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. KeywordsTLR; SLE; B cell; autoimmune disease IntroductionSystemic autoimmune diseases are frequently associated with the production of autoantibodies reactive with nuclear or cytosolic cellular components. These autoantibodies play an important role in the pathogenesis of various autoimmune diseases, as they can form immune complexes (ICs) that can deposit in the kidneys, skin, or vessel endothelium driving local pathology. Antibodies can contribute to disease pathogenesis through a variety of mechanisms, including complement fixation, engagement of activating Fc receptors, and direct cell activation. Each autoimmune disease is associated with a particular spectrum of autoantibody specificities. For example, Systemic Lupus Erythematosis (SLE) is frequently b To whom correspondence should be addressed. Division of Rheumatology, Department of Medicine, University of Massachusetts Medical School, UMass Memorial Medical Center, Lazare Research Building, 364 Plantation Street, Worcester, MA 01605, USA. 508-856-8089. Ann.Rothstein@umassmed.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [4]. The availability of autoantigens may drive the autoantibody repertoire, as described in the autoimmune myopathy polymyositis (PM): stria...

Yersinia pestis, the causative agent of plague, secretes LcrV (low-calcium-response V or V antigen) during infection. LcrV triggers the release of interleukin 10 (IL-10) by host immune cells and suppresses proinflammatory cytokines such as tumor necrosis factor alpha and gamma interferon as well as innate defense mechanisms required to combat the pathogenesis of plague. Although immunization of animals with LcrV elicits protective immunity, the associated suppression of host defense mechanisms may preclude the use of LcrV as a human vaccine. Here we show that short deletions within LcrV can reduce its immune modulatory properties. An LcrV variant lacking amino acid residues 271 to 300 (rV10) elicited immune responses that protected mice against a lethal challenge with Y. pestis. Compared to full-length LcrV, rV10 displayed a reduced ability to release IL-10 from mouse and human macrophages. Furthermore, the lipopolysaccharide-stimulated release of proinflammatory cytokines by human or mouse macrophages was inhibited by full-length LcrV but not by the rV10 variant. Thus, it appears that LcrV variants with reduced immune modulatory properties could be used as a human vaccine to generate protective immunity against plague.Plague epidemics have likely killed more people worldwide than any other infectious disease (36,54). Mammals, in particular rats, prairie dogs, and gerbils, are the primary reservoir of Yersinia pestis, and human disease transmission occurs by flea bites, aerosol, or contact (15, 17). Flea bite-transmitted Y. pestis infection leads to bubonic plague, with characteristic lymph node swellings and disease symptoms that progress frequently to systemic infection and pneumonia (7,14). Aerosol transmission of virulent Y. pestis, whether during plague epidemics or deliberate dissemination, precipitates pneumonic plague, a rapidly fatal disease with few characteristic symptoms (28). Considering the ubiquitous zoonotic nature of the disease and the possible illegitimate use of Y. pestis as a weapon, there is an urgent need for vaccine development to protect humans against bubonic and pneumonic plague (36).Some, but not all, bubonic plague victims survive the disease, even without therapy, and appear to develop immunity (12,48). Burrows discovered Y. pestis LcrV as a protective antigen which stimulates humoral immune responses in experimental animals that afford protection against plague infection (10, 11). Based on these observations, several laboratories developed recombinant LcrV subunit vaccines, either alone or in combination with other Y. pestis proteins, and demonstrated that a humoral immune response to LcrV confers plague protection in experimental animals (2,21,25,31,32,47). Brubaker and colleagues first showed that LcrV injection of animals stimulates the release of interleukin 10 (IL-10), a cytokine that suppresses innate immune functions (8, 34). LcrV injection also prevents the release of proinflammatory cytokines, such as gamma interferon (IFN-␥) or tumor necrosis factor alpha (TNF-␣), during ...

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.

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.

Endosomal Toll-like receptors (TLRs) play an important role in systemic autoimmune diseases such as SLE, where DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune prone mice develop more severe clinical disease, while TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we have now directly compared the functional properties of autoantigen activated WT, TLR9-deficient and TLR7-deficient B cells, in an experimental system where proliferation depends on BCR/TLR co-engagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than either WT or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, and TLR7 to promote, the clinical features of SLE.