Lipopolysaccharide from Porphyromonas gingivalis, but Not from Porphyromonas endodontalis, Induces Macrophage M1 Profile

Abstract

Apical Lesions of Endodontic Origin (ALEO) are initiated by polymicrobial endodontic canal infection. Porphyromonas gingivalis (Pg) and Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS) can induce a pro-inflammatory macrophage response through their recognition by TLR2 and TLR4. However, polarization responses induced by Pg and/or Pe LPS in macrophages are not fully understood. We aimed to characterize the polarization profiles of macrophages differentiated from THP-1 cells following Pg and/or Pe LPS stimulation from reference strain and clinical isolates. A modified LPS purification protocol was implemented and the electrophoretic LPS profiles were characterized. THP-1 human monocytes differentiated to macrophages were stimulated with Pg and Pe LPS. Polarization profiles were characterized through cell surface markers and secreted cytokines levels after 24 h of stimulation. TLR2 and TLR4 cell surfaces and transcriptional levels were determined after 24 or 2 h of LPS stimulation, respectively. LPS from Pg induced a predominant M1 profile in macrophages evidenced by changes in the expression of the surface marker CD64 and pro-inflammatory cytokine profiles, TNF-α, IL-1β, IL-6, and IL-12. Pe LPS was unable to induce a significant response. TLR2 and TLR4 expressions were neither modified by Pg or Pe LPS. Pg LPS, but not Pe LPS, induced a macrophage M1 Profile.

Lipopolysaccharides from Porphyromonas endodontalis and Porphyromonas gingivalis promote angiogenesis via Toll‐like‐receptors 2 and 4 pathways in vitro

“…Bacteria were inoculated in brain heart infusion supplemented with hemin and menadione (5 μg/mL), and cells were obtained in the late exponential phase. LPS were purified by a modified TRIzol extraction protocol (Veloso et al, 2022). In brief, the protocol involves additional steps, which included ultrasonication of bacterial pellets, incubation with 0.1 mg/mL of DNase (Sigma‐Aldrich), 0.2 mg/mL of RNase (Thermo Fisher) at 37°C overnight and 1 mg/mL Proteinase K at 60°C for 1.5 h (Veloso et al, 2022).…”

“…LPS were purified by a modified TRIzol extraction protocol (Veloso et al, 2022). In brief, the protocol involves additional steps, which included ultrasonication of bacterial pellets, incubation with 0.1 mg/mL of DNase (Sigma‐Aldrich), 0.2 mg/mL of RNase (Thermo Fisher) at 37°C overnight and 1 mg/mL Proteinase K at 60°C for 1.5 h (Veloso et al, 2022). The presence of proteins was ruled out through Bradford assay (detection limit, 0.1 mg/mL) and nucleic acids by agarose gel red.…”

“…Interestingly, these variable regions seem to participate in the progression of inflammatory diseases, triggering a variety of signalling pathways in periodontal cells (Olsen & Singhrao, 2018). In this context, the LPS from P. endodontalis and P. gingivalis have been shown to exacerbate the host's inflammatory response (Ogura et al, 1994; Veloso et al, 2022) and promote osteoclastogenesis and bone resorption (Guo et al, 2014).…”

Porphyromonas gingivalis regulates atherosclerosis through an immune pathway

“…P. gingivalis LPS 1690 with penta-acylated lipid A mainly activates the NF-κB signaling pathway, while P. gingivalis LPS 1435/1449 with tetra-acylated lipid A mainly induces p38/MAPK and ERK1/2 signaling pathways ( 74 ). P. gingivalis LPS induces M1-type polarization in macrophages and promotes the expression of several pro-inflammatory factors, such as TNF-α, IL-1β, and IL-6 ( 75 ). P. gingivalis LPS promotes the progression of periodontal inflammation by inducing pyroptosis in gingival fibroblasts ( 76 ).…”

“…Animal studies have shown that oral inoculation of P. gingivalis can activate NLRP3 inflammasomes in macrophages in AS plaques in a gingipains-independent manner, promote the release of IL-1β and TNF-α, and accelerate AS progression ( 47 , 148 ). In vitro studies have revealed that P. gingivalis LPS may induce M1-type polarization of macrophages via pattern recognition receptor triggering receptors expressed on myeloid cells-1(TREM-1) and its downstream signaling pathways to promote the secretion of multiple inflammatory factors and accelerate AS progression ( 75 , 156 , 157 ) ( Figure 1 ). However, recent studies have also indicated that P. gingivalis LPS-induced tolerant macrophages represent an intermediate state between M1/M2 polarization and function as M2-like cells to limit the inflammatory response ( 158 ), suggesting that P. gingivalis LPS tolerant macrophages may be closely related to tissue repair.…”