Susceptibility to experimental Lyme arthritis correlates with KC and monocyte chemoattractant protein-1 production in joints and requires neutrophil recruitment via CXCR2

The development of experimental Lyme arthritis has been correlated with the expression of a number of chemokines and cytokines, however, none of these have been measured directly from the arthritic joint. We examined the temporal expression of IL-1beta, IL-4, IL-6, IL-10, IL-12p70, GM-CSF, IFN-gamma, TNF-alpha, macrophage inflammatory protein-2, KC, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1 directly from the tibiotarsal joint in arthritis-resistant C57BL/6 (B6) and -susceptible C3H/He (C3H) mice. Only the chemokines KC and monocyte chemoattractant protein-1 were differentially expressed in joints of B6 and C3H mice and correlated with the development of Lyme arthritis. Infection of CXCR2(-/-) mice on either genetic background resulted in a significant decrease in the development of pathology, although infection of CCR2(-/-) mice had little or no effect. Neutrophils in CXCR2(-/-) mice were marginalized within blood vessels and could not enter the joint tissue. These results suggest that chemokine-mediated recruitment of neutrophils into the infected joint is a key requirement for the development of experimental Lyme arthritis.     

5-Lipoxygenase-deficient mice infected with Borrelia burgdorferi develop 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.     

CD28 deficiency exacerbates joint inflammation upon Borrelia burgdorferi infection, resulting in the development of chronic Lyme arthritis

Lyme disease, caused by the tick-borne spirochete Borrelia burgdorferi (Bb), is a multisystem illness, affecting many organs, such as the heart, the nervous system, and the joints. Months after Bb infection, approximately 60% of patients experience intermittent arthritic attacks, a condition that in some individuals progresses to chronic joint inflammation. Although mice develop acute arthritis in response to Bb infection, the joint inflammation clears after 2 wk, despite continuous infection, only very rarely presenting with chronic Lyme arthritis. Thus, the lack of an animal system has so far prevented the elucidation of this persistent inflammatory process that occurs in humans. In this study, we report that the majority of Bb-infected CD28-/- mice develop chronic Lyme arthritis. Consistent with observations in chronic Lyme arthritis patients, the infected mutant, but not wild-type mice present recurring monoarticular arthritis over an extended time period, as well as anti-outer surface protein A of Bb serum titers. Furthermore, we demonstrate that anti-outer surface protein A Abs develop in these mice only after establishment of chronic Lyme arthritis. Thus, the Bb-infected CD28-/- mice provide a murine model for studying chronic Lyme arthritis.     

Bone-marrow chimeras reveal hemopoietic and nonhemopoietic control of resistance to experimental Lyme arthritis

Both genetic resistance and susceptibility to development of experimental Lyme arthritis are mediated by the innate immune response. To determine whether this process is mainly controlled by hemopoietic or nonhemopoietic cells, we created bone marrow (BM) chimeric mice between arthritis-resistant DBA/2J (DBA) and arthritis-susceptible C3H/HeJ (C3H) mice and infected them with Borrelia burgdorferi. Both sets of BM chimeric mice, C3H donors into DBA recipients (C-->D) and DBA donors into C3H recipients (D-->C), as well as DBA sham chimeric mice (D-->D) were resistant to the development of experimental Lyme arthritis as measured by ankle swelling and arthritis severity scores. Only the C3H sham chimeric mice (C-->C) developed severe arthritis. These results indicate that independent and nonoverlapping mechanisms exist in hemopoietic and nonhemopoietic cellular compartments that can provide protection against arthritic pathology.     

The cell-mediated immune response to Borrelia afzelii, garinii and burgdorferi in C57BL/6 mice is dependent on antigen specificity

Inbred C57BL/6 mice were challenged with Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen-specific T-cell response in vitro. The sonicated preparations of in vitro grown spirochetes were capable of stimulating polyclonal proliferation and specific cell-mediated response, depending on duration of the cell culture. Murine splenocytes previously sensitized to B. burgdorferi sensu lato (s.l. ), but not those from control mice, could be induced for antigen-specific proliferation in vitro. Moreover, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies as revealed by the [(3)H]thymidine incorporation assay. The current study considers that the strict B. burgdorferi s.l. antigen-specific response may also be expected in infections in humans and contributes to the explanation of the frequently poor antibody- and cell-mediated immune response observed in patients diagnosed with Lyme disease.     

Induction of a B-cell-dependent chronic arthritis with glucose-6-phosphate isomerase

Antibodies specific for glucose-6-phosphate isomerase (G6PI) from T-cell receptor transgenic K/BxN mice are known to induce arthritis in mice, and immunization of DBA/1 mice with G6PI led to acute arthritis without permanent deformation of their joints. Because rheumatoid arthritis is a chronic disease, we set out to identify the capacity of G6PI to induce chronic arthritis in mice. Immunization with recombinant human G6PI induced a chronically active arthritis in mice with a C3H genomic background, whereas the DBA/1 background allowed only acute arthritis and the C57BL/10 background permitted no or very mild arthritis. The disease was associated with the major histocompatibility region sharing an allelic association similar to that of collagen-induced arthritis (i.e. q > p > r). All strains developed a strong antibody response to G6PI that correlated only in the C3H.NB strain with arthritis severity. Similarly, a weak response to type II collagen in a few mice was observed, which was associated with arthritis in C3H.NB mice. Mice on the C3H background also developed ankylosing spondylitis in the vertebrae of the tail. Both C3H.Q and B10.Q mice deficient for B cells were resistant to arthritis. We conclude that G6PI has the ability to induce a chronic arthritis, which is MHC associated and B-cell dependent. Thus, there are striking similarities between this and the collagen-induced arthritis model.     

Transfer of Borrelia burgdorferi s.s. infection via blood transfusion in a murine model

Without antibiotic treatment, the Lyme-disease-causing bacterium, Borrelia burgdorferi can be cultured from the peripheral blood of human patients nearly 6 wk post-tick bite. To determine if Lyme disease spirochetes can be transmitted from a spirochetemic donor mouse to a naive recipient during blood transfusion, blood taken from immunocompetent infected mice was transfused into either immunodeficient (SCID) mice, inbred immunocompetent animals (C3H/HeJ), or outbred mice. Nine of 19 (47.7%) immunodeficient mice, 7 of 15 (46.8%) inbred immunocompetent mice, and 6 of 10 (60.0%) outbred mice became infected with B. burgdorferi after transfusion. Our results indicate that it is possible to acquire B. burgdoferi infection via transfused blood in a mouse model of Lyme borreliosis.     

Cyclooxygenase 2 activity modulates the severity of murine Lyme arthritis

Cyclooxygenase (Cox) is a key enzyme in the biosynthetic metabolism of prostaglandins. The inducible isoform of Cox-2 has been implicated in inflammation and its specific inhibition can be used to treat noninfectious inflammatory diseases, such as rheumatoid arthritis. Borrelia burgdorferi, the agent of Lyme disease, can induce joint inflammation. Here we show that B. burgdorferi induced the upregulation of cox-2 gene expression in murine joints at the onset of arthritis in infected mice. The level of mRNA expression correlated with the degree of inflammation. The specific inhibition of Cox-2 diminished the degree of joint inflammation, without affecting B. burgdorferi-specific antibody or cytokine responses. Cox-2 activity is therefore associated with the genesis of infectious arthritis caused by B. burgdorferi.     

Borrelia burgdorferi complement regulator-acquiring surface protein 2 does not contribute to complement resistance or host infectivity

Borrelia burgdorferi, the pathogen of Lyme disease, cycles in nature through Ixodes ticks and mammalian hosts. At least five Complement Regulator-Acquiring Surface Proteins (BbCRASPs) are produced by B. burgdorferi, which are thought to assist spirochetes in host immune evasion. Recent studies established that BbCRASP-2 is preferentially expressed in mammals, and elicits robust antibody response in infected hosts, including humans. We show that BbCRASP-2 is ubiquitously expressed in diverse murine tissues, but not in ticks, reinforcing a role of BbCRASP-2 in conferring B. burgdorferi defense against persistent host immune threats, such as complement. BbCRASP-2 immunization, however, fails to protect mice from B. burgdorferi infection and does not modify disease, as reflected by the development of arthritis. An infectious BbCRASP-2 mutant was generated, therefore, to examine the precise role of the gene product in spirochete infectivity. Similar to wild type B. burgdorferi, BbCRASP-2 mutants remain insensitive to complement-mediated killing in vitro, retain full murine infectivity and induce arthritis. Quantitative RT-PCR assessment indicates that survivability of BbCRASP-2-deficient B. burgdorferi is not due to altered expression of other BbCRASPs. Together, these results suggest that the function of a selectively expressed B. burgdorferi gene, BbCRASP-2, is not essential for complement resistance or infectivity in the murine host.     

Characterization of Ross River virus tropism and virus-induced inflammation in a mouse model of viral arthritis and myositis

Mosquito-borne alphaviruses are a significant cause of both encephalitic and arthritic disease in humans worldwide. In contrast to the encephalitic alphaviruses, the pathogenesis of alphavirus-induced arthritic disease is not well understood. Utilizing a mouse model of Ross River virus (RRV) disease, we found that the primary targets of RRV infection are bone, joint, and skeletal muscle tissues of the hind limbs in both outbred CD-1 mice and adult C57BL/6J mice. Moreover, histological analyses demonstrated that RRV infection resulted in severe inflammation of these tissues. Characterization of the inflammatory infiltrate within the skeletal muscle tissue identified inflammatory macrophages, NK cells, and CD4+ and CD8+ T lymphocytes. To determine the contribution of the adaptive immune system, the outcome of RRV-induced disease was examined in C57BL/6J RAG-1(-/-) mice, which lack functional T and B lymphocytes. RAG-1(-/-) and wild-type mice developed similar disease signs, infiltration of inflammatory macrophages and NK cells, and muscle pathology, suggesting that the adaptive immune response does not play a critical role in the development of disease. These results establish the mouse model of RRV disease as a useful system for the identification of viral and host factors that contribute to alphavirus-induced arthritis and myositis.     

Impaired host defense to infection and Toll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice

To investigate the role of Toll-like receptor 2 (TLR2)-mediated signaling in host innate defense and development of Lyme disease, the pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing two distinct genotypes (RST1 and RST3A) was assessed in TLR2(-/-) C3H/HeJ mice. All TLR2(-/-) mice infected with a B. burgdorferi RST1 isolate developed severe arthritis. The numbers of spirochetes in heart, joint and ear biopsy specimens were significantly higher in TLR2(-/-) mice than in wild-type mice similarly infected as determined by real-time quantitative polymerase chain reaction. Interestingly, despite the higher spirochete levels in heart tissues, milder carditis was observed in TLR2(-/-) than in wild-type mice infected with this RST1 isolate (P=0.02). By contrast, no positive cultures were obtained from any of the blood and tissue specimens from TLR2(-/-) mice inoculated with two RST3A clinical isolates. The data suggest that there is impaired host innate defense against infection and TLR2-independent killing of B. burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice.     

Gestational attenuation of Lyme arthritis is mediated by progesterone and IL-4

Infection of different strains of laboratory mice with the agent of Lyme disease, Borrelia burgdorferi, results in arthritis, the severity of which has been correlated with the dominance of Th1 cytokines. In this study, we demonstrate that changes in B. burgdorferi-specific immunologic responses associated with pregnancy can alter the outcome of Lyme arthritis in mice. Whereas nonpregnant female C3H mice consistently developed severe Lyme arthritis, pregnant mice had a marked reduction in arthritis severity that was associated with a slight reduction in IFN-gamma and markedly increased levels of IL-4 production by B. burgdorferi-specific T cells. Similar reductions in arthritis severity and patterns of cytokine production were observed in nonpregnant, progesterone-implanted mice. Ab neutralization of IL-4 in progesterone-implanted mice resulted in severe arthritis. Our results are consistent with the known shift toward Th2 cytokine expression at the maternal-fetal interface, and are the first to show a pregnancy-related therapeutic effect in an infectious model.     

Fc gamma R expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA)

STATEMENT OF FINDINGS: We investigated the role of Fc gamma receptors (Fc gamma Rs) on synovial macrophages in immune-complex-mediated arthritis (ICA). ICA elicited in knee joints of C57BL/6 mice caused a short-lasting, florid inflammation and reversible loss of proteoglycans (PGs), moderate chondrocyte death, and minor erosion of the cartilage. In contrast, when ICA was induced in knee joints of Fc receptor (FcR) gamma-chain(-/-) C57BL/6 mice, which lack functional Fc gamma RI and RIII, inflammation and cartilage destruction were prevented. When ICA was elicited in DBA/1 mice, a very severe, chronic inflammation was observed, and significantly more chondrocyte death and cartilage erosion than in arthritic C57BL/6 mice. The synovial lining and peritoneal macrophages of na?ve DBA/1 mice expressed a significantly higher level of Fc gamma Rs than was seen in C57BL/6 mice. Moreover, elevated and prolonged expression of IL-1 was found after stimulation of these cells with immune complexes. Zymosan or streptococcal cell walls caused comparable inflammation and only mild cartilage destruction in all strains. We conclude that Fc gamma R expression on synovial macrophages may be related to the severity of synovial inflammation and cartilage destruction during ICA.     

FcgammaR expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA)

We investigated the role of Fcγ receptors (FcγRs) on synovial macrophages in immune-complex-mediated arthritis (ICA). ICA elicited in knee joints of C57BL/6 mice caused a short-lasting, florid inflammation and reversible loss of proteoglycans (PGs), moderate chondrocyte death, and minor erosion of the cartilage. In contrast, when ICA was induced in knee joints of Fc receptor (FcR) γ-chain^-/- C57BL/6 mice, which lack functional FcγRI and RIII, inflammation and cartilage destruction were prevented. When ICA was elicited in DBA/1 mice, a very severe, chronic inflammation was observed, and significantly more chondrocyte death and cartilage erosion than in arthritic C57BL/6 mice. The synovial lining and peritoneal macrophages of na?ve DBA/1 mice expressed a significantly higher level of FcγRs than was seen in C57BL/6 mice. Moreover, elevated and prolonged expression of IL-1 was found after stimulation of these cells with immune complexes. Zymosan or streptococcal cell walls caused comparable inflammation and only mild cartilage destruction in all strains. We conclude that FcγR expression on synovial macrophages may be related to the severity of synovial inflammation and cartilage destruction during ICA.     

Characterization of Bglu3, a mouse fasting glucose locus, and identification of Apcs as an underlying candidate gene

Bglu3 is a quantitative trait locus for fasting glucose on distal chromosome 1 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE(-/-)) mice. This locus was subsequently replicated in two separate mouse intercrosses. The objective of this study was to characterize Bglu3 through construction and analysis of a congenic strain and identify underlying candidate genes. Congenic mice were constructed by introgressing a genomic region harboring Bglu3 from C3H.apoE(-/-) into B6.apoE(-/-) mice. Mice were started with a Western diet at 6 wk of age and maintained on the diet for 12 wk. Gene expression in the liver was analyzed by microarrays. Congenic mice had significantly higher fasting glucose levels and developed more significant glucose intolerance compared with B6.apoE(-/-) mice on the Western diet. Microarray analysis revealed 336 genes to be differentially expressed in the liver of congenic mice. Further pathway analysis suggested a role for acute phase response signaling in regulating glucose intolerance. Apcs, encoding an acute phase response protein serum amyloid P (SAP), is located underneath the linkage peak of Bglu3. Multiple single nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Apcs. Apcs expression in the liver was significantly higher in congenic and C3H mice compared with B6 mice. The Western diet consumption led to a gradual rise in plasma SAP levels, which was accompanied by rising fasting glucose in both B6 and C3H apoE(-/-) mice. Expression of C3H Apcs in B6.apoE(-/-) mice aggravated glucose intolerance. Bglu3 is confirmed to be a locus affecting diabetes susceptibility, and Apcs is a probable candidate gene.