Lipopolysaccharides-Induced Suppression of Innate-Like B Cell Apoptosis Is Enhanced by CpG Oligodeoxynucleotide and Requires Toll-Like Receptors 2 and 4

Abstract

Innate-like B lymphocytes play an important role in innate immunity in periodontal disease through Toll-like receptor (TLR) signaling. However, it is unknown how innate-like B cell apoptosis is affected by the periodontal infection-associated innate signals. This study is to determine the effects of two major TLR ligands, lipopolysaccharide (LPS) and CpG-oligodeoxynucleotides (CpG-ODN), on innate-like B cell apoptosis. Spleen B cells were isolated from wild type (WT), TLR2 knockout (KO) and TLR4 KO mice and cultured with E. coli LPS alone, P. gingivalis LPS alone, or combined with CpG-ODN for 2 days. B cell apoptosis and expressions of specific apoptosis-related genes were analyzed by flow cytometry and real-time PCR respectively. P. gingivalis LPS, but not E. coli LPS, reduced the percentage of AnnexinV+/7-AAD- cells within IgMhighCD23lowCD43-CD93- marginal zone (MZ) B cell sub-population and IgMhighCD23lowCD43+CD93+ innate response activator (IRA) B cell sub-population in WT but not TLR2KO or TLR4KO mice. CpG-ODN combined with P. gingivalis LPS further reduced the percentage of AnnexinV+/7-AAD- cells within MZ B cells and IRA B cells in WT but not TLR2 KO or TLR4 KO mice. Pro-apoptotic CASP4, CASP9 and Dapk1 were significantly down-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT but not TLR2 KO or TLR4 KO mice. Anti-apoptotic IL-10 was significantly up-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT and TLR2 KO but not TLR4 KO mice. These results suggested that both TLR2 and TLR4 signaling are required for P. gingivalis LPS-induced, CpG-ODN-enhanced suppression of innate-like B cell apoptosis.

Fig 1. B cell proliferation after E. coli LPS, P. gingivalis LPS and CpG-ODN treatment.

Splenocyte B cells were separated from WT, TLR2 KO and TLR4 KO mice and cultured 48 hours with E. coli LPS (10μg/ml), P. gingivalis LPS (10μg/ml), E. coli LPS (10μg/ml) + CpG (10μM), and P. gingivalis LPS (10μg/ml) + CpG (10μM). Viable cells quantities were measured by absorbance at 490 nm reading from each group of WT, TLR2 KO and TLR4 KO mice respectively (A) (mean±SE, n = 6, *p<0.05, **p<0.01). Proliferation cells quantities were also measured by CellTrace CSFE staining and presented as percentages of total cells (B). (mean±SE, n = 3, *p<0.05, **p<0.01).

Fig 2. B cell early apoptosis and late apoptosis after E. coli LPS, P. gingivalis LPS and CpG-ODN treatment.

Splenocyte B cells were separated from WT, TLR2 KO and TLR4 KO mice and cultured 48 hours with E. coli LPS (10μg/ml), P. gingivalis LPS (10μg/ml), E. coli LPS (10μg/ml) + CpG (10μM), and P. gingivalis LPS (10μg/ml) + CpG (10μM). Cells were then stained by FITC-conjugated AnnexinV mAb and 7-AAD and measured by flow cytometry (A). Percentage of Annexin V⁺7-AAD^- cells (B) and Annexin V⁺7-AAD⁺ cells (C) in different treatment groups of WT, TLR2KO and TLR4KO mice were analyzed and compared respectively (mean±SE, n = 6, *p<0.05, **p<0.01).

Fig 3. Frequencies of Innate-like B cell subsets after E. coli LPS, P. gingivalis LPS and CpG-ODN treatment.

Splenocyte B cells were separated from WT mice and cultured 48 hours with E. coli LPS (10μg/ml), P. gingivalis LPS (10μg/ml), E. coli LPS (10μg/ml) + CpG (10μM), and P. gingivalis LPS (10μg/ml) + CpG (10μM). Cells were then stained by PE-conjugated anti IgM, PerCP-Cy5.5-conjugated anti CD23, APC-conjugated anti CD93, Pacific Blue-conjugated anti CD43 and measured by flow cytometry (A). Within overall innate-like B cell (IgM^highCD23^low B cells) population, the percentage of CD43^-CD93^- marginal zone B cells (B), CD43^-CD93⁺ transitional B cells (C), CD43⁺CD93⁺ innate response activator B cells (D) and CD43⁺CD93^- B1 B cells (E) in different treatment groups were analyzed and compared respectively (mean±SE, n = 4, *p<0.05, **p<0.01).

Fig 4. Early apoptosis analysis of innate-like B cell subsets after E. coli LPS, P. gingivalis LPS and CpG-ODN treatment.

Splenocyte B cells were separated from WT, TLR2 KO and TLR4 KO mice and cultured 48 hours with E. coli LPS (10μg/ml), P. gingivalis LPS (10μg/ml), E. coli LPS (10μg/ml) + CpG (10μM), and P. gingivalis LPS (10μg/ml) + CpG (10μM). Cells were then stained by FITC-conjugated AnnexinV, 7-AAD, PE-conjugated anti IgM, PerCP-Cy5.5-conjugated anti CD23, APC-conjugated anti CD93, Pacific Blue-conjugated anti CD43 and measured by flow cytometry. In different innate-like B cell subsets including CD43^-CD93^- marginal zone B cells (A), CD43^-CD93⁺ transitional B cells (B), CD43⁺CD93⁺ innate response activator B cells (C) and CD43⁺CD93^- B1 B cells (D), the percentage of AnnexinV⁺/7-AAD^- (early apoptotic) B cells in different treatment groups of WT, TLR2 KO and TLR4 KO mice were analyzed and compared respectively (mean±SE, n = 5, *p<0.05, **p<0.01).

Fig 5. Differential mRNA levels of apoptosis-related genes in B cells after P. gingivalis LPS and CpG-ODN treatment.

Splenocyte B cells were separated from WT, TLR2 KO and TLR4 KO mice and cultured 48 hours with P. gingivalis LPS (10μg/ml) and P. gingivalis LPS (10μg/ml) + CpG (10μM). Total RNA was extracted from these cells and used for RT² Profiler^™ PCR Array Mouse Apoptosis (A). mRNA levels of Casp4 (B), Casp9 (C), Dapk1 (D) and IL-10 (E) in different groups of WT, TLR2 KO and TLR4 KO mice were determined by real-time PCR and the ratio of each treatment group to control group were analyzed and compared respectively (mean±SE, n = 3, *p<0.05, **p<0.01). Total cell lysis were used to detect Casp4 activity (F) and Casp9 activity (G) using fluorometric Assay kits. The ratio of each treatment group to control group were analyzed and compared respectively (mean±SE, n = 3, *p<0.05, **p<0.01).