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.
Highlights
Innate immune system recognizes pathogen-associated molecular patterns with a set of germline-encoded pattern-recognition receptors including Toll-like receptors (TLRs) [1, 2]
To test the innate proliferative property of B cells in response to the TLRs stimulation, purified B cells were cultured under 5 different conditions and cell proliferation assays were performed after 48 hours
To confirm the MTS proliferation results, cell proliferations of each groups were measured by CellTrace CSFE cell proliferation assay and similar results were observed, demonstrating that the addition of CpG oligodeoxynucleotides (CpG-ODN) together with LPS significantly elevated the proliferation of B cells as compared to those treated with LPS alone (Fig 1B)
Summary
Innate immune system recognizes pathogen-associated molecular patterns with a set of germline-encoded pattern-recognition receptors including Toll-like receptors (TLRs) [1, 2]. As multiple TLRs could be activated simultaneously by their corresponding ligands during immune response to pathogens in diseases, the effect of co-activation of these TLR pathways on B cell apoptosis has not been investigated. Different from E. coli LPS, which is a definitive TLR4 ligand, P. gingivalis LPS has been shown to be able to activate both TLR2 and TLR4 [11, 12]. Together with the ligation between bacterial DNA component CpG oligodeoxynucleotides (CpG-ODN) and its receptor TLR9 during P. gingivalis infection, it is valuable to determine the effects of multiple TLR activation (TLR2, TLR4 and TLR9) in the regulation of immune B cell functions in order to understand the role of TLR signaling in infection-associated periodontal pathogenesis
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