Porphyromonas gingivalis lipopolysaccharide promotes T-hel per17 cell differentiation by upregulating Delta-like ligand 4 expression on CD14+ monocytes

Abstract

Backgroud: To investigate the effect and mechanism of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on Th17 cell differentiation mediated by CD14⁺ monocytes.

Methods: P. gingivalis LPS-activated CD14⁺ monocytes were co-cultured with CD4⁺T cells in different cell ratios. An indirect co-culture system was also established using transwell chambers. Furthermore, anti- Delta-like ligand 4 (Dll-4) antibody was used to investigate the role of Dll-4 in Th17 cell response. The mRNA expression was analyzed using quantitative reverse transcription-polymerase chain reaction, and secreted cytokines in culture supernatant were detected using enzyme-linked immunosorbent assay. Flow cytometry was used to determine the frequencies of Th17 cells. IL-17 protein expression levels were determined using western blotting assay.

Results: P. gingivalis LPS increased the expressions of interleukin (IL)-1β, IL-6, IL-23 and transforming growth factor (TGF)-β in CD14⁺ monocytes. Th17 cell frequency upregulated, which is not solely cytokine-dependent but rather requires cell-cell contact with activated monocytes, particularly in the 1:10 cell ratio. Furthermore, P. gingivalis LPS increased t he expression of Dll-4 on CD14⁺ monocytes, whereas the anti- Dll-4 a ntibody decreased the response of Th17 cells. The results suggest that P. gingivalis LPS enhances Th17 cell response via Dll-4 upregulation on CD14⁺ monocytes.

Keywords: Delta-like ligand 4; Lipopolysaccharide; Monocytes; Porphyromonas gingivalis; Th17 cells.

Figure 1. Effect of P. gingivalis lipopolysaccharide-treated CD14⁺ monocytes on Th17 cell differentiation.

mRNA expression levels of IL-17 (A-C) and ROR C (D-F) in the presence or absence of P. gingivalis lipopolysaccharide were determined using qRT-PCR. (G) Dot plots show the proportion of Th17 cell differentiation determined by using flow cytometry. (H-J) Frequencies of Th17 cells in each of the three co-culture ratios analyzed using flow cytometry. Data are presented as mean ±SD of triplicate assays for six independent experiments (n = 6). CD14⁺ monocytes (Mo), lipopolysaccharide (LPS), retinoid-related orphan receptor C (RORC), no significant difference (ns), * p < 0.05, ** p < 0.01.

Figure 2. Expression of IL-1β, IL-6, TGF-β, and IL-23 in P. gingivalis lipopolysaccharide-treated CD14⁺ monocytes.

mRNA expression levels of (A) IL-1β (A), IL-6 (B), TGF-β (C), and IL-23 (D) determined using qRT-PCR. Levels of IL-1β (E), IL-6 (F), TGF-β (G), and IL-23 (H) secreted by CD14⁺ monocytes in the presence or absence of P. gingivalis lipopolysaccharide detected using ELISA. Data are presented as mean ± SD of triplicate assays for six independent experiments (n = 6). P. gingivalis lipopolysaccharide (Pg-LPS), * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3. Role of cell–cell contact in the Th17 cell response induced by P. gingivalis lipopolysaccharide-activated CD14⁺ monocytes.

CD14⁺ monocytes and CD4⁺ T cells were cultured in transwells for 3 days, and the expression of RORC (A) and IL-17 (B) mRNA in the presence or absence of P. gingivalis lipopolysaccharide was determined. (C) Dot plots show the proportion of Th17 cell differentiation determined using flow cytometry. (D) Frequency of Th17 cells in each group determined by using flow cytometry. Data are presented as mean ± SD of triplicate assays for six independent experiments (n = 6). Transwell (TW), lipopolysaccharide (LPS), retinoid-related orphan receptor C (RORC), no significant difference (ns).

Figure 4. Expression of Jagged-1 (A) and Dll-4 (B) mRNA in the presence or absence of P. gingivalis lipopolysaccharide.

Data are presented as mean ± SD of triplicate assays for six independent experiments (n = 6). P. gingivalis lipopolysaccharide (Pg-LPS), Delta-like ligand 4 (Dll-4), no significant difference (ns), ** p < 0.01.

Figure 5. Role of Dll-4 in the Th17 cell differentiation induced by P. gingivalis lipopolysaccharide-activated CD14⁺ monocytes.

CD14⁺ monocytes were pretreated with 5 and 10 µg/mL anti-Dll-4 antibody, for 90 min prior to addition of the P. gingivalis lipopolysaccharide. The cells were then cocultured with the activated CD4⁺ T cells for 5 days. (A) Dot plots show the proportion of Th17 cell differentiation determined using flow cytometry. (B) Frequency of Th17 cells in each group determined using flow cytometry. (C) Protein level of IL-17 detected using western blotting. GAPDH was used as the protein loading control. (D) The intensified bands of IL-17 were measured using ImageJ software and were normalized to GADPH. (E) IL-17 concentration in the supernatant of the co-cultured cells measured after 5 days using ELISA. Data are presented as mean ± SD of triplicate assays for six independent experiments (n = 6). P. gingivalis lipopolysaccharide (Pg-LPS), Delta-like ligand 4 (Dll-4), * p < 0.05, ** p < 0.01, *** p < 0.001.