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1.  FAS mediates non-canonical IL-1β and IL-18 maturation via caspase-8 in a Rip3-independent manner1 
Fas, a tumor necrosis factor family receptor, is activated by the membrane protein Fas ligand (FasL) expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas induced cytokines, the IL-1β family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1β cytokines via caspase-1. The mechanisms, by which Fas regulates IL-1β activation, however, remain unresolved. Here, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells leading to the maturation of IL-1β and IL-18 independently of inflammasomes or Rip3. Hence, Fas controls a novel non-canonical IL-1β activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance and control of infectious diseases.
doi:10.4049/jimmunol.1202121
PMCID: PMC3518757  PMID: 23144495
2.  Natural antibody to apoptotic cell membranes inhibits the proinflammatory properties of lupus autoantibody immune complexes 
Arthritis and rheumatism  2012;64(10):3388-3398.
Objective
Naturally-arising IgM antibodies (NAbs) to apoptotic cell (AC) determinants are present from birth and can be further induced by AC challenge. In systemic lupus erythematosus, lower anti-AC NAb levels have been associated with higher disease activity. We have recently shown that a prototypic AC-specific NAb IgM can suppress pro-inflammatory responses to purified agonists for Toll-like receptors, and also block the in vivo induction of IgG-immune complex (IC)-induced arthritis. IgG autoantibody-complexes with nuclear antigens, which activate dendritic cells (DCs), have been implicated in autoimmune pathogenesis. Here, we sought to investigate potential roles of such NAbs for regulating immune-complex mediated DC activation, which is believed to be involved in disease initiation and perpetuation.
Methods
Bone-marrow-derived myeloid DCs were stimulated with ICs composed of IgG-autoantibody-chromatin or IgG-autoantibody-RNA. Outcome was evaluated based on production of inflammatory cytokines by ELISA, and expression of co-stimulatory molecules, which are markers of DC activation, by flow cytometry. MAPK activation was evaluated by phospho-flow and immunofluorescence microscopy.
Results
Anti-AC NAb IgM dose-dependently suppressed both DNA- and RNA-IC-induced IL-6 and DNA-IC-induced TNF-α production, as well as RNA-IC-induced upregulation of CD86 and CD40 on DCs. NAb IgM-mediated inhibition was associated with suppression of IC-mediated p38-MAPK activation and nuclear localization.
Conclusions
We demonstrated a direct in vitro inhibitory effect of the NAb IgM on inflammatory responses induced by IgG-nucleic acid ICs. These findings contribute to emerging evidence that regulatory NAbs to apoptotic-cell determinants may oppose the influence of pathogenic lupus autoantibody-ICs and thereby play roles in the maintenance of immune homeostasis.
doi:10.1002/art.34537
PMCID: PMC3462267  PMID: 22577035
3.  Role of type I interferons in the activation of autoreactive B cells 
Immunology and cell biology  2012;90(5):498-504.
Type I interferons (IFNs) are a family of cytokines involved in the defense against viral infections that play a key role in the activation of both the innate and adaptive immune system. IFNs both directly and indirectly enhance the capacity of B lymphocytes to respond to viral challenge and produce cytotoxic and neutralizing antibodies. However, prolonged type I IFN exposure is not always beneficial to the host. If not regulated properly IFN can drive autoantibody production as well as other parameters of systemic autoimmune disease. Type I IFNs impact B-cell function through a variety of mechanisms, including effects on receptor engagement, Toll-like receptor expression, cell migration, antigen presentation, cytokine responsiveness, cytokine production, survival, differentiation and class-switch recombination. Type I IFNs are also cytotoxic for a variety of cell types and thereby contribute to the accumulation of cell debris that serves as a potential source for autoantigens. Type I IFN engagement of a variety of accessory cells further promotes B-cell survival and activation, as exemplified by the capacity of type I IFNs to increase the level of B-cell survival factors, such as B lymphocyte stimulator, produced by dendritic cells. Therefore, it is not surprising that the loss of expression of the type I IFN receptor can have dramatic effects on the production of autoantibodies and on the clinical features of systemic autoimmune diseases such as systemic lupus erythematosus.
doi:10.1038/icb.2012.10
PMCID: PMC3701256  PMID: 22430248
type I IFN; B cell; autoimmunity; SLE
4.  Selective Binding of anti-DNA Antibodies to Native dsDNA Fragments of Differing Sequence 
Immunology letters  2012;143(1):85-91.
Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs) has been shown. Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.
doi:10.1016/j.imlet.2012.01.003
PMCID: PMC3655556  PMID: 22285306
Autoantibody; Systemic lupus erythematosus; Anti-DNA; TLR9; B-cell; CpG-rich DNA
5.  Beyond transitional selection: New roles for BLyS in peripheral tolerance 
Drug development research  2011;72(8):779-787.
B cell targeted therapies have enjoyed recent success in the treatment of systemic autoimmune diseases. Among these, Belimumab, which blocks the B cell survival cytokine BLyS, was recently approved for the treatment of Systemic Lupus Erythematosus. It is therefore important to consider the roles BLyS plays in B cell tolerance. Herein, we review how BLyS contributes to the negative selection of autoreactive B cell clones from the preimmune repertoire as well as its role in regulating both germinal center and extrafollicular peripheral B cell responses. We further examine the complex role of Toll-like receptors (TLRs) in humoral autoimmunity, pointing out potential crosstalk between BLyS and TLR pathways.
doi:10.1002/ddr.20487
PMCID: PMC3272642  PMID: 22323842
BLyS; BAFF; B lymphocyte; Autoimmunity; Tolerance; Toll like receptors; Belimumab
6.  Thymic stromal lymphopoietin (TSLP)-mediated dermal inflammation aggravates experimental asthma 
Mucosal immunology  2012;5(3):342-351.
Individuals with one atopic disease are far more likely to develop a second. Approximately half of all atopic dermatitis (AD) patients subsequently develop asthma, particularly those with severe AD. This association, suggesting a role for AD as an entry point for subsequent allergic disease, is a phenomenon known as the ‘atopic march’. While the underlying cause of the atopic march remains unknown, recent evidence suggests a role for the cytokine TSLP. We have established a mouse model to determine whether TSLP plays a role in this phenomenon, and in this study show that mice exposed to the antigen OVA in the skin in the presence of TSLP develop severe airway inflammation when later challenged with the same antigen in the lung. Interestingly, neither TSLP production in the lung nor circulating TSLP is required to aggravate the asthma that was induced upon subsequent antigen challenge. However, CD4 T cells are required in the challenge phase of the response, as was challenge with the sensitizing antigen, demonstrating that the response was antigen-specific. This study, which provides a clean mouse model to study human atopic march, indicates that skin-derived TSLP may represent an important factor that triggers progression from atopic dermatitis to asthma.
doi:10.1038/mi.2012.14
PMCID: PMC3328620  PMID: 22354320
TSLP; atopic dermatitis; atopic march; mouse model
7.  Differential Cytokine Production and Bystander Activation of Autoreactive B Cells in Response to CpG-A and CpG-B ODNs1 
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.
doi:10.4049/jimmunol.0901941
PMCID: PMC3426913  PMID: 19864612
8.  Response to self antigen imprints regulatory memory in tissues 
Nature  2011;480(7378):538-542.
Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathologic autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration1. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression2. In the practice of antigen-specific immunotherapy (antigen-SIT), allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure3. Although these findings suggest that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (Treg cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity. After resolution of the inflammatory response, activated Treg cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, Treg cells function to confer ‘regulatory memory’ to the target tissue. These findings provide a framework for understanding how Treg cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.
doi:10.1038/nature10664
PMCID: PMC3263357  PMID: 22121024
9.  Response to self antigen imprints regulatory memory in tissues 
Nature  2011;480(7378):538-542.
Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathologic autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration1. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression2. In the practice of antigen-specific immunotherapy (antigen-SIT), allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure3. Although these findings suggest that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (Treg cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity. After resolution of the inflammatory response, activated Treg cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, Treg cells function to confer ‘regulatory memory’ to the target tissue. These findings provide a framework for understanding how Treg cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.
doi:10.1038/nature10664
PMCID: PMC3263357  PMID: 22121024
10.  Toll-like receptor driven B cell activation in the induction of systemic autoimmunity 
Seminars in immunology  2011;23(2):106-112.
Studies over the past decade have demonstrated a key role for pattern recognition receptors in the activation of autoreactive B cells. Self reactive B cells that manage to escape negative selection often express relatively low affinity receptors for self antigens (ignorant B cells), and can only be activated by integrating a relatively weak BCR signal with signals from additional receptors. Members of the toll-like receptor (TLR) gene family, and especially the nucleic acid binding receptors TLR 7, 8 and 9, appear to play a key role in this regard and promote the production of autoantibodies reactive with DNA- or RNA-associated autoantigens. These autoantibodies are able to form immune complexes with soluble or cell-bound ligands, and these immune complexes can in turn activate a second round of proinflammatory cells that further contribute to the autoimmune disease process. Recent data have emerged showing a pathogenic role for TLR7, with an opposing, protective role for TLR9. Targeting these disregulated pathways offers a therapeutic opportunity to treat autoimmune diseases without crippling the entire immune system. Further understanding of the role of specific receptors, cell subsets, and inhibitory signals that govern these TLR-associated pathways will enable future therapeutics to be tailored to specific categories of autoimmune disease.
doi:10.1016/j.smim.2011.01.016
PMCID: PMC3070769  PMID: 21306913
TLR; SLE; B cell; autoimmune disease
11.  FcγRIIB Regulation of BCR/TLR-Dependent Autoreactive B Cell Responses 
European journal of immunology  2010;40(10):2692-2698.
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.
doi:10.1002/eji.200940184
PMCID: PMC3060940  PMID: 20809520
Autorective B cells; endogenous TLR ligands; inhibitory Fc receptor
12.  Poly(I:C) drives type I IFN- and TGFβ-mediated inflammation and dermal fibrosis simulating altered gene expression in systemic sclerosis 
Immune activation of fibrosis likely plays a crucial role in the pathogenesis of systemic sclerosis (SSc). The goal of this study was to better understand innate immune regulation and associated IFN- and TGFβ-responsive gene expression in SSc skin and dermal fibroblasts, in particular the effect of different Toll-like receptor (TLR) ligands. To better understand the relationship between inflammation and fibrosis in vivo we developed a murine model for chronic innate immune stimulation. We found that expression of both IFN- and TGFβ-responsive genes are increased in SSc skin and in SSc fibroblasts when stimulated by TLR ligands. In contrast, cutaneous lupus skin showed much more highly upregulated IFN-responsive and much less highly upregulated TGFβ-responsive gene expression. The TLR3 ligand, Poly(I:C), mostly highly increased fibroblast expression of both IFN- and TGFβ-responsive genes as well as TLR3. Chronic subcutaneous immune stimulation by Poly(I:C) stimulated inflammation, and IFN- and TGFβ-responsive gene expression. However, in this model type I IFNs played no apparent role regulating TGFβ activity in the skin. These results suggest that TLR agonists may be important stimuli of dermal fibrosis, potentially mediated by TLR3 or other innate immune receptors.
doi:10.1038/jid.2010.200
PMCID: PMC3086558  PMID: 20613770
13.  Opposing Roles for Membrane Bound and Soluble Fas Ligand in Glaucoma-Associated Retinal Ganglion Cell Death 
PLoS ONE  2011;6(3):e17659.
Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.
doi:10.1371/journal.pone.0017659
PMCID: PMC3066205  PMID: 21479271
14.  Regulation of Autoreactive B Cell Responses to Endogenous TLR Ligands 
Autoimmunity  2010;43(1):76-83.
Immune complexes (ICs) containing DNA and RNA are responsible for disease manisfestations found in patients with Systemic Lupus Erythematosus (SLE). B cells contribute to SLE pathology through BCR recognition of endogenous DNA- and RNA-associated autoantigens and delivery of these self-constitutents to endosomal TLR9 and TLR7, respectively. B cell activation by these pathways leads to production of class-switched DNA- and RNA reactive autoantibodies, contributing to an inflammatory amplification loop characteristic of disease. Intriguinly, self-DNA and RNA are typically non-stimulatory for TLR9/7 due to absence of stimulatory sequences or presence of molecular modifications. Recent evidence from our lab and others suggests that B cell activation by BCR/TLR pathways is tightly regulated by surface-expressed receptors on B cells, and the outcome of activation depends on the balance of stimulatory and inhibitory signals. Either IFNα engagement of the type I IFN receptor, or loss of IgG ligation of the inhibitory FcγRIIB receptor promotes B cell activation by weakly-stimulatory DNA and RNA TLR ligands. In this context, autoreactive B cells can respond robustly to common autoantigens. These findings have important implications for the role of B cells in vivo in the pathology of SLE.
doi:10.3109/08916930903374618
PMCID: PMC3059585  PMID: 20014959
Systemic Lupus Erythematosus; AM14 B cells; TLR9; TLR7; FcγRIIB; Type I IFN
15.  Gαq-containing G proteins regulate B cell selection and survival and are required to prevent B cell–dependent autoimmunity 
The Journal of Experimental Medicine  2010;207(8):1775-1789.
Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the Gαq subunit of trimeric G proteins (Gnaq−/− mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq−/− B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq−/− chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq−/− B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.
doi:10.1084/jem.20092735
PMCID: PMC2916136  PMID: 20624888
16.  RAGE-Independent Autoreactive B Cell Activation In Response To Chromatin And HMGB1/DNA Immune Complexes 
Autoimmunity  2010;43(1):103-110.
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.
doi:10.3109/08916930903384591
PMCID: PMC2929824  PMID: 20014975
HMGB1; RAGE; AM14 B cells; TLR9; Systemic Lupus Erythematosus; autoreactive B cell activation
17.  Interferon Regulatory Factor 5 Is Required For Disease Development In The FcγRIIB−/−Yaa and FcγRIIB−/− Mouse Models Of Systemic Lupus Erythematosus 1 
Polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study we show that IRF5 is absolutely required for disease development in the FcγRIIB−/−Yaa and FcγRIIB−/− lupus models. In contrast to IRF5-sufficient FcγRIIB−/−Yaa mice, IRF5-deficient FcγRIIB−/−Yaa mice do not develop lupus manifestations and have a phenotype comparable to wildtype mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity as IRF5-heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I interferon IFN-α, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcγRIIB−/−Yaa mice lacking the type I interferon receptor IFNAR1. Unlike the IRF5-deficient and IRF5-heterozygous FcγRIIB−/−Yaa mice, IFNAR1-deficient FcγRIIB−/−Yaa mice maintained a substantial level of residual disease. Furthermore, in FcγRIIB−/− mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations indicating that the beneficial effects of IRF5 deficiency in FcγRIIB−/−Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I interferon production.
doi:10.4049/jimmunol.0901748
PMCID: PMC2858062  PMID: 20007534
18.  Requirement for DNA CpG Content in TLR9-dependent Dendritic Cell Activation Induced by DNA-containing Immune Complexes1 
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 (SLE), a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DC), is through uptake as a DNA-containing immune complex (IC). In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated cytosine-guanosine (CpG) dinucleotides within dsDNA are required for murine DC TLR9 activation induced by DNA-containing IC. 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 which 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 IC. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as SLE and psoriasis in which activation of TLR9 in DC by self-DNA has been implicated in disease pathogenesis.
doi:10.4049/jimmunol.0900399
PMCID: PMC2860771  PMID: 19648272
autoimmunity; cytokines; dendritic cells; rodent; systemic lupus erythematosus
19.  Murine B Cell Response to TLR7 Ligands Depends on an IFN-β Feedback Loop1 
Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR−/− B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1−/− B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-β feedback loop and constitutively low expression of TLR7 in the IFNAR1−/− B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.
doi:10.4049/jimmunol.0803899
PMCID: PMC2929820  PMID: 19587008
20.  Autoreactive B Cells Discriminate CpG-rich and CpG-poor DNA and This Response is Modulated by IFN-α1 
Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs)6 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. By 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. 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 (SLE).
PMCID: PMC2584609  PMID: 18941176
21.  DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Faslpr/lpr mice in vivo 
Introduction
B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion.
Methods
We have developed a new generation of DNA-like compounds named class R inhibitory oligonucleotides (INH-ODNs). We tested their effectiveness in autoimmune B cells and interferon-alpha-producing dendritic cells in vitro and in lupus-prone MRL-Faslpr/lpr mice in vivo.
Results
Class R INH-ODNs have 10- to 30-fold higher inhibitory potency when autoreactive B cells are synergistically activated through the BCR and associated TLR7 or 9 than when stimulation occurs via non-BCR-engaged TLR7/9. Inhibition of TLR9 requires the presence of both CCT and GGG triplets in an INH-ODN, whereas the inhibition of the TLR7 pathway appears to be sequence-independent but dependent on the phosphorothioate backbone. This difference was also observed in the MRL-Faslpr/lpr mice in vivo, where the prototypic class R INH-ODN was more effective in curtailing abnormal autoantibody secretion and prolonging survival.
Conclusions
The increased potency of class R INH-ODNs for autoreactive B cells and dendritic cells may be beneficial for lupus patients by providing pathway-specific inhibition yet allowing them to generate protective immune response when needed.
doi:10.1186/ar2710
PMCID: PMC2714127  PMID: 19476613
22.  Toll-like receptors and innate immune responses in systemic lupus erythematosus 
A series of discoveries over the past several years has provided a new paradigm for understanding autoimmunity in systemic lupus erythematosus. The discoveries of pattern recognition receptors and of how these receptors can be recruited into autoimmune responses underpin this paradigm. The implications of these observations continue to unfold with ongoing investigation into the range and specificity of pattern recognition receptors, into how immune complexes containing nucleic acids trigger these receptors, into how endogenous macromolecular 'danger signals' stimulate innate immune responses, and into the effect of pattern recognition receptor activation on various cell types in initiating and perpetuating autoimmunity. The development of clinical trials using therapeutic agents that target components of the innate immune system suggests that these advances may soon culminate in new medications for treating patients with systemic lupus erythematosus.
doi:10.1186/ar2321
PMCID: PMC2246252  PMID: 18086320
23.  RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement 
The Journal of Experimental Medicine  2005;202(9):1171-1177.
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.
doi:10.1084/jem.20050630
PMCID: PMC2213226  PMID: 16260486
24.  Toll-like Receptor 9–Dependent and –Independent Dendritic Cell Activation by Chromatin–Immunoglobulin G Complexes 
The Journal of Experimental Medicine  2004;199(12):1631-1640.
Dendritic cell (DC) activation by nucleic acid–containing immunoglobulin (Ig)G complexes has been implicated in systemic lupus erythematosus (SLE) pathogenesis. However, the mechanisms responsible for activation and subsequent disease induction are not completely understood. Here we show that murine DCs are much more effectively activated by immune complexes that contain IgG bound to chromatin than by immune complexes that contain foreign protein. Activation by these chromatin immune complexes occurs by two distinct pathways. One pathway involves dual engagement of the Fc receptor FcγRIII and Toll-like receptor (TLR)9, whereas the other is TLR9 independent. Furthermore, there is a characteristic cytokine profile elicited by the chromatin immune complexes that distinguishes this response from that of conventional TLR ligands, notably the induction of BAFF and the lack of induction of interleukin 12. The data establish a critical role for self-antigen in DC activation and explain how the innate immune system might drive the adaptive immune response in SLE.
doi:10.1084/jem.20031942
PMCID: PMC2212813  PMID: 15197227
systemic lupus erythematosus; innate immunity; Fc receptor; BAFF; tumor necrosis factor
25.  Opposing Effects of Transmembrane and Soluble FAS Ligand Expression on Inflammation and Tumor Cell Survival 
The Journal of Experimental Medicine  2000;191(7):1209-1220.
Fas ligand (FasL) has been shown to mediate both apoptotic and inflammatory reactions. To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL. First, the ability of these lines to induce an inflammatory response was assessed in vivo by injecting the transfectants intraperitoneally and measuring subsequent neutrophil extravasation into the peritoneal cavity. Second, lines were assessed by injecting the transfectants subcutaneously and monitoring their growth as solid tumors. Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities. A neutrophil response could only be elicited in mice with intact Fas death domains although Fas expression by the neutrophils was not essential. Lymphoma cells expressing the soluble FasL form corresponding to the natural cleavage product could not trigger apoptosis and did not induce a neutrophil response. In contrast to the other FasL transfectants, these cells survived as tumor transplants. However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.
PMCID: PMC2193164  PMID: 10748238
Fas ligand; apoptosis; neutrophil; chemotaxis; lymphoma

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