Development Of Lyme Arthritis Research Articles

Sleep fragmentation disrupts Lyme arthritis resolution in mice

Study objectivesLyme arthritis is a common late-stage complication of infection by Borrelia burgdorferi, the agent of Lyme disease. Patients with Lyme arthritis report increased levels of sleep disturbance associated with pain. Using a mouse model of experimental Lyme arthritis, we investigated the effect of disrupted sleep on the development and resolution of joint inflammation. MethodsLyme arthritis-susceptible C3H/HeJ mice (n = 10/group) were infected with B. burgdorferi and were left either alone (control) or subjected to sleep fragmentation (SF). Arthritis development or resolution were monitored. The impact of SF on immune and inflammatory parameters such as arthritis severity scores, anti-borrelia antibody production, and bacterial clearance was measured. We also determined the effect of SF on arthritis resolution in C3H mice deficient in leukotriene (LT) B4 signaling (BLT1/2−/−) who display delayed Lyme arthritis resolution. ResultsSF had no significant impact on Lyme arthritis development or inflammatory parameters regardless of whether SF treatment began 1 week prior to or congruent with infection. However, initiation of SF at the peak of arthritis resulted in a significant delay in arthritis resolution as measured by joint edema, arthritis severity scores, and decreased bacterial clearance from the joint. This was accompanied by significant changes in joint cytokine transcription levels (e.g., increased TNFα and decreased IL-4). SF has no significant impact on Lyme arthritis resolution in the BLT1/2−/− mice. ConclusionsPoor sleep, especially near the peak of arthritis inflammation, may delay initiation of resolution programs possibly through altering cytokine production and host immune responses, leading to defects in spirochete clearance and prolonged disease.

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

Abstract Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi, with a spectrum of clinical disorders. Infected C3H mice develop severe arthritis while B6 mice display mild arthritis, allowing analysis of host genetic contribution to disease. B6 mice introgressed with the C3H allele of Bbaa1 on Chr4, B6.C3-Bbaa1, display increased severity of arthritis, and heightened expression of Type I IFN. IFNβ, was identified as the key effector for arthritis severity, acting through the muscle regulatory protein myostatin. To identify regulators of IFNβ in the Bbaa1 locus, candidate genes were subjected to siRNA silencing in macrophages. The Cdkn2a gene encoded protein p19ARF was identified as the modulator of IFNβ expression. B6 Arf−/− mice reconstituted with cells expressing the C3H allele of p19ARF developed severe arthritis, whereas mice reconstituted with cells expressing the B6 allele developed mild disease. IFNβ induction by p19ARF is inducible by other pathogens and a variety of PAMPs. P19ARF regulation of IFNβ involved the tumor suppressor p53 and transcription repressor BCL6 in myeloid cells. Indeed, targeted blocking of BCL6 enhanced IFNβ activation in the joint tissue of B6 mice and resulted in increased severity of Lyme arthritis. Similar responses in B6 Rag1−/− mice, indicate dependence on myeloid cells expression of BCL6. Thus, we have identified novel involvement of p19ARF in modulating IFNβ expression in Lyme arthritis development. Supported by R01 AR043521

The Cdkn2a gene product p19 alternative reading frame (p19ARF) is a critical regulator of IFNβ-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 IFNβ 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 IFNβ and IFN-stimulated genes (ISGs) in bone marrow-derived macrophages (BMDMs). The Cdkn2a-encoded p19 alternative reading frame (p19ARF) protein regulates IFNβ induction in BMDMs as shown by siRNA silencing and overexpression of ARF. In vivo studies demonstrated that p19ARF contributes to joint-specific induction of IFNβ and arthritis severity in B. burgdorferi-infected mice. p19ARF regulates B. burgdorferi-induced IFNβ 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 IFNβ-induced Lyme arthritis in vivo and indicate potential novel roles for p19ARF, p53, and BCL6 in Lyme disease and other IFN hyperproduction syndromes.

IFNβ is a critical regulator of Lyme arthritis severity in mice and is regulated by the tumor suppressor gene Cdkn2a on murine Chr4

Abstract Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi, and results in a spectrum of clinical disorders. Infected C3H mice develop severe Lyme arthritis while B6 mice display mild disease, allowing analysis of host genetic contribution to disease. We used forward genetics to identify a region on Chr4, Bbaa1, that includes the Type I IFN cluster and regulates arthritis severity. The development of congenic mice, B6.C3-Bbaa1, isolating the C3H allele of Bbaa1 onto the background of B6 mice demonstrated that Bbaa1 regulates arthritis severity by modulating Type I IFN expression. IFNβ was identified as the key effector for Lyme arthritis severity, however, paradoxically there are no polymorphisms between C3H and B6 in this gene. To identify regulators of IFNβ in the Bbaa1 locus, candidate genes were subjected to siRNA silencing. A single gene, Cdkn2a, was identified as the modulator of IFNβ expression, and its encoded protein ARF was found to regulate the magnitude of IFNβ expression. Reconstitution of Arf−/− mice with hematopoietic cells expressing the C3H allele of Arf developed severe arthritis, whereas mice reconstituted with cells expressing the B6 Arf allele developed mild arthritis. ARF was found to regulate B. burgdorferi induced IFNβ through p53 and BCL6 in myeloid cells. Indeed, targeted blocking of BCL6 enhanced IFNβ activation in the joint tissue of B6 mice and resulted in increased severity of Lyme arthritis. Results were similar in Rag-1-deficient mice, indicating dependence on myeloid cells expression of BCL6. Thus, we have identified novel pathways modulating IFNβ expression in Lyme arthritis development which could lead to therapeutic discovery.

Genetic mechanisms contributing to IFNβ regulation of Lyme Arthritis: involvement of the Borrelia burgdorferi arthritis associated locus 1 (Bbaa1)

Abstract Lyme disease shows a spectrum of clinical disorders in patients that are infected with the tick borne spirochete Borrelia burgdorferi. Studies have shown that C3H mice develop severe arthritis while C57BL/6 (B6) mice display mild arthritis when infected with the same isolate of B. burgdorferi. In order to study the role of the host genetics in regulating the severity of Lyme arthritis development, we used forward genetics and identified a genetic region, Bbaa1, that includes the Type I IFN cluster on Chr4. We further developed congenic mice isolating the C3H allele of Bbaa1 onto the background of B6 mice and determined that Bbaa1 regulates arthritis severity by upregulating Type I IFN. Antibody blocking revealed IFNβ, but not IFNα, as the key effector that drives severe Lyme arthritis. Because there are no SNPs between the C3H and B6 IFNβ genes, we hypothesized that other genetic factors in the Bbaa1 locus regulate the differential expression of IFNβ and Lyme arthritis severity. RNA silencing was used to identify candidate genes within Bbaa1 that regulate IFNβ expression in myeloid cells. Cdkn2a was identified as a strong regulator of IFNβ expression. Although Cdkn2a encodes two proteins, p16 and ARF, only the silencing of Arf resulted in the suppression of IFNβ expression. Additionally, the overexpression of ARF led to enhancement of IFNβ. We propose that ARF regulates IFNβ by two pathways involving the proteins p53 and BCL6. Indeed, blocking of BCL6 by a small molecule inhibitor induced IFNβ activation in the joint tissue of B6 mice and enhanced the severity of Lyme arthritis. Future work will focus on the impact of the two pathways on IFNβ expression and arthritis severity in mice.

A joint effort: The interplay between the innate and the adaptive immune system in Lyme arthritis.

Articular joints are a major target of Borrelia burgdorferi, the causative agent of Lyme arthritis. Despite antibiotic treatment, recurrent or persistent Lyme arthritis is observed in a significant number of patients. The host immune response plays a crucial role in this chronic arthritic joint complication of Borrelia infections. During the early stages of B. burgdorferi infection, a major hinder in generating a proper host immune response is the lack of induction of a strong adaptive immune response. This may lead to a delayed hyperinflammatory reaction later in the disease. Several mechanisms have been suggested that might be pivotal for the development of Lyme arthritis and will be highlighted in this review, from molecular mimicry of matrix metallopeptidases and glycosaminoglycans, to autoimmune responses to live bacteria, or remnants of Borrelia spirochetes in joints. Murine studies have suggested that the inflammatory responses are initiated by innate immune cells, but this does not exclude the involvement of the adaptive immune system in this dysregulated immune profile. Genetic predisposition, via human leukocyte antigen‐DR isotype and microRNA expression, has been associated with the development of antibiotic‐refractory Lyme arthritis. Yet the ultimate cause for (antibiotic‐refractory) Lyme arthritis remains unknown. Complex processes of different immune cells and signaling cascades are involved in the development of Lyme arthritis. When these various mechanisms are fully been unraveled, new treatment strategies can be developed to target (antibiotic‐refractory) Lyme arthritis more effectively.

Genetic Control of Lyme Arthritis by Borrelia burgdorferi Arthritis-Associated Locus 1 Is Dependent on Localized Differential Production of IFN-β 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-β in Lyme arthritis development in B6.C3-Bbaa1 mice. Genetic control of IFN-β 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-β 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.

Borrelia-primed and -infected mice deficient of interleukin-17 develop arthritis after neutralization of gamma-interferon.

The immune mechanisms responsible for development of Lyme arthritis are partially understood with interleukin-17 (IL-17) and gamma-interferon (IFN-γ) playing a generally accepted role. Elevated levels of IL-17 and/or IFN-γ have been reported in samples from human Lyme arthritis patients and experimental mice. In addition, IL-17 and IFN-γ have been implicated in the onset of arthritis in Borrelia-primed and -infected C57BL/6 mice. Recently, we showed that IL-17-deficient mice developed swelling and histopathological changes consistent with arthritis in the presence of high levels of IFN-γ. We hypothesized that neutralization of IFN-γ in IL-17-deficient mice would inhibit Borrelia-induced arthritis. Our results, however, showed that swelling of the hind paws and histopathological changes of arthritis did not differ between Borrelia-primed and -infected IL-17-deficient and wild-type mice with or without neutralization of IFN-γ. We also found higher levels of tumor necrosis factor alpha (TNF-α) and IL-6 in the popliteal lymph node cells of Borrelia-primed and -infected IL-17-deficient mice after neutralization of IFN-γ. These results suggest that multiple cytokines interact in the development of Borrelia-induced arthritis.

T Cells Exacerbate Lyme Borreliosis in TLR2-Deficient Mice.

Infection of humans with the spirochete, Borrelia burgdorferi, causes Lyme borreliosis and can lead to clinical manifestations such as arthritis, carditis, and neurological conditions. Experimental infection of mice recapitulates many of these symptoms and serves as a model system for the investigation of disease pathogenesis and immunity. Innate immunity is known to drive the development of Lyme arthritis and carditis, but the mechanisms driving this response remain unclear. Innate immune cells recognize B. burgdorferi surface lipoproteins primarily via toll-like receptor (TLR)2; however, previous work has demonstrated TLR2−/− mice had exacerbated disease and increased bacterial burden. We demonstrate increased CD4 and CD8 T cell infiltrates in B. burgdorferi-infected joints and hearts of C3H TLR2−/− mice. In vivo depletion of either CD4 or CD8 T cells reduced Borrelia-induced joint swelling and lowered tissue spirochete burden, whereas depletion of CD8 T cells alone reduced disease severity scores. Exacerbation of Lyme arthritis correlated with increased production of CXCL9 by synoviocytes, and this was reduced with CD8 T cell depletion. These results demonstrate T cells can exacerbate Lyme disease pathogenesis and prolong disease resolution possibly through dysregulation of inflammatory responses and inhibition of bacterial clearance.

Arthritis is developed in Borrelia-primed and -infected mice deficient of interleukin-17.

Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis in experimental mice. For example, neutralization of IL-17 with antibodies inhibits induction of arthritis in Borrelia-primed and -infected C57BL/6 wild-type mice. We hypothesized that mice lacking IL-17 would fail to develop Borrelia-induced arthritis. IL-17-deficient and wild-type C57BL/6 mice were primed with heat-inactivated Borrelia and then infected with viable spirochetes 3 weeks later. No swelling or major histopathological changes of the hind paws were detected in IL-17-deficient or wild-type mice that were primed with Borrelia or infected with viable spirochetes. By contrast, IL-17-deficient and wild-type mice that were primed and subsequently infected with heterologous Borrelia developed severe swelling and histopathological changes of the hind paws. In addition, Borrelia-primed and -infected IL-17-deficient mice exhibited elevated gamma-interferon (IFN-γ) levels in sera and increased frequencies of IFN-γ-expressing lymphocytes in popliteal lymph nodes compared to Borrelia-primed and -infected wild-type mice. These results demonstrate that IL-17 is not required for development of severe pathology in response to infection with Borrelia burgdorferi, but may contribute to disease through an interaction with IFN-γ.

Infection of Interleukin 17 Receptor A-Deficient C3H Mice with Borrelia burgdorferi Does Not Affect Their Development of Lyme Arthritis and Carditis.

Recently, a number of studies have reported the presence of interleukin 17 (IL-17) in patients with Lyme disease, and several murine studies have suggested a role for this cytokine in the development of Lyme arthritis. However, the role of IL-17 has not been studied using the experimental Lyme borreliosis model of infection of C3H mice with Borrelia burgdorferi. In the current study, we investigated the role of IL-17 in the development of experimental Lyme borreliosis by infecting C3H mice devoid of the common IL-17 receptor A subunit (IL-17RA) and thus deficient in most IL-17 signaling. Infection of both C3H and C3H IL-17RA(-/-) mice led to the production of high levels of IL-17 in the serum, low levels in the heart tissue, and no detectable IL-17 in the joint tissue. The development and severity of arthritis and carditis in the C3H IL-17RA(-/-) mice were similar to what was seen in wild-type C3H mice. In addition, development of antiborrelia antibodies and clearance of spirochetes from tissues were similar for the two mouse strains. These results demonstrate a limited role for IL-17 signaling through IL-17RA in the development of disease following infection of C3H mice with B. burgdorferi.

Interferon gamma is involved in Borrelia-induced arthritis in mice lacking interleukin-17 (MPF7P.702)

Abstract Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis. Neutralization of IL-17 with antibodies inhibits the progression of arthritis in immunocompetent mice. Our study is the first to examine if Borrelia-induced arthritis occurs with IL-17-deficiency. We hypothesized that mice lacking IL-17 would have low to no Borrelia-induced arthritic symptoms. IL-17 knockout C57BL/6 mice were vaccinated with heat-inactivated Borrelia and/or infected with live spirochetes. Hind paw swelling, histopathology of ankle joints, serum cytokine levels, and T cell populations in tissues were measured. We found insignificant hind paw swelling and pathological changes in mice that were only vaccinated or infected when compared to control naïve mice, due to the Borrelia-resistant C57BL/6 background. However, mice that were both vaccinated and infected had more severe swelling and pathological changes, eliciting a robust immune response disregarding resistance. Also, elevated serum interferon gamma (IFNγ) levels and increasing frequencies of IFNγ-expressing lymphocytes with CD4 or CD8 were also observed in Borrelia-vaccinated and -infected mice. Therefore in these mice, IFNγ is directly involved in Borrelia-induced arthritis without IL-17. Future studies include identifying the role of IFNγ and other understudied IFNγ-expressing immune cells that induce Lyme arthritis in the absence of IL-17.

Follistatin-like protein 1 is a critical mediator of experimental Lyme arthritis and the humoral response to Borrelia burgdorferi infection

Follistatin-like protein 1 (FSTL-1) has recently been described as a critical mediator of CIA and a marker of disease activity. Lyme arthritis, caused by Borrelia burgdorferi, shares similarities with autoimmune arthritis and the experimental murine model collagen-induced arthritis (CIA). Because FSTL-1 is important in CIA and autoimmune arthritides, and Lyme arthritis shares similarities with CIA, we hypothesized that FSTL-1 may be an important mediator of Lyme arthritis. We demonstrate for the first time that FSTL-1 is induced by B. burgdorferi infection and is required for the development of Lyme arthritis in a murine model, utilizing a gene insertion to generate FSTL-1 hypomorphic mice. Using qPCR and qRT-PCR, we found that despite similar early infectious burden, FSTL-1 hypomorphic mice have improved spirochetal clearance in the face of attenuated arthritis and inflammatory cytokine production. Further, FSTL-1 mediates pathogen-specific antibody production and antigen recognition when assessed by ELISA and one- and two-dimensional immunoblotting. This study is the first to describe a role for FSTL-1 in the development of Lyme arthritis and anti-Borrelia response, and the first to demonstrate a role for FSTL-1 in response to infection, highlighting the potential for FSTL-1 as a target in the treatment of B. burgdorferi infection.

Interleukin-10 (IL-10) Inhibits Borrelia burgdorferi-Induced IL-17 Production and Attenuates IL-17-Mediated Lyme Arthritis

Previous studies have shown that cells and cytokines associated with interleukin-17 (IL-17)-driven inflammation are involved in the arthritic response to Borrelia burgdorferi infection. Here, we report that IL-17 is a contributing factor in the development of Lyme arthritis and show that its production and histopathological effects are regulated by interleukin-10 (IL-10). Spleen cells obtained from B. burgdorferi-infected, "arthritis-resistant" wild-type C57BL/6 mice produced low levels of IL-17 following stimulation with the spirochete. In contrast, spleen cells obtained from infected, IL-10-deficient C57BL/6 mice produced a significant amount of IL-17 following stimulation with B. burgdorferi. These mice developed significant arthritis, including erosion of the bones in the ankle joints. We further show that treatment with antibody to IL-17 partially inhibited the significant hind paw swelling and histopathological changes observed in B. burgdorferi-infected, IL-10-deficient mice. Taken together, these findings provide additional evidence of a role for IL-17 in Lyme arthritis and reveal an additional regulatory target of IL-10 following borrelial infection.

Dietary Fish Oil Substitution Alters the Eicosanoid Profile in Ankle Joints of Mice during Lyme Infection1–3

AbstractDietary ingestion of (n-3) PUFA alters the production of eicosanoids and can suppress chronic inflammatory and autoimmune diseases. The extent of changes in eicosanoid production during an infection of mice fed a diet high in (n-3) PUFA, however, has not, to our knowledge, been reported. We fed mice a diet containing either 18% by weight soybean oil (SO) or a mixture with fish oil (FO), FO:SO (4:1 ratio), for 2 wk and then infected them with Borrelia burgdorferi. We used an MS-based lipidomics approach and quantified changes in eicosanoid production during Lyme arthritis development over 21 d. B. burgdorferi infection induced a robust production of prostanoids, mono-hydroxylated metabolites, and epoxide-containing metabolites, with 103 eicosanoids detected of the 139 monitored. In addition to temporal and compositional changes in the eicosanoid profile, dietary FO substitution increased the accumulation of 15-deoxy PGJ2, an antiinflammatory metabolite derived from arachidonic acid. Chiral analysis of the mono-hydroxylated metabolites revealed they were generated from primarily nonenzymatic mechanisms. Although dietary FO substitution reduced the production of inflammatory (n-6) fatty acid-derived eicosanoids, no change in the host inflammatory response or development of disease was detected.

5-Lipoxygenase–Deficient Mice Infected withBorrelia burgdorferiDevelop Persistent Arthritis

The enzyme 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid into the leukotrienes, which are critical regulators of inflammation and inflammatory diseases, such as asthma and arthritis. Although leukotrienes are present in the synovial fluid of Lyme disease patients, their role in the development of Lyme arthritis has not been determined. In the current study, we used a murine model of Lyme arthritis to investigate the role 5-LO products might have in the development of this inflammatory disease. After infection of Lyme arthritis-susceptible C3H/HeJ mice with Borrelia burgdorferi, mRNA expression of 5-LO and 5-LO-activating protein was induced in the joints, and the 5-LO product leukotriene B(4) was produced. Using C3H 5-LO-deficient mice, we demonstrated that 5-LO activity was not necessary for the induction of Lyme arthritis, but that its deficiency resulted in earlier joint swelling and an inability to resolve arthritis as demonstrated by sustained arthritis pathology through day 60 postinfection. Although production of anti-Borrelia IgG was decreased in 5-LO-deficient mice, bacterial clearance from the joints was unaffected. Phagocytosis of B. burgdorferi and efferocytosis of apoptotic neutrophils was defective in macrophages from 5-LO-deficient mice, and uptake of opsonized spirochetes by neutrophils was reduced. These results demonstrate that products of the 5-LO metabolic pathway are not required for the development of disease in all models of arthritis and that caution should be used when targeting 5-LO as therapy for inflammatory diseases.

CD14 Signaling Reciprocally Controls Collagen Deposition and Turnover to Regulate the Development of Lyme Arthritis

CD14 is a glycosylphosphatidylinositol-anchored protein expressed primarily on myeloid cells (eg, neutrophils, macrophages, and dendritic cells). CD14(-/-) mice infected with Borrelia burgdorferi, the causative agent of Lyme disease, produce more proinflammatory cytokines and present with greater disease and bacterial burden in infected tissues. Recently, we uncovered a novel mechanism whereby CD14(-/-) macrophages mount a hyperinflammatory response, resulting from their inability to be tolerized by B. burgdorferi. Paradoxically, CD14 deficiency is associated with greater bacterial burden despite the presence of highly activated neutrophils and macrophages and elevated levels of cytokines with potent antimicrobial activities. Killing and clearance of Borrelia, especially in the joints, depend on the recruitment of neutrophils. Neutrophils can migrate in response to chemotactic gradients established through the action of gelatinases (eg, matrix metalloproteinase 9), which degrade collagen components of the extracellular matrix to generate tripeptide fragments of proline-glycine-proline. Using a mouse model of Lyme arthritis, we demonstrate that CD14 deficiency leads to decreased activation of matrix metalloproteinase 9, reduced degradation of collagen, and diminished recruitment of neutrophils. This reduction in neutrophil numbers is associated with greater numbers of Borrelia in infected tissues. Variation in the efficiency of neutrophil-mediated clearance of B. burgdorferi may underlie differences in the severity of Lyme arthritis observed in the patient population and suggests avenues for development of adjunctive therapy designed to augment host immunity.

Significant differences between the Borrelia-infection and Borrelia-vaccination and -infection models of Lyme arthritis in C3H/HeN mice

The immunological events leading to the development of Lyme arthritis in humans are partially understood. Much of this information has been gained by studying the course of infection of naïve or vaccinated mice with Borrelia burgdorferi. However, the Borrelia-vaccination and -infection model has not been described using the organismal parameters commonly used in the widely accepted Borrelia-infection model. This is the first comparison between the Borrelia-infection and the Borrelia-vaccination and -infection models of arthritis. Borrelia-vaccinated and -infected C3H/HeN mice develop acute inflammation comparable to that of nonvaccinated, Borrelia-infected C3H/HeN mice. The duration and severity of arthritis in Borrelia-vaccinated and -infected mice was slightly increased compared with Borrelia-infected mice. Significantly, Borrelia-vaccinated and -infected C3H/HeN mice produce interleukin-17 (IL-17), while Borrelia-infected mice that had not been previously vaccinated do not. Neutralization of IL-17 in Borrelia-vaccinated and -infected C3H/HeN mice decreased the severity of arthritis, although not to the degree we observed previously in C57BL/6 mice. Collectively, these findings show that the Borrelia-vaccination and -infection model of Lyme arthritis incorporates elements of adaptive immunity that likely have relevance to human disease, but may not be observed in Borrelia-infected C3H/HeN mice.

The Lyme Disease SpirocheteBorrelia burgdorferiUtilizes 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.

Lipidomic Analysis of Dynamic Eicosanoid Responses during the Induction and Resolution of Lyme Arthritis

Eicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2(-/-) mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies.