Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

Abstract IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.


Introduction
IL-10 expressing regulatory B cells (B10) is a humans 27 . B10 cell down-regulates autoimmune IL-10 expression, playing crucial regulatory roles in innate and adaptive immunity 27 . Though mouse B10 they have been found to be predominantly enriched in spleen CD1d high CD5 + B cells 27 . recognition receptors, are specialized transmembrane proteins that mediate innate immunity through detecting common structures of many microbial species such as bacterial lipopolysaccharides (LPS) or viral nucleic acids 17,25 . Upon recognition of a pathogen, TLRs initiate a signaling cascade that 8,21 .
Porphyromonas gingivalis (P. gingivalis) LPS has been shown to be able to activate both TLR2 and TLR4 due to its unique structure and function 2,6 , and CpG responses 9,16 .
Interaction between CD40 Ligand (CD40L) and CD40 plays an important role in the initiation and progression of cellular and humoral adaptive immunity 15 . The activation of CD40 on B cells by CD40L is crucial for T cell-dependent B cell proliferation, differentiation, and antibody isotype switching 11,13,14 .
Recent studies demonstrated that culturing spleen B cells with LPS or CD40L for 48 h induced 10 production in B cells than control in vitro 20 . LPS stimulation of spleen B cells for 24 h induced more IL-10 than unstimulated cells 28

R21DE021837 and R56DE023807.
Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells