Mechanism and Regulatory Function of CpG Signaling via Scavenger Receptor B1 in Primary B Cells

Abstract

It is well established that CpG promotes pro-inflammatory cytokine and antibody production by B cells via the Toll-like receptor 9 (TLR9)-dependent pathway. However, scavenger receptors (SRs) are also capable of binding such pathogen-derived molecules, yet their contribution to CpG-induced signaling events has not yet been evaluated. Here we identified a novel TLR9-independent mechanism of CpG-induced signaling and immune function that is mediated by the scavenger B1 receptor (SR-B1). Specifically, we show that CpG/SR-B1 triggers calcium entry into primary B lymphocytes via phospholipase C␥-1-mediated activation of TRPC3 channels and also B cell adhesion to vascular cell adhesion molecule-1. CpG-induced calcium signals and vascular cell adhesion molecule-1 adhesion are TLR9-independent and are mediated exclusively by SR-B1. Although proinflammatory cytokine and Ig production induced by CpG require TLR9 expression, we also found that SR-B1 negatively regulates TLR9-dependent production of interleukin-6, interleukin-10, and IgM. Thus, our results provide a novel perspective on the complexity of CpG signaling within B cells by demonstrating that SR-B1 is an alternative pathway for nucleic acid-induced signaling that provides feedback inhibition on specific TLR9-dependent responses of B cells. Consequently, these results have wide implications for understanding the mechanisms regulating immune tolerance to nucleic acids and pathogen-associated molecules.Stimulus-induced dynamic changes in the concentration of cytoplasmic calcium are primary determinants of the activation, immunological function, and developmental fate of lymphocytes. Calcium signaling through the B cell antigen receptor (BCR) 2 complex is initiated by the activation of proximal tyrosine kinases Lyn and Syk, which phosphorylate the adaptor BLNK to facilitate its association with and activation of PLC␥-2. PLC␥2 hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and 1,4,5-inositol trisphosphate (IP 3 ) (for review see Ref. 1), which activates IP 3 receptor/channels that mediate Ca 2ϩ release from endoplasmic reticulum into the cytosol (2) (for review see Refs. 3, 4). Ca 2ϩ release from endoplasmic reticulum stores and the resulting depletion of Ca 2ϩ(not an increase in cytoplasmic [Ca 2ϩ ]) are the central and prerequisite events required to activate plasma membrane "storeoperated" calcium release-activated calcium (CRAC) channels.CRAC channels are responsible for antigen receptor-triggered calcium entry; however, a growing body of evidence suggests that CRAC channels do not underlie all the diverse calcium-regulated responses of lymphocytes, particularly those triggered by innate stimuli. For example, we previously identified several calcium-permeant non-selective cation channels (NSCCs) that are uniquely activated by distinct arachidonic acid-derived (eicosanoid) inflammatory mediators and by mechanical stimuli (5-7). Thus, multiple calcium-permeant channels with distinct activation mechanisms may underlie stimulus-specific calciu...

CpG-Oligodeoxynucleotide Inhibits Smad-Dependent Bone Morphogenetic Protein Signaling: Effects on Myeloma Cell Apoptosis and In Vitro Osteoblastogenesis

“…D, Levels of phosphorylated Smads 1/5/8 in total cell extracts from INA-6 cells treated with BMP-6 (50 ng/ml), PTO-CpG-ODN (2 mM), fucoidan (1 mg/ml), poly (I) (10 mg/ml), and dextran sulfate (20 mg/ml) as indicated for 1 h. Percentages of viable nonstained cells are presented, and error bars represent 1 SD of duplicate measurements (A, C). TLR9 is considered the major receptor for CpG-ODN, although a few studies have described TLR9-independent effects of CpG-ODN (10,11,28,52). We found that PTO-CpG-ODN and PTOGpC-ODN to the same extent inhibited BMP-induced osteoblast differentiation, BMP-induced apoptosis of myeloma cells and Smadsignaling downstream of BMP.…”

Exploiting scavenger receptors in cancer immunotherapy: Lessons from CD5 and SR‐B1

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“…This is the case of CpG oligonucleotides that trigger SR-B1-dependent Ca 2+ entry ( Fig. 2A) [90]. On the other hand, the signaling mediated by SR-B1 can lead to the opposite immune reaction with the release of potent pro-inflammatory cytokines and chemokines such as tumor necrosis factor-α, IL-1β, and IL-8, thus contributing to the fine-tuning of the immune response (Fig.…”

Benzo-fused lactams from a diversity-oriented synthesis (DOS) library as inhibitors of scavenger receptor BI (SR-BI)-mediated lipid uptake

“…Though it is now recognized as an important target for the study of lipid metabolism and infectious disease, as well as immune response 12–14 and female fertility, 3 the structure and mechanism of SR-BI is not understood in full detail. 15–18 In an effort to identify novel small molecule modulators of this receptor that may exert unique effects on lipid transport, as well as more potent and less toxic compounds, we embarked on a high-throughput screen (HTS) supported by the NIH Molecular Libraries Program (MLP).…”