Recognition of nucleic acids by TLR9 requires its trafficking from the endoplasmic reticulum to endolysosomal compartments and its subsequent proteolytic processing. Both processes depend on interactions of TLR9 with the polytopic endoplasmic reticulum–resident protein UNC93B1. To examine the intracellular behavior of TLR9 in primary APCs, we generated transgenic mice expressing a TLR9-GFP fusion. The TLR9-GFP transgene is functional and is proteolytically processed in resting bone marrow–derived macrophages (BMDMs), dendritic cells, and B cells. Inhibition of cleavage impairs TLR9-dependent responses in all primary APCs analyzed. The kinetics of TLR9-GFP processing in BMDMs and B cells differs: in B cells, proteolysis occurs at a faster rate, consistent with an almost exclusive localization to endolysosomes at the resting state. In contrast to the joint requirement for cathepsins L and S for TLR9 cleavage in macrophages, TLR9-GFP cleavage depends on cathepsin L activity in B cells. As expected, in BMDMs and B cells from UNC93B1 (3d) mutant mice, cleavage of TLR9-GFP is essentially blocked, and the expression level of UNC93B1 appears tightly correlated with TLR9-GFP cleavage. We conclude that proteolysis is a universal requirement for TLR9 activation in the primary cell types tested, however the cathepsin requirement, rate of cleavage, and intracellular behavior of TLR9 varies. The observed differences in trafficking indicate the possibility of distinct modes of endosomal content sampling to facilitate initiation of TLR-driven responses in APCs.

Synthetic oligonucleotides containing CpG motifs (CpG ODNs) have been shown to induce proliferation, differentiation and cytokine production in B cells, macrophages and DCs through a TLR9-dependent mechanism. A class (CpG-A) and B class (CpG-B) ODNs display distinct physical properties. CpG-A, but not CpG-B, can multimerize to form exceedingly large lattices. CpG-A cannot effectively activate B cells but does induce pDCs to produce high levels of IFNα, while CpG-B is a potent B cell mitogen. Here we report that CpG-A is internalized by B cells, and CpG-A and CpG-B accumulate to distinct intracellular compartments. When present in the form of an immune complex (CpG-A IC), CpG-A is taken up more efficiently by AM14 IgG2a-specific B cells, and elicits a robust TLR9-dependent B cell proliferative response. B cells proliferating comparably and in a TLR9-dependent fashion in response to CpG-A IC and CpG-B exhibited distinct cytokine profiles. CpG-A IC induced enhanced production of RANTES and markedly reduced levels of IL-6 when compared to CpG-B. We also found that engagement of the AM14 BCR by a protein IC, which cannot by itself induce proliferation, promoted TLR9-dependent but BCR-independent proliferation by bystander CpG-A or fragments of mammalian dsDNA. These data identify direct and indirect mechanisms by which BCR engagement facilitates access of exogenous ligands to TLR9-associated compartments and subsequent B cell activation.

Summary

Crosslinking of FcγRIIB and the BCR by immune complexes (ICs) can downregulate antigen-specific B cell responses. Accordingly, FcγRIIB deficiencies have been associated with B cell hyperactivity in patients with SLE and mouse models of lupus. However, we have previously shown that murine IgG2a-autoreactive AM14 B cells respond robustly to chromatin- associated ICs through a mechanism dependent on both the BCR and endosomal TLR9, despite FcγRIIB coexpression. To further evaluate the potential contribution of FcγRIIB to the regulation of autoreactive B cells, we have now compared the IC-triggered responses of FcγRIIB-deficient and FcγRIIB-sufficient AM14 B cells. We find that FcγRIIB-deficient cells respond significantly better than FcγRIIB-sufficient cells when stimulated with DNA ICs that incorporate low affinity TLR9 ligand (CG-poor dsDNA fragments). AM14 B cells also respond to RNA-associated ICs through BCR/TLR7 coengagement, but such BCR/TLR7 dependent responses are normally highly dependent on IFNα costimulation. However, we now show that AM14 FcγRIIB-/- B cells are very effectively activated by RNA ICs without supplemental IFNα priming. These results demonstrate that FcγRIIB can effectively modulate both BCR/TLR9 and BCR/TLR7 endosomal-dependent activation of autoreactive B cells.

Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive BCR transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1/RAGE interaction was important for this response [1]. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1/DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE+ and RAGE− AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.