6 heterodimer complexes were triggered more readily instead of TLR4 in response to _E. coli_ LPS stimulation. Further in-depth studies, including experiments utilizing LPS from periodontaltopathogens, are warranted to clarify the current observations. In general, hypoxia and _E. coli_ LPS conferred somehow similar effects on HGK/HGF expression of DAMP sensors of interests (Table 4, Figure 3). However, there are limited reports documenting whether hypoxia and LPS act on periodontal resident cells independently or in any synergistic fashion [50; 51]. Further studies are, therefore, needed to decipher if hypoxia and LPS, the two elements likely to coexist in periodontal inflammation/infection, could influence each other.