Gregory A. Viglianti

RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603–607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837–847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-α, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.

Activation of autoreactive B cells by CpG dsDNA.

The proliferative response of autoreactive rheumatoid factor (RF) B cells to mammalian chromatin-containing immune complexes (ICs) results from the sequential engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9). We have used ICs constructed from anti-hapten antibodies and defined haptenated dsDNA fragments to determine the form of mammalian DNA that mediates this process. Despite their relatively low abundance in mammalian DNA, we found that inclusion of hypomethylated CpG motifs in these ICs was necessary for effective activation. In the absence of antibody, the same fragments could efficiently stimulate low-affinity hapten-specific and DNA-reactive 3H9 B cells, but not RF B cells. These results extend the BCR/TLR9 coengagement paradigm to a second major class of autoreactive B cells, further confirm the critical role of the BCR in chromatin ligand delivery to TLR9, and implicate hypomethylated CpG motifs as ligand elements necessary for the initiation of systemic autoimmune disease.

Toll-like receptors, endogenous ligands, and systemic autoimmune disease.

Summary:  The critical role of Toll‐like receptors (TLRs) as mediators of pathogen recognition by the innate immune system is now firmly established. Such recognition results in the initiation of an inflammatory immune response and subsequent instruction of the adaptive immune system, both of which are designed to rid the host of the invading pathogen. More controversial is the potential role of TLRs in the recognition of endogenous ligands and what effect this might have on the consequent development of autoimmune or other chronic sterile inflammatory disorders. An increasing number of studies implicate TLRs as being involved in the immune response to self‐molecules that have in some way been altered from their native state or accumulate in non‐physiologic sites or amounts, although questions have been raised about possible contaminants in certain of these studies. In this review, we discuss the evidence for endogenous ligand–TLR interactions with particular emphasis on mammalian chromatin, systemic lupus erythematosus, and atherosclerosis. Overall, the data support the general concept of a role for TLRs in the recognition of endogenous ligands. However, the precise details of the interactions and the extent to which they may contribute to the pathogenesis of human disease remain to be clarified.

Requirement for DNA CpG Content in TLR9-Dependent Dendritic Cell Activation Induced by DNA-Containing Immune Complexes

Although TLR9 was originally thought to specifically recognize microbial DNA, it is now evident that mammalian DNA can be an effective TLR9 ligand. However, the DNA sequence required for TLR9 activation is controversial, as studies have shown conflicting results depending on the nature of the DNA backbone, the route of DNA uptake, and the cell type being studied. In systemic lupus erythematosus, a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DCs), is through uptake as a DNA-containing immune complex. In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated CpG dinucleotides within dsDNA are required for murine DC TLR9 activation induced by a DNA-containing immune complex. The strongest activation is seen with dsDNA fragments containing optimal CpG motifs (purine-purine-CpG-pyrimidine-pyrimidine) that are common in microbial DNA but rare in mammalian DNA. Importantly, however, activation can also be induced by CpG-rich DNA fragments that lack these optimal CpG motifs and that we show are plentiful in CpG islands within mammalian DNA. No activation is induced by DNA fragments lacking CpG dinucleotides, although this CpG-free DNA can induce DC activation if internalized by liposomal transfection instead of as an immune complex. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis in which activation of TLR9 in DCs by self DNA has been implicated in disease pathogenesis.

Autoreactive B Cells Discriminate CpG-Rich and CpG-Poor DNA and This Response Is Modulated by IFN-α

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs) through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a-reactive B cells. In contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR crosslinking alone is insufficient to activate low-affinity autoreactive B cells. Importantly, priming B cells with IFN-α lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Taken together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-α can contribute to the pathogenesis of systemic lupus erythematosus.

PPARγ and LXR Signaling Inhibit Dendritic Cell-Mediated HIV-1 Capture and trans-Infection

Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARγ and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARγ and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARγ and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission.

Comparison of CpG s-ODNs, chromatin immune complexes, and dsDNA fragment immune complexes in the TLR9-dependent activation of rheumatoid factor B cells

Synthetic single-stranded oligodeoxynucleotides (15—30 bp) containing CpG motifs and phosphorothioate backbones (CpG s-ODN), immune complexes consisting of anti-nucleosome mAbs and mammalian chromatin (chromatin IC), and immune complexes consisting of anti-hapten mAbs and haptenated-double stranded DNA fragments (~600 bp) can all effectively stimulate transgenic B cells expressing a rheumatoid factor receptor by a TLR9-dependent process. However, differential sensitivity to both s-ODN and small molecule inhibitors suggests that stimulatory CpG sODN and chromatin IC may either access TLR9 via different routes or depend on discrete activation parameters. These data have important implications regarding the therapeutic application of TLR9 inhibitors to the treatment of systemic autoimmune diseases.

Nuclear Receptor Signaling Inhibits HIV-1 Replication in Macrophages through Multiple trans-Repression Mechanisms

ABSTRACT Sexually transmitted pathogens activate HIV-1 replication and inflammatory gene expression in macrophages through engagement of Toll-like receptors (TLRs). Ligand-activated nuclear receptor (NR) transcription factors, including glucocorticoid receptor (GR), peroxisome proliferator-activated receptor gamma (PPARγ), and liver X receptor (LXR), are potent inhibitors of TLR-induced inflammatory gene expression. We therefore hypothesized that ligand-activated NRs repress both basal and pathogen-enhanced HIV-1 replication in macrophages by directly repressing HIV-1 transcription and by ameliorating the local proinflammatory response to pathogens. We show that the TLR2 ligand PAM3CSK4 activated virus transcription in macrophages and that NR signaling repressed both basal and TLR-induced HIV-1 transcription. NR ligand treatment repressed HIV-1 expression when added concurrently with TLR ligands and in the presence of cycloheximide, demonstrating that they act independently of new cellular gene expression. We found that treatment with NR ligands inhibited the association of AP-1 and NF-κB subunits, as well as the coactivator CBP, with the long terminal repeat (LTR). We show for the first time that the nuclear corepressor NCoR is bound to HIV-1 LTR in unstimulated macrophages and is released from the LTR after TLR engagement. Treatment with PPARγ and LXR ligands, but not GR ligands, prevented this TLR-induced clearance of NCoR from the LTR. Our data demonstrate that both classical and nonclassical trans-repression mechanisms account for NR-mediated HIV-1 repression. Finally, NR ligand treatment inhibited the potent proinflammatory response induced by PAM3CSK4 that would otherwise activate HIV-1 expression in infected cells. Our findings provide a rationale for studying ligand-activated NRs as modulators of basal and inflammation-induced HIV-1 replication.

Retinoid-Dependent Restriction of Human Immunodeficiency Virus Type 1 Replication in Monocytes/Macrophages

ABSTRACT Vitamin A deficiency has been correlated with increased severity of human immunodeficiency virus type 1 (HIV-1)-associated disease. Moreover, vitamin A supplementation can reduce AIDS-associated morbidity and mortality. Our group and others have shown that retinoids, the bioactive metabolites of vitamin A, repress HIV-1 replication in monocytic cell lines and primary macrophages by blocking long-terminal-repeat (LTR)-directed transcription. Based on these studies, we hypothesize that retinoids are natural repressors of HIV-1 in vivo. We show here that all-trans-retinoic acid (RA)-mediated repression of HIV-1 activation requires pretreatment for at least 12 h and is blocked by the protein synthesis inhibitors cycloheximide and puromycin. Studies of the kinetics of RA-mediated repression in U1 cells and primary monocyte-derived macrophages (MDMs) reveal that the repressive effects of RA on HIV-1 expression are long-lasting but reversible. We demonstrate that HIV-1 expression is activated when U1 cells or MDMs are cultured in retinoid-free synthetic medium and show that physiological concentrations of RA repress this activation. In addition, the synthetic pan-retinoic acid receptor antagonist BMS-204 493 activates HIV-1 replication in U1 cells in a dose-dependent manner, suggesting that RA-induced transactivation of cellular gene expression is required for HIV-1 repression. Together, these data support the hypothesis that retinoids present in tissue culture media in vitro and serum in vivo maintain HIV-1 in a transcriptionally repressed state in monocytes/macrophages.

Retinoic Acid Inhibition of Chromatin Remodeling at the Human Immunodeficiency Virus Type 1 Promoter UNCOUPLING OF HISTONE ACETYLATION AND CHROMATIN REMODELING

All-trans retinoic acid (RA) represses HIV-1 transcription and replication in cultured monocytic cells and in primary monocyte-derived macrophages. Here we examine the role of histone acetylation and chromatin remodeling in RA-mediated repression. RA pretreatment of latently infected U1 promonocytes inhibits HIV-1 expression in response to the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). TSA is thought to activate HIV-1 transcription by inducing histone hyperacetylation within a regulatory nucleosome, nuc-1, positioned immediately downstream from the transcription start site. Acetylation of nuc-1 is thought to be a critical step in activation that precedes nuc-1 remodeling and, subsequently, transcriptional initiation. Here we demonstrate that TSA treatment induces H3 and H4 hyperacetylation and nuc-1 remodeling. Although RA pretreatment inhibits nuc-1 remodeling and HIV-1 transcription, it has no effect on histone acetylation. This suggests that acetylation and remodeling are not obligatorily coupled. We also show that growth of U1 cells in retinoid-deficient medium induces nuc-1 remodeling and HIV-1 expression but does not induce histone hyperacetylation. These findings suggest that remodeling, not histone hyperacetylation, is the limiting step in transcriptional activation in these cells. Together, these data suggest that RA signaling maintains the chromatin structure of the HIV-1 promoter in a transcriptionally non-permissive state that may contribute to the establishment of latency in monocyte/macrophages.