High concentrations of Porphyromonas gingivalis-LPS downregulate Tlr4 and modulate phosphorylation of ERK and AKT in murine cementoblasts

Periodontitis is characterized as an inflammatory condition of the periodontium, the supportive structure of teeth consisting of gingiva, periodontal ligament (PDL), alveolar bone and cementum (Nanci and B., 2006, Chapple et al., 2018). Emerging from gingivitis, especially biofilm-induced gingivitis, periodontitis is associated with an irreversible loss of periodontal tissue. Surrounding oral bacteria, and especially Porphyromonas gingivalis (PG) as a key-pathobiont, represent a particular risk factor for periodontitis (Chapple et al., 2018, Socransky et al., 1998). At note, in the periodontitis, PG or other single bacteria are not the only cause of an exaggerated immune response. It can also be triggered by a dysbiosis in the microbial composition of oral biofilms.

Porphyromonas gingivalis (PG), a gram-negative anaerobic bacterial species of the phylum Bacteroidetes, is involved in the development and progression of periodontitis (Fiorillo et al., 2019, Mysak et al., 2014). Its released lipopolysaccharide (PG-LPS) acts as a potent virulence factor regarding gene expression in different cell types such as periodontal ligament (PDL) fibroblasts (Yiemwattana et al., 2018, Yu et al., 2019), gingival fibroblasts (Bozkurt et al., 2017) and cementoblasts (Bozkurt et al., 2021; Nemoto et al., 2006; Nociti et al., 2004).

Cementoblasts, which are located at the roots of teeth, are essential for cementogenesis. Cementum represents a mineralized tissue where periodontal ligament fibers insert to attach the tooth to alveolar bone (Arzate et al., 2015). PDL cells have been shown to regulate periodontal remodeling, a process which can be affected by PG-LPS. Current discussions also indicate that PDL cells may regulate tissue regeneration by controlling resorption though macrophage migration and osteoclastic differentiation (Meikle, 2006).

There are some studies which show the effects of PG-LPS in murine cementoblasts (S. B. Bozkurt et al., 2021; Nemoto et al., 2006; Nociti et al., 2004). Transcripts of inflammation-associated cytokines such as IL-1ß, IL-6, IL-8, IL-10, and IL-17 were (differentially-)regulated and metalloproteases MMP-1, MMP-2, MMP-3, TIMP-1, TIMP-2, with exception of MMP-9, were upregulated (Bozkurt et al., 2021). According to other studies, PG-LPS with penta- (PgLPS1690) acylated lipid A could rapidly and dramatically induce RANKL, IL-6, RANTES, and MIP1 upon stimulation, whereas tetra-(PgLPS1435/1449) could only induce slight changes. Moreover, PgLPS1690 significantly induced transcripts for NF-kB. The Nuclear Factor-kappa B (NF-κB) family of transcription factors regulates the expression of a wide range of genes critical for immune and inflammatory responses, cell survival, immune development, and cell proliferation (Solt and May, 2008). A pivotal regulator of all inducible NF-κB signaling pathways is the IκB kinase (IKK) complex that consists of two kinases (IKKα and IKKβ) and a regulatory subunit named NF-κB essential modulator (NEMO) (Solt and May, 2008). This activation was inhibited by pre-treatment with anti-toll like receptor (Tlr)-2 but not with Tlr-4 antibodies (Nemoto et al., 2006). Additionally, PG-LPS downregulated the expression of transcripts for osteocalcin (OCN) and RANKL, but dose-dependently upregulated the mRNA expression of osteopontin (OPN) and osteoprotegerin (OPG). Monoclonal antibody specific for mouse Tlr-4/MD-2 parcially neutralized the effect by PG-LPS on cementoblasts (Nociti et al., 2004). However, information about the intracellular signaling pathway including protein expression levels remain unknown.

Murine macrophages are known to be involved in pathogen recognition by the innate immune system (Akira and Takeda, 2004, Janeway and Medzhitov, 2002). Toll like receptors which are located on the surface of macrophages, monocytes and dendritic cells, work as pattern recognition receptors (PRR) modulating inflammatory immune responses during the initial sensing of infection (Kawai and Akira, 2010). Tlr4 functions as a receptor for LPS (Akira and Hoshino, 2003, Kawai and Akira, 2010, Nativel et al., 2017). After Tlr4 activation by LPS complex downstream pathways are initiated (Fang et al., 2017, Kawai and Akira, 2007). Of note, for PG-LPS a Tlr4-agonistic function might need higher concentrations compared to the regular e.g., E. coli-LPS. This is mainly due to the reduced number of Lipid A fatty acid chains and variable phosphate groups in PG-LPS (Qiu et al., 2021). Lipid A provides the anchor securing the molecule in the bacterial outer membrane. As such, Lipid A is a lipid component of an endotoxin held responsible for the toxicity of gram-negative bacteria. It is the innermost of the LPS and its hydrophobic nature allows it to anchor the LPS to the outer membrane. In an in-vivo situation with many other regular hexa- and penta-acylated LPS molecules, the less acylated and phosphorylated PG-LPS will even function as Tlr4-antagonist, as blocking LBP-, CD14- and Tlr4-MD-2 complex- binding sites. This leads to reduced killing of pathobionts and survival/selection of PG in particular (G. Hajishengallis, 2013). In RAW 264.7 macrophages, LPS stimulates the expression of Tlr4, myeloid differentiation factor 88 (MyD88) and phospho-AKT (Fang et al., 2017) as well as phospho-IκBα and mitogen-activated protein kinases (MAPK) phospho-ERK, phospho-JNK and phospho-p38 (Akanda et al., 2018, Ryu et al., 2012, Sharma et al., 2017, Youn et al., 2013).

Here, we want to explore the effect of PG-LPS focusing on intracellular signaling in cementoblasts via Tlr4. We follow potentially different downstream effects in the Tlr4-pathway of cementoblasts induced by PG-LPS via Tlr4 compared to the well-described intracellular signaling in RAW 264.7 macrophages which we used for control conditions. We explore the effect of high concentrations of PG-LPS on cell viability and morphology of cementoblasts. To understand the intracellular signaling in cementoblasts, we also investigate the possible downstream targets of Tlr4 in expression and phosphorylation status including IκBα, MyD88, pan AKT and the MAPK family consisting of ERK1/2, p38 and JNK. Although cementoblasts are not related to macrophages, we used RAW 264.7 cells as control cells because the effect of LPS in macrophages is well described.

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