Lipopolysaccharide Preparation Derived From Porphyromonas gingivalis Induces a Weaker Immuno-Inflammatory Response in BV-2 Microglial Cells Than Escherichia coli by Differentially Activating TLR2/4-Mediated NF-κB/STAT3 Signaling Pathways

Alzheimer’s disease (AD) is a degenerative disease of the central nervous system with unclear etiology and pathogenesis. In recent years, as the infectious theory and endotoxin hypothesis of AD has gained substantial attention, several studies have proposed that Porphyromonas gingivalis (P. gingivalis), one of the main pathogenic bacteria of chronic periodontitis, and the lipopolysaccharide (LPS) of P. gingivalis may lead to AD-like pathological changes and cognition impairment. However, research on the relationship between P. gingivalis-LPS and neuroinflammation is still lacking. Our study aimed to investigate the effects of P. gingivalis-LPS preparation on immuno-inflammation in microglial cells and further compared the differential inflammatory response induced by P. gingivalis-LPS and Escherichia coli (E. coli) LPS preparations. The results showed that P. gingivalis-LPS could upregulate the gene expression and release of pro-inflammatory factors in BV-2 microglial cells, including IL-1β, IL-6, TNF-α, IL-17, and IL-23. We also observed an increase in the level of Toll-like receptor 2/4 (TLR2/4) and NF-κB/STAT3 signaling. Moreover, the changes mentioned above were more significant in the E. coli-LPS group and the effects of both kinds of LPS could be differentially reversed by the administration of the TLR2 inhibitor C29 and TLR4 inhibitor TAK-242. The molecular simulation showed that the binding affinity of P. gingivalis-lipid A to TLR4-MD-2 was weaker than E. coli-lipid A, which was probably due to the presence of fewer acyl chains and phosphate groups of P. gingivalis-lipid A than E. coli-lipid A. We conclude that P. gingivalis-LPS could activate TLR2/4-mediated NF-κB/STAT3 signaling pathways, which ultimately resulted in an immune-inflammatory response in BV-2 microglia. In contrast to E. coli-LPS, P. gingivalis-LPS is a weaker TLR2/4 agonist and NF-κB/STAT3 signaling activator. Furthermore, the different fatty acid chains and phosphate groups between P. gingivalis-lipid A and E. coli-lipid A may be the reason for the weaker activating properties of P. gingivalis-LPS.


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To explore the effects of Ultrapure-LPS, Pam3CSK4 and heat-killed bacteria on IL-1β, IL-6, and TNFα levels in BV-2 microglial cells, an enzyme-linked immunosorbent assay (ELISA) was performed.
The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane after separation. A wide range of protein markers was run in parallel to detect the molecular weight of proteins. A 5% skimmed milk solution was used for membrane blockage to reduce nonspecific binding. Proteins were probed with specific antibodies and images were quantified using ImageJ 1.52a software (National Instituted of Health, Bethesda, MD, USA).

Effects of Ultrapure-LPS on the expression of Inflammatory cytokine and phosphorylation
of NF-κB p65 in BV-2 microglial cells.
After 1µg/mL Ultrapure-LPS simulation for 6h, the P. gingivalis-LPS Ultrapure group showed an approximately 1.30-, 1.37-, or 1.25-fold increase in IL-1β, IL-6, or TNF-α gene upregulation in comparison with the control group, respectively ( Figure 1S A-C). Meanwhile, the expression of pro-IL-1β, IL-6, and TNF-α protein in the P. gingivalis-LPS Ultrapure group was significantly higher than that in the control group ( Figure 1S D-F). In contrast, the E. coli-LPS Ultrapure group showed an 3 approximately 85-, 57-, or 16-fold increase in IL-1β, IL-6, or TNF-α gene upregulation compared with the control group, and the expression of pro-IL-1β, IL-6, and TNF-α protein in the E. coli-LPS Ultrapure group was significantly higher than that of the control group. Significant differences were observed between the P. gingivalis-LPS Ultrapure group and E. coli-LPS Ultrapure group as mentioned above ( Figure 1S A-F). Furthermore, the Ultrapure-LPS plus TAK-242 groups showed downregulation of IL-1β, IL-6, TNF-α mRNA and protein expression in comparison to the Ultrapure-LPS group (Figure 1S A-F).
Moreover, the elevated expression of phosphor(p)-p65/p65 protein induced by LPS-Ultrapure was attenuated by the TLR4 inhibitor TAK-242 rather than the TLR2 inhibitor C29 at 6h (Figure 1S G). Meanwhile, significant differences of p-p65/p65 protein expression were observed between the P. gingivalis-LPS Ultrapure group and E. coli-LPS Ultrapure group at 6h (Figure 1S G).

NF-κB p65 in BV-2 microglial cells.
After 1µg/mL Pam3CSK4 simulation for 6h, the Pam3CSK4 group showed an approximately 60-, 57-, or 15-fold increase in IL-1β, IL-6, or TNF-α gene upregulation in comparison with the control group, respectively ( Figure 2S A-C). Meanwhile, the expression of pro-IL-1β, IL-6, and TNF-α protein in the Pam3CSK4 group was significantly higher than that in the control group ( Figure  Moreover, the elevated expression of p-p65/p65 protein induced by Pam3CSK4 was attenuated by the TLR2 inhibitor C29 rather than the TLR4 inhibitor TAK-242 at 6h (Figure 2S G).

Effects of heat-killed bacteria on the expression of Inflammatory cytokine and phosphorylation of NF-κB p65 in BV-2 microglial cells.
After heat-killed bacteria (MOI of 100) simulation for 6h, the heat-killed P. gingivalis group showed an approximately 13-, 34-, or 3-fold increase in IL-1β, IL-6, or TNF-α gene upregulation in comparison with the control group, respectively ( Figure 3S A-C). Meanwhile, the expression of pro-IL-1β, IL-6, and TNF-α protein in the heat-killed P. gingivalis group was significantly higher than that in the control group ( Figure 3S D-F). In contrast, the heat-killed E. coli group showed an approximately 238-, 247-, or 10-fold increase in IL-1β, IL-6, or TNF-α gene upregulation compared with the control group, and the expression of pro-IL-1β, IL-6, and TNF-α protein in the heat-killed E. coli group was significantly Supplementary Material 4 higher than that of the control group. Significant differences were observed between the heat-killed P.
gingivalis group and heat-killed E. coli group as mentioned above ( Figure 3S A-F). Furthermore, the heat-killed bacteria plus TAK-242 groups and heat-killed bacteria plus C29 groups showed downregulation of IL-1β, IL-6, TNF-α mRNA and protein expression in comparison to the heat-killed bacteria group. (Figure 3S A-F).
Moreover, the elevated expression of p-p65/p65 protein induced by heat-killed bacteria was attenuated by the TLR4 inhibitor TAK-242 or TLR2 inhibitor C29 at 6h (Figure 3S G). Meanwhile, significant differences of p-p65/p65 protein expression were observed between the heat-killed P.
gingivalis group and heat-killed E. coli group at 6h (Figure 3S G).

Mature-IL-1β release in Standard LPS-stimulated BV-2 microglial cells
As shown in Figure 5S, ELISA was used to measure levels of mature-IL-1β using the cell medium.
The result indicates that mature IL-1β was not visible in cell medium.

Figure S4. Inflammatory cytokine gene expression in P. gingivalis-LPS Standard and E. coli-LPS
Standard stimulated BV-2 microglial cells. BV-2 microglial cells were treated with 1µg/ml Standard-LPS for 0-24h, and RT-PCR was performed; two-way ANOVA, *p<0.05, **p<0.01, and ***p<0.001 compared to the 0hr group. Data from three independent experiments are presented as mean ± SD. (100µM) or serum-free medium for 60min, following by treatment with 1µg/ml Standard-LPS or serum-free medium for 6h, and mature-IL-1β level were measured using ELISA kits. Data from three independent experiments are presented as mean ± SD.
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