The Cdkn2a gene product p19 alternative reading frame (p19ARF) is a critical regulator of IFNß-mediated Lyme arthritis.

Type I interferon (IFN) has been identified in patients with Lyme disease, and its abundant expression in joint tissues of C3H mice precedes development of Lyme arthritis. Forward genetics using C3H mice with severe Lyme arthritis and C57BL/6 (B6) mice with mild Lyme arthritis identified the Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1) on chromosome 4 (Chr4) as a regulator of B. burgdorferi-induced IFNß expression and Lyme arthritis severity. B6 mice introgressed with the C3H allele for Bbaa1 (B6.C3-Bbaa1 mice) displayed increased severity of arthritis, which is initiated by myeloid lineage cells in joints. Using advanced congenic lines, the physical size of the Bbaa1 interval has been reduced to 2 Mbp, allowing for identification of potential genetic regulators. Small interfering RNA (siRNA)-mediated silencing identified Cdkn2a as the gene responsible for Bbaa1 allele-regulated induction of IFNß and IFN-stimulated genes (ISGs) in bone marrow-derived macrophages (BMDMs). The Cdkn2a-encoded p19 alternative reading frame (p19ARF) protein regulates IFNß induction in BMDMs as shown by siRNA silencing and overexpression of ARF. In vivo studies demonstrated that p19ARF contributes to joint-specific induction of IFNß and arthritis severity in B. burgdorferi-infected mice. p19ARF regulates B. burgdorferi-induced IFNß in BMDMs by stabilizing the tumor suppressor p53 and sequestering the transcriptional repressor BCL6. Our findings link p19ARF regulation of p53 and BCL6 to the severity of IFNß-induced Lyme arthritis in vivo and indicate potential novel roles for p19ARF, p53, and BCL6 in Lyme disease and other IFN hyperproduction syndromes.

Association of Persistent Symptoms after Lyme Neuroborreliosis and Increased Levels of Interferon-α in Blood.

Patients who have Lyme neuroborreliosis (LNB) might experience lingering symptoms that persist despite antibiotic drug therapy. We tested whether those symptoms are caused by maladaptive immune responses by measuring 20 immune mediators in serum and cerebrospinal fluid (CSF) in 79 LNB patients followed for 1 year. At study entry, most mediators were highly concentrated in CSF, the site of the infection. Those responses resolved with antibiotic therapy, and associations between CSF cytokines and signs and symptoms of LNB were no longer observed. In contrast, subjective symptoms that persisted after use of antibiotics were associated with increased levels of serum interferon-α (IFN-α), which were already observed at study entry, and remained increased at each subsequent timepoint. Highest IFN-α levels corresponded with severe disease. Although the infection serves as the initial trigger, sequelae after antibiotic therapy are associated with unremitting systemic IFN-α levels, consistent with the pathogenic role of this cytokine in interferonopathies in other conditions.

Bystander T Cells: A Balancing Act of Friends and Foes.

T cell responses are essential for appropriate protection against pathogens. T cell immunity is achieved through the ability to discriminate between foreign and self-molecules, and this relies heavily on stringent T cell receptor (TCR) specificity. Recently, bystander activated T lymphocytes, that are specific for unrelated epitopes during an antigen-specific response, have been implicated in diverse diseases. Numerous infection models have challenged the classic dogma of T cell activation as being solely dependent on TCR and major histocompatibility complex (MHC) interactions, indicating an unappreciated role for pathogen-associated receptors on T cells. We discuss here the specific roles of bystander activated T cells in pathogenesis, shedding light on the ability of these cells to modulate disease severity independently from TCR recognition.

IL-10 Deficiency Reveals a Role for TLR2-Dependent Bystander Activation of T Cells in Lyme Arthritis.

T cells predominate the immune responses in the synovial fluid of patients with persistent Lyme arthritis; however, their role in Lyme disease remains poorly defined. Using a murine model of persistent Lyme arthritis, we observed that bystander activation of CD4+ and CD8+ T cells leads to arthritis-promoting IFN-γ, similar to the inflammatory environment seen in the synovial tissue of patients with posttreatment Lyme disease. TCR transgenic mice containing monoclonal specificity toward non-Borrelia epitopes confirmed that bystander T cell activation was responsible for disease development. The microbial pattern recognition receptor TLR2 was upregulated on T cells following infection, implicating it as marker of bystander T cell activation. In fact, T cell-intrinsic expression of TLR2 contributed to IFN-γ production and arthritis, providing a mechanism for microbial-induced bystander T cell activation during infection. The IL-10-deficient mouse reveals a novel TLR2-intrinsic role for T cells in Lyme arthritis, with potentially broad application to immune pathogenesis.

Genetic Control of Lyme Arthritis by Borrelia burgdorferi Arthritis-Associated Locus 1 Is Dependent on Localized Differential Production of IFN-ß and Requires Upregulation of Myostatin.

Previously, using a forward genetic approach, we identified differential expression of type I IFN as a positional candidate for an expression quantitative trait locus underlying Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1). In this study, we show that mAb blockade revealed a unique role for IFN-ß in Lyme arthritis development in B6.C3-Bbaa1 mice. Genetic control of IFN-ß expression was also identified in bone marrow-derived macrophages stimulated with B. burgdorferi, and it was responsible for feed-forward amplification of IFN-stimulated genes. Reciprocal radiation chimeras between B6.C3-Bbaa1 and C57BL/6 mice revealed that arthritis is initiated by radiation-sensitive cells, but orchestrated by radiation-resistant components of joint tissue. Advanced congenic lines were developed to reduce the physical size of the Bbaa1 interval, and confirmed the contribution of type I IFN genes to Lyme arthritis. RNA sequencing of resident CD45- joint cells from advanced interval-specific recombinant congenic lines identified myostatin as uniquely upregulated in association with Bbaa1 arthritis development, and myostatin expression was linked to IFN-ß production. Inhibition of myostatin in vivo suppressed Lyme arthritis in the reduced interval Bbaa1 congenic mice, formally implicating myostatin as a novel downstream mediator of the joint-specific inflammatory response to B. burgdorferi.

ß-Glucuronidase, a Regulator of Lyme Arthritis Severity, Modulates Lysosomal Trafficking and MMP-9 Secretion in Response to Inflammatory Stimuli.

The lysosomal enzyme ß-glucuronidase (Gusb) is a key regulator of Lyme-associated and K/B×N-induced arthritis severity. The luminal enzymes present in lysosomes provide essential catabolic functions for the homeostatic degradation of a variety of macromolecules. In addition to this essential catabolic function, lysosomes play important roles in the inflammatory response following infection. Secretory lysosomes and related vesicles can participate in the inflammatory response through fusion with the plasma membrane and release of bioactive contents into the extracellular milieu. In this study, we show that GUSB hypomorphism potentiates lysosomal exocytosis following inflammatory stimulation. This leads to elevated secretion of lysosomal contents, including glycosaminoglycans, lysosomal hydrolases, and matrix metalloproteinase 9, a known modulator of Lyme arthritis severity. This mechanistic insight led us to test the efficacy of rapamycin, a drug known to suppress lysosomal exocytosis. Both Lyme and K/B×N-associated arthritis were suppressed by this treatment concurrent with reduced lysosomal release.

MicroRNA-146a provides feedback regulation of lyme arthritis but not carditis during infection with Borrelia burgdorferi.

MicroRNAs have been shown to be important regulators of inflammatory and immune responses and are implicated in several immune disorders including systemic lupus erythematosus and rheumatoid arthritis, but their role in Lyme borreliosis remains unknown. We performed a microarray screen for expression of miRNAs in joint tissue from three mouse strains infected with Borrelia burgdorferi. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling, in all three strains, suggesting it plays an important role in the in vivo response to B. burgdorferi. Infection of B6 miR-146a-/- mice with B. burgdorferi revealed a critical nonredundant role of miR-146a in modulating Lyme arthritis without compromising host immune response or heart inflammation. The impact of miR-146a was specifically localized to the joint, and did not impact lesion development or inflammation in the heart. Furthermore, B6 miR-146a-/- mice had elevated levels of NF-κB-regulated products in joint tissue and serum late in infection. Flow cytometry analysis of various lineages isolated from infected joint tissue of mice showed that myeloid cell infiltration was significantly greater in B6 miR-146a-/- mice, compared to B6, during B. burgdorferi infection. Using bone marrow-derived macrophages, we found that TRAF6, a known target of miR-146a involved in NF-κB activation, was dysregulated in resting and B. burgdorferi-stimulated B6 miR-146a-/- macrophages, and corresponded to elevated IL-1ß, IL-6 and CXCL1 production. This dysregulated protein production was also observed in macrophages treated with IL-10 prior to B. burgdorferi stimulation. Peritoneal macrophages from B6 miR-146a-/- mice also showed enhanced phagocytosis of B. burgdorferi. Together, these data show that miR-146a-mediated regulation of TRAF6 and NF-κB, and downstream targets such as IL-1ß, IL-6 and CXCL1, are critical for modulation of Lyme arthritis during chronic infection with B. burgdorferi.

Zfp318 regulates IgD expression by abrogating transcription termination within the Ighm/Ighd locus.

The protein Zfp318 is expressed during the transition of naive B cells from an immature to mature state. To evaluate its role in mature B cell functions, a conditional gene deficiency in Zfp318 was created and deleted in bone marrow lineages via Vav-Cre. B cell development was minimally altered in the absence of the protein, although transitional 2 (T2) B cell populations were depressed in the absence of Zfp318. Intriguingly, the analysis of IgM and IgD expression by maturing and mature naive B cells demonstrated an elevated level of IgM gene products and a virtual loss of IgD products. Transcriptome analysis of Zfp318-deficient B cells revealed that only two gene products showed altered expression in the absence of Zfp318 (Ighd and Sva), demonstrating a remarkable specificity of Zfp318 action. In the absence of Zfp318, Ighm/Ighd transcripts, which would normally encode IgM and IgD from heterogeneous nuclear RNA transcripts via alternative splicing, lack intron and exon sequences from the IgD (Ighd)-encoding region. This finding indicates that Zfp318, in a novel manner, functions by repressing recognition of the transcriptional termination site at the 3' end of the terminal IgM-encoding exon, allowing for synthesis of the complete Ighm/Ighd heterogeneous nuclear RNA.

Borrelia burgdorferi arthritis-associated locus Bbaa1 regulates Lyme arthritis and K/B×N serum transfer arthritis through intrinsic control of type I IFN production.

Localized upregulation of type I IFN was previously implicated in development of Borrelia burgdorferi-induced arthritis in C3H mice, and was remarkable due to its absence in the mildly arthritic C57BL/6 (B6) mice. Independently, forward genetics analysis identified a quantitative trait locus on Chr4, termed B. burgdorferi-associated locus 1 (Bbaa1), that regulates Lyme arthritis severity and includes the 15 type I IFN genes. Involvement of Bbaa1 in arthritis development was confirmed in B6 mice congenic for the C3H allele of Bbaa1 (B6.C3-Bbaa1), which developed more severe Lyme arthritis and K/B×N model of rheumatoid arthritis (RA) than did parental B6 mice. Administration of a type I IFN receptor blocking mAb reduced the severity of both Lyme arthritis and RA in B6.C3-Bbaa1 mice, formally linking genetic elements within Bbaa1 to pathological production of type I IFN. Bone marrow-derived macrophages from Bbaa1 congenic mice implicated this locus as a regulator of type I IFN induction and downstream target gene expression. Bbaa1-mediated regulation of IFN-inducible genes was upstream of IFN receptor-dependent amplification; however, the overall magnitude of the response was dependent on autocrine/paracrine responses to IFN-ß. In addition, the Bbaa1 locus modulated the functional phenotype ascribed to bone marrow-derived macrophages: the B6 allele promoted expression of M2 markers, whereas the C3H allele promoted induction of M1 responses. This report identifies a genetic locus physically and functionally linked to type I IFN that contributes to the pathogenesis of both Lyme and RA.

Fatal autoimmunity results from the conditional deletion of Snai2 and Snai3.

Transcriptional regulation of gene expression is a key component of orchestrating proper immune cell development and function. One strategy for maintaining these transcriptional programs has been the evolution of transcription factor families with members possessing overlapping functions. Using the germ line deletion of Snai2 combined with the hematopoietic specific deletion of Snai3, we report that these factors function redundantly to preserve the development of B and T cells. Such animals display severe lymphopenia, alopecia and dermatitis as well as profound autoimmunity manifested by the production of high levels of autoantibodies as early as 3 weeks of age and die by 30 days after birth. Autoantibodies included both IgM and IgG isotypes and were reactive against cytoplasmic and membranous components. A regulatory T cell defect contributed to the autoimmune response in that adoptive transfer of wild type regulatory T cells alleviated symptoms of autoimmunity. Additionally, transplantation of Snai2/Snai3 double deficient bone marrow into Snai2 sufficient Rag2(-/-) recipients resulted in autoantibody generation. The results demonstrated that appropriate expression of Snai2 and Snai3 in cells of hematopoietic derivation plays an important role in development and maintenance of immune tolerance.

Optimal germinal center B cell activation and T-dependent antibody responses require expression of the mouse complement receptor Cr1.

Follicular dendritic cells (FDCs) and complement receptor (Cr)1 and complement receptor (Cr)2 are important for the generation of humoral immunity. Cr1/2 expression on B cells and FDCs was shown to provide a secondary signal for B cell activation, to facilitate transport of Ag in immune follicles, and to enhance retention of immune complexes by FDCs. We show in this study that murine B cells predominantly express the Cr2 product from the Cr2 gene, whereas FDCs almost exclusively express the Cr1 isoform generated from the Cr2 gene. To define the specific role of Cr1, we created an animal that maintains normal cell-restricted expression of Cr2 but does not express Cr1. Cr1-deficient (Cr1KO) mice develop normal B1 and B2 immature and mature B cell subsets and have normal levels of naive serum Abs but altered levels of natural Abs. Immunization of the Cr1KO animal demonstrates deficient Ab responses to T-dependent, but not T-independent, Ags. Germinal centers from the immunized Cr1KO animal possess a deficiency in activated B cells, similar to that seen for animals lacking both Cr1 and Cr2 or C3. Finally, animals lacking only Cr1 respond similarly to wild-type animals to infections with Streptococcus pneumoniae, a pathogen to which animals lacking C3 or both Cr1 and Cr2 are particularly sensitive. Altogether, these data suggest that the production of Cr1, primarily by FDCs, is critical in the generation of appropriately activated B cells of the germinal center and the generation of mature Ab responses.

Bone marrow-induced Mef2c deficiency delays B-cell development and alters the expression of key B-cell regulatory proteins.

The Mef2 family transcriptional regulator Mef2c (myocyte enhancer factor 2c) is highly expressed in maturing bone marrow and peripheral mature B-cells. To evaluate the role of this transcription factor in B-cell development, we generated a B-cell-specific conditional deletion of Mef2c using the Mb-1-Cre transgene that is expressed during the early stages of immunoglobulin rearrangement. Young mice possessing this defect demonstrated a significant impairment in B-cell numbers in bone marrow and spleen. This phenotype was evident in all B-cell subsets; however, as the animals mature, the deficit in the peripheral mature B-cell compartments was overcome. The absence of Mef2c in mature B-cells led to unique CD23+ and CD23- subsets that were evident in Mef2c knockout primary samples as well as Mef2c-deficient cultured, differentiated B-cells. Genome-wide expression analysis of immature and mature B-cells lacking Mef2c indicated altered expression for a number of key regulatory proteins for B-cell function including Ciita, CD23, Cr1/Cr2 and Tnfsf4. Chromatin immunoprecipitation analysis confirmed Mef2c binding to the promoters of these genes indicating a direct link between the presence (or absence) of Mef2c and altered transcriptional control in mature B-cells.

Localized production of IL-10 suppresses early inflammatory cell infiltration and subsequent development of IFN-γ-mediated Lyme arthritis.

IL-10 is a nonredundant inflammatory modulator that suppresses arthritis development in Borrelia burgdorferi-infected mice. Infected C57BL/6 (B6) IL-10(-/-) mice were previously found to have a prolonged IFN-inducible response in joint tissue. Infection of B6 IL-10 reporter mice identified macrophages and CD4(+) T cells as the primary sources of IL-10 in the infected joint tissue, suggesting that early local production of IL-10 dampened the proarthritic IFN response. Treatment of B6 IL-10(-/-) mice with anti-IFN-γ reduced the increase in arthritis severity and suppressed IFN-inducible transcripts to wild-type levels, thereby linking dysregulation of IFN-γ to disease in the B6 IL-10(-/-) mouse. Arthritis in B6 IL-10(-/-) mice was associated with elevated numbers of NK cell, NKT cell, α/ß T cell, and macrophage infiltration of the infected joint. FACS lineage sorting revealed NK cells and CD4(+) T cells as sources of IFN-γ in the joint tissue of B6 IL-10(-/-) mice. These findings suggest the presence of a positive-feedback loop in the joint tissue of infected B6 IL-10(-/-) mice, in which production of inflammatory chemokines, infiltration of IFN-γ-producing cells, and additional production of inflammatory cytokines result in arthritis. This mechanism of arthritis is in contrast to that seen in C3H/He mice, in which arthritis development is linked to transient production of type I IFN and develops independently of IFN-γ. Due to the sustained IFN response driven by NK cells and T cells, we propose the B6 IL-10(-/-) mouse as a potential model to study the persistent arthritis observed in some human Lyme disease patients.

Endothelial cells and fibroblasts amplify the arthritogenic type I IFN response in murine Lyme disease and are major sources of chemokines in Borrelia burgdorferi-infected joint tissue.

Localized elevation in type I IFN has been uniquely linked to the severe Lyme arthritis that develops in C3H mice infected with the spirochete Borrelia burgdorferi. In this study, the dynamic interactions that result in generation of these responses were further examined in C3H mice carrying the type I IFN receptor gene ablation, which effectively blocks all autocrine/paracrine signaling crucial to induction of downstream effectors. Reciprocal radiation chimeras between C3H and IFNAR1⁻/⁻ mice implicated both radiation-sensitive and radiation-resistant cells of the joint tissue in the proarthritic induction of type I IFN. Ex vivo analysis of cells from the naive joint revealed CD45⁺ cells residing in the tissue to be uniquely capable of initiating the type I IFN response to B. burgdorferi. Type I IFN responses were analyzed in real time by lineage sorting of cells from infected joint tissue. This demonstrated that myeloid cells, endothelial cells, and fibroblasts were responsible for propagating the robust IFN response, which peaked at day 7 postinfection and rapidly resolved. Endothelial cells and fibroblasts were the dominant sources of IFN signature transcripts in the joint tissue. Fibroblasts were also the major early source of chemokines associated with polymorphonuclear leukocyte and monocyte/macrophage infiltration, thus providing a focal point for arthritis development. These findings suggest joint-localized interactions among related and unrelated stromal, endothelial, and myeloid cell lineages that may be broadly applicable to understanding the pathogeneses of diseases associated with type I IFN signature, including systemic lupus erythematosus and some rheumatoid arthritides.

Overexpression of Snai3 suppresses lymphoid- and enhances myeloid-cell differentiation.

The altered expression of transcription factors in hematopoietic stem cells and their subsequent lineages can alter the development of lymphoid and myeloid lineages. The role of the transcriptional repressor Snai3 protein in the derivation of cells of the hemato-poietic system was investigated. Snai3 is expressed in terminal T-cell and myeloid lineages, therefore, we chose to determine if expressing Snai3 in the early stages of hematopoietic development would influence cell-lineage determination. Expression of Snai3 by retroviral transduction of hematopoietic stem cells using bone marrow chimera studies demonstrated a block in lymphoid-cell development and enhanced expansion of myeloid-lineage cells. Analysis of Snai3-expressing hematopoietic precursor cells showed normal numbers of immature cells, but a block in the development of cells committed to lymphoid lineages. These data indicate that the overexpression of Snai3 does alter bone marrow cell development and that the identification of genes whose expression is altered by the presence of Snai3 would aid in our understanding of these developmental pathways.

The in vitro derivation of phenotypically mature and diverse B cells from immature spleen and bone marrow precursors.

The capacity of immature B cells of the spleen and bone marrow to differentiate in vitro into cells representing mature end stage cells was investigated using B-cell activating factor belonging to the TNF family (BAFF) and Notch pathway activators. Immature splenic and bone marrow B cells were found, in the presence of both of these activators, to mature into cells with follicular mature (FM) and marginal zone (MZ) cell phenotypes. Such cells were functionally responsive to B-cell-specific activation. The derivation in vitro of cells with an MZ phenotype was more robust from CD23(-) populations than CD23(+) immature/transitional B cells, suggesting a direct immature/T1 B cell to MZ cell differentiation pathway. Transcript analysis of the in vitro-derived B-cell populations demonstrated expression profiles similar to maturing B cells in vivo. FACS-purified populations of B220(+)CD19(+)CD21(-)CD23(-) cells from bone marrow of 2-wk-old mice gave rise to populations of CD21(+)CD23(-) cells with MZ cell phenotypes as well as CD21(+)CD23(+) cells with FM cell phenotypes in percentages similar to those found in vivo. These data suggest that the commitment to an MZ and FM B cell phenotype is set prior to immature B-cell release from the marrow.

Toll-like receptors mediate induction of hepcidin in mice infected with Borrelia burgdorferi.

Hepcidin is the major regulator of systemic iron homeostasis in mammals. Hepcidin is produced mainly by the liver and is increased by inflammation, leading to hypoferremia. We measured serum levels of bioactive hepcidin and its effects on serum iron levels in mice infected with Borrelia burgdorferi. Bioactive hepcidin was elevated in the serum of mice resulting in hypoferremia. Infected mice produced hepcidin in both liver and spleen. Both intact and sonicated B burgdorferi induced hepcidin expression in cultured mouse bone marrrow macrophages. Hepcidin production by cultured macrophages represents a primary transcriptional response stimulated by B burgdorferi and not a secondary consequence of cytokine elaboration. Hepcidin expression induced by B burgdorferi was mediated primarily by activation of Toll-like receptor 2.

Regulation of murine splenic B cell CR3 expression by complement component 3.

Complement component C3 has established roles in both innate and adaptive immune responses. C3 cleavage products function in B cell activation through the complement receptors CD21/35. Phenotypes of Ab production between CD21/35(-/-) and C3(-/-) mice are not always congruent, implicating additional roles for C3 in B cell responses. To further characterize complement and complement receptors, we have identified a role for C3 in the regulation of CR3 on splenic B cells. Splenic B2 cells are not defined as expressing CR3, yet the analysis of splenic B cells from C3(-/-) animals demonstrate cell surface expression of CR3. B cells from both wild-type (WT) and C3(-/-) animals express CR3/CD11b/Itgam (integrin alpha M) gene transcripts although the level of such transcripts is 2- to 3-fold higher in B cells from the C3(-/-) animal vs WT cells. C3(-/-) and WT animals have similar B cell subpopulations with identical CR3 expression on B220(-) cells from the spleen, marrow, and lymph nodes. The C3-deficient environment is responsible for altered CR3 expression as WT splenic B cells transferred into C3(-/-) animals expressed cell surface CR3 within 48 h while transfer of C3(-/-) splenic B cells into WT animals depressed surface expression of CR3. Furthermore, transfer of C3-producing splenic macrophages into C3(-/-) mice depressed CR3 expression by resident B cells. These data suggest a role for C3 in influencing the level of expression of CR3 by modulating the transcript levels encoding the CD11b alpha integrin protein.

Lyme arthritis: linking infection, inflammation and autoimmunity.

Infectious agents can trigger autoimmune responses in a number of chronic inflammatory diseases. Lyme arthritis, which is caused by the tick-transmitted spirochaete Borrelia burgdorferi, is effectively treated in most patients with antibiotic therapy; however, in a subset of patients, arthritis can persist and worsen after the spirochaete has been killed (known as post-infectious Lyme arthritis). This Review details the current understanding of the pathogenetic events in Lyme arthritis, from initial infection in the skin, through infection of the joints, to post-infectious chronic inflammatory arthritis. The central feature of post-infectious Lyme arthritis is an excessive, dysregulated pro-inflammatory immune response during the infection phase that persists into the post-infectious period. This response is characterized by high amounts of IFNγ and inadequate amounts of the anti-inflammatory cytokine IL-10. The consequences of this dysregulated pro-inflammatory response in the synovium include impaired tissue repair, vascular damage, autoimmune and cytotoxic processes, and fibroblast proliferation and fibrosis. These synovial characteristics are similar to those in other chronic inflammatory arthritides, including rheumatoid arthritis. Thus, post-infectious Lyme arthritis provides a model for other chronic autoimmune or autoinflammatory arthritides in which complex immune responses can be triggered and shaped by an infectious agent in concert with host genetic factors.

The Lyme disease spirochete Borrelia burgdorferi utilizes multiple ligands, including RNA, for interferon regulatory factor 3-dependent induction of type I interferon-responsive genes.

We recently discovered a critical role for type I interferon (IFN) in the development of murine Lyme arthritis. Borrelia burgdorferi-mediated induction of IFN-responsive genes by bone marrow-derived macrophages (BMDMs) was dependent upon a functional type I IFN receptor but independent of Toll-like receptor 2 (TLR2), TLR4, TLR9, and the adapter molecule MyD88. We now demonstrate that induction of the IFN transcriptional profile in B. burgdorferi-stimulated BMDMs occurs independently of the adapter TRIF and of the cytoplasmic sensor NOD2. In contrast, B. burgdorferi-induced transcription of these genes was dependent upon a rapid STAT1 feedback amplification pathway. IFN profile gene transcription was IRF3 dependent but did not utilize B. burgdorferi-derived DNA or DNase-sensitive ligands. Instead, IFN-responsive gene expression could be induced by B. burgdorferi-derived RNA. Interferon regulatory factor 3 (IRF3)-dependent IFN profile gene transcription was also induced by sonicated bacteria, by the lipoprotein OspA, and by factors released into the BSKII medium during culture of B. burgdorferi. The IFN-stimulatory activity of B. burgdorferi culture supernatants was not destroyed by nuclease treatment. Nuclease digestion also had no effect on IFN profile induction mediated by sonicated B. burgdorferi. Thus, B. burgdorferi-derived RNA, OspA, and non-nucleic acid ligands present in both sonicated bacteria and B. burgdorferi culture medium contribute to type I IFN-responsive gene induction. These findings suggest that B. burgdorferi invasion of joint tissue and the resultant type I IFN induction associated with Lyme arthritis development may involve multiple triggering ligands.