Adenoviral delivery of interleukin-10 fails to attenuate experimental Lyme disease

Production of interleukin-10 (IL-10) by C57BL/6 mice following infection with Borrelia burgdorferi has been proposed as a mechanism whereby resistance to the development of experimental Lyme arthritis is maintained. In the current study, we sought to determine the role of IL-10 during infection of arthritis- and carditis-susceptible C3H mice. Infection of C3H IL-10^−/− mice led to increased joint swelling and arthritis severity scores over those of wild-type C3H mice. Measurement of B. burgdorferi numbers in joints or disseminated tissues indicated a more efficient clearance of spirochetes in the absence of IL-10, similar to that reported in C57BL/6 IL-10^−/− mice. However, in contrast to previous in vitro work, infection of C3H IL-10^−/− mice led to decreased in vivo expression of the cytokines KC, IL-1β, IL-4, and IL-12p70 in the infected joints. Finally, adenoviral expression of IL-10 in the infected joints of C3H mice was unable to modulate the development of severe Lyme arthritis and had no effect on spirochete clearance or Borrelia-specific antibody production. Development of Lyme carditis appeared to be independent of modulation by IL-10. These results suggest that IL-10 limits the development of joint inflammation in both arthritis-resistant and -susceptible mouse strains infected with B. burgdorferi and that increased IL-10 production cannot rescue genetic susceptibility to development of pathology in this model.     

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.     

Macrophage Polarization during Murine Lyme Borreliosis

Infection of C3H mice with Borrelia burgdorferi, the causative agent of Lyme disease, reliably produces an infectious arthritis and carditis that peak around 3 weeks postinfection and then spontaneously resolve. Macrophage polarization has been suggested to drive inflammation, the clearance of bacteria, and tissue repair and resolution in a variety of infectious disease models. During Lyme disease it is clear that macrophages are capable of clearing Borrelia spirochetes and exhausted neutrophils; however, the role of macrophage phenotype in disease development or resolution has not been studied. Using classical (NOS2) and alternative (CD206) macrophage subset-specific markers, we determined the phenotype of F4/80⁺ macrophages within the joints and heart throughout the infection time course. Within the joint, CD206⁺ macrophages dominated throughout the course of infection, and NOS2⁺ macrophage numbers became elevated only during the peak of inflammation. We also found dual NOS2⁺ CD206⁺ macrophages which increased during resolution. In contrast to findings for the ankle joints, numbers of NOS2⁺ and CD206⁺ macrophages in the heart were similar at the peak of inflammation. 5-Lipoxygenase-deficient (5-LOX^−/−) mice, which display a failure of Lyme arthritis resolution, recruited fewer F4/80⁺ cells to the infected joints and heart, but macrophage subset populations were unchanged. These results highlight differences in the inflammatory infiltrates during Lyme arthritis and carditis and demonstrate the coexistence of multiple macrophage subsets within a single inflammatory site.     

Outer Surface Protein OspC Is an Antiphagocytic Factor That Protects Borrelia burgdorferi from Phagocytosis by Macrophages

Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface of Borrelia burgdorferi during tick feeding and the early phase of mammalian infection. OspC is required for B. burgdorferi to establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses. However, the exact biological function of OspC remains elusive. In this study, we showed that the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγ^null mice that lack B cells, T cells, NK cells, and lytic complement. The ospC mutant also could not establish infection in anti-Ly6G-treated SCID and C3H/HeN mice (depletion of neutrophils). However, depletion of mononuclear phagocytes at the skin site of inoculation in SCID and C3H/HeN mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted mice, the ospC mutant was able to colonize the joints and triggered neutrophilia during dissemination. Furthermore, we found that phagocytosis of green fluorescent protein (GFP)-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 macrophage-like cells, but not in PMN-HL60, was significantly higher than parental wild-type B. burgdorferi strains, suggesting that OspC has an antiphagocytic property. In addition, overproduction of OspC in spirochetes also decreased the uptake of spirochetes by murine peritoneal macrophages. Together, our findings provide evidence that mononuclear phagocytes play a key role in clearance of the ospC mutant and that OspC promotes spirochetes'' evasion of macrophages during early Lyme borreliosis.     

The Chemokine Receptor CXCR2 Ligand KC (CXCL1) Mediates Neutrophil Recruitment and Is Critical for Development of Experimental Lyme Arthritis and Carditis

Deletion of the chemokine receptor CXCR2 prevents the recruitment of neutrophils into tissues and subsequent development of experimental Lyme arthritis. Following footpad inoculation of Borrelia burgdorferi, the agent of Lyme disease, expression of the CXCR2 ligand KC (CXCL1) is highly upregulated in the joints of arthritis-susceptible mice and is likely to play an important role in the recruitment of neutrophils to the site of infection. To test this hypothesis, we infected C3H KC^−/− mice with B. burgdorferi and followed the development of arthritis and carditis. Ankle swelling was significantly attenuated during the peak of arthritis in the KC^−/− mice. Arthritis severity scores were significantly lower in the KC^−/− mice on days 11 and 21 postinfection, with fewer neutrophils present in the inflammatory lesions. Cardiac lesions were also significantly decreased in KC^−/− mice at day 21 postinfection. There were, however, no differences between C3H wild-type and KC^−/− mice in spirochete clearance from tissues. Two other CXCR2 ligands, LIX (CXCL5) and MIP-2 (CXCL2), were not increased to compensate for the loss of KC, and the production of several innate cytokines was unaltered. These results demonstrate that KC plays a critical nonredundant role in the development of experimental Lyme arthritis and carditis via CXCR2-mediated recruitment of neutrophils into the site of infection.The recruitment of neutrophils to the site of infection is a critical first step in the immune response to pathogens. Neutrophils are highly phagocytic and are capable of engulfing and destroying many microbial invaders; however, they are also linked to tissue damage, which can occur upon the extracellular release of neutrophil granule contents. In experimental Lyme arthritis, neutrophils are the primary cell type present during the acute inflammatory phase, while macrophages predominate during the resolution phase of the disease (6). The presence of live Borrelia burgdorferi spirochetes is required for the development of disease, as indicated by the failure of injecting dead spirochetes or borrelial antigen to cause arthritis (7, 26). The presence of spirochetes in the joint alone, however, is not sufficient to induce disease, as some inbred mouse strains are resistant to the development of Lyme arthritis even though they harbor as many spirochetes in the joint as arthritis-susceptible strains (12, 34). Thus, experimental Lyme arthritis is an immunopathology, most likely resulting from an overly exuberant or inappropriately regulated inflammatory response.Neutrophils are required for the development of arthritis in many if not most experimental animal models. Depletion of neutrophils has been shown to ameliorate or attenuate disease severity in adjuvant-induced arthritis (AIA) (46), collagen-induced arthritis (CIA) (47), streptococcal cell wall-induced arthritis (48), K/BxN serum transfer arthritis (54), and collagen antibody-induced arthritis (CAIA) (56). Similarly, treatment modalities that alter the recruitment of neutrophils or their ability to extravasate into the joint tissue can also have profound effects on both the incidence and severity of arthritis. For example, blocking E-selectin or junctional adhesion molecule C (JAM-C) can inhibit AIA (23, 42), and blocking VCAM-1 or CD147 or inhibiting granulocyte colony-stimulating factor (G-CSF)-mediated upregulation of adhesion molecules can reduce the development of CIA (13, 16, 18). Thus, the development of arthritis in general appears to require the presence of neutrophils within the joint tissue, and inhibition of neutrophil extravasation, and perhaps activation, can prevent the onset and progression of disease.Chemokines are chemotactic cytokines that guide the homeostatic movement of cells, as well as leukocyte recruitment during immune responses. Chemokines mediate their biologic effects via G protein-coupled receptors, including the receptors CXCR1 and CXCR2, which appear to play major roles in neutrophil recruitment during arthritis (27). The human neutrophil chemoattractant ligands for these receptors, GROα (CXCL1) (29), ENA-78 (CXCL5) (28), and interleukin-8 (IL-8; CXCL8) (30), are abundantly present in the synovial fluid of rheumatoid arthritis (RA) patients. Studies in animal models have demonstrated that antagonism or genetic depletion of CXCR2 can block the development of arthritis in CAIA (36), AIA (5, 15, 20), and K/BxN serum-induced arthritis (24); however, studies targeting CXCR2 ligands have been less successful at blocking disease progression (8). This may stem from the perceived redundancy of chemokines and their receptors or from the difficulty of targeting the chemokines themselves in vivo, e.g., via antibody-mediated blockade or depletion.In a previous study, we measured the production over time of 12 cytokines and chemokines directly from the ankle joints of B. burgdorferi-infected mouse strains in order to gain insight into the mechanisms of disease resistance and susceptibility to experimental Lyme arthritis (10). Of the cytokines and chemokines tested, only the production of KC and monocyte chemoattractant protein 1 (MCP-1; CCL2) correlated with the development of arthritis. KC is a major neutrophil chemoattractant and binds to CXCR2 in mice, while MCP-1 is a major monocyte chemoattractant and binds to CCR2 in mice. Infection of C3H CCR2^−/− mice had little effect on the development of Lyme arthritis, but C3H CXCR2^−/− mice were fully protected from the development of disease. However, whether KC alone was responsible for the CXCR2-mediated recruitment of neutrophils into the infected joint or whether other chemokines or neutrophil chemoattractants also played a role was unclear. In the current study, we have used mice genetically deficient in KC (KC^−/−) to determine the role of KC-mediated CXCR2 signaling in the pathogenesis of Lyme borreliosis. Our results demonstrate that KC plays a critical role in the development of both arthritis and carditis in the murine model of Lyme disease.     

The DBA/1 Strain Is a Novel Mouse Model for Experimental Borrelia burgdorferi Infection

Lyme arthritis, caused by Borrelia burgdorferi, has similarities to rheumatoid arthritis and its experimental murine model, collagen-induced arthritis (CIA). Currently, no common strain exists for examination of arthritis models of Lyme arthritis and CIA, which are typically studied in C3H/HeJ and DBA/1 mice, respectively. The aim of this study was to define the characteristics of Borrelia burgdorferi infection and arthritis in the DBA/1 murine strain. Murine Lyme arthritis was induced in C3H/HeJ and DBA/1 mice by subcutaneous infection with B. burgdorferi. Tibiotarsal joints were measured during infection, and mice were sacrificed for histologic, microbiologic, and serologic analysis on days 14 and 42 postinfection. All bladder cultures obtained from C3H/HeJ and DBA/1 mice at 14 days postinfection grew Borrelia. There was no significant difference in spirochetal burdens in hearts and tibiotarsal joints at days 14 and 42 postinfection. Tibiotarsal joint swelling and histologic scoring were not significantly different between the two strains. Serologic analysis revealed increased IgG2a production in C3H/HeJ mice compared to DBA/1 mice. Analysis of 2-dimensional immunoblots revealed several specific antigens (LA7, BBA03, BBA64, BBA73, OspA, and VlsE) which were not recognized by DBA/1 sera. We conclude that the DBA/1 murine strain is a suitable model for the study of Lyme arthritis and experimental B. burgdorferi infection, allowing direct comparison between Lyme arthritis and collagen-induced arthritis. The specificity of the humoral immune response differs between the two strains, further study of which may reveal important findings about how individual strains respond to B. burgdorferi infection.     

The Caspase 1 Inflammasome Is Not Required for Control of Murine Lyme Borreliosis

The contribution of the inflammasome to the development of immune responses and disease during infection with the Lyme disease spirochete, Borrelia burgdorferi, is not well defined. Host defense against the spirochete is severely impaired in mice deficient in the adaptor molecule myeloid differentiation antigen 88 (MyD88), which is required not only for Toll-like receptor-mediated responses but also for the production of the proforms of interleukin 1β (IL-1β) and IL-18. These cytokines are released in active forms after cleavage by the inflammasome-associated enzyme caspase 1. To investigate the contribution of the inflammasome to host defense against B. burgdorferi, we examined Lyme borreliosis in mice deficient in either caspase 1 or apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), a molecule upstream of caspase 1 in the inflammasome signaling cascade. We found that caspase 1-deficient mice had a mild transient elevation in pathogen burden and a trend toward an increase in the prevalence of arthritis early in infection, but these differences resolved by day 14 postinfection. Caspase 1 deficiency had no effect on B. burgdorferi-induced humoral immunity, T-cell responses, or the abilities of macrophages to ingest and degrade spirochetes. The absence of the ASC protein had no effect on the control of the spirochete or the development of immune responses and disease. These findings reveal that the caspase 1 inflammasome is not critical to host defense against the extracellular pathogen Borrelia burgdorferi.Infection of humans with the Lyme disease spirochete, Borrelia burgdorferi, results in a characteristic pattern of skin lesions, arthritis, carditis, and neurologic abnormalities that reflect the immune response to the spirochete as it invades and disseminates in the mammalian host (7). In the murine model of Lyme borreliosis, spirochetes inoculated into the skin disseminate within days to infect all organ systems, but disease is primarily manifested in the joints and heart (4). Disease in the animal model is due largely to the innate immune response to spirochetes because histopathology reveals mainly neutrophils and macrophages within inflamed joints and hearts, respectively (5, 28, 36, 43), and occurs in the absence of adaptive (T- and B-cell-mediated) immunity (8, 28, 43).Recent studies have further defined the role of innate immunity in Lyme borreliosis. B. burgdorferi lipoproteins activate innate immune cells through the pattern recognition molecule Toll-like receptor 2 (TLR2), which is required for innate but not adaptive immune responses to the spirochete (2, 19, 49). Spirochete components also stimulate murine cells through TLR5 and TLR9 (44). The TLR cytosolic domains contain a Toll/interleukin 1 (IL-1) receptor domain (TIR) that interacts with myeloid differentiation antigen 88 (MyD88) and results in the activation of NF-κB and the production of proinflammatory cytokines, chemokines, and costimulatory molecules that are important for host defense (6, 12, 14). We and others have previously shown that B. burgdorferi-infected MyD88-deficient (MyD88^−/−) mice have significantly elevated pathogen burdens that persist through 90 days of infection despite the presence of high titers of anti-B. burgdorferi antibodies (9, 25). The elevated level of pathogen DNA in tissues was explained in part by our finding that MyD88^−/− peritoneal macrophages ingested spirochetes at the same rate as wild-type (WT) cells, but the kinetics of degradation was slower, with internalized spirochetes remaining in an elongated form for a longer period. Others have found that bone marrow-derived MyD88^−/− macrophages do not efficiently ingest spirochetes (44). The MyD88^−/− mice developed carditis and arthritis similar to the disease in WT mice analyzed at its peak (days 14 and 28) and during regression (day 45) (9, 25). Together, these results showed that MyD88-dependent signaling pathways are not required for B. burgdorferi-induced inflammation or disease regression but are necessary for efficient control of the pathogen burden by phagocytes. These studies did not distinguish whether interruption of MyD88-dependent TLR signaling pathways was solely responsible for the impaired control of the pathogen or whether other MyD88-dependent pathways also play a role.In addition to being a crucial signaling molecule for TLRs involved in B. burgdorferi recognition, MyD88 is required for IL-1 receptor (IL-1R)- and IL-18R-associated kinase signaling. TLR activation is a key inducer of the proforms of IL-1β and IL-18, and the secreted forms of these two cytokines require MyD88 for their receptors to mediate their effects (1, 34, 38). Behera et al. (6) have shown that IL-18 alone does not significantly contribute to host immunity in Lyme borreliosis because IL-18^−/− mice exhibit no defects in pathogen clearance or the development of disease. IL-1β, however, may play a role because human peripheral blood mononuclear cells secrete IL-1β after ingestion of live B. burgdorferi spirochetes (15). In support of this hypothesis, serum levels of IL-1β were reported to be elevated in Lyme disease patients, and the levels decreased significantly after doxycycline treatment (35). IL-1β mRNA levels in erythema migrans lesions were also shown to be elevated (31).To further delineate the role of MyD88-dependent signaling pathways in host defense against B. burgdorferi, we examined the course of Lyme borreliosis in mice deficient in either the intracellular cysteine protease caspase 1 or apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). Caspase 1 plays a key role in inflammatory responses by cleaving pro-IL-1β and pro-IL-18 into their active secreted forms (16, 22). These cytokines are matured in a large caspase 1-containing protein complex called the inflammasome (37). ASC, a component of the inflammasome, is required for eliciting the enzymatic activity of caspase 1. Caspase 1 contains an N-terminal caspase recruitment domain (CARD) shown to be involved in the assembly of protein platforms that promote proteolytic activation of recruited caspases in the context of apoptosis and inflammation (14). In addition to cleaving pro-IL-1 and pro-IL-18, caspase 1 is also involved in other proinflammatory pathways, including NF-κB signaling pathways associated with innate and adaptive immune responses (21, 23, 41). In contrast, ASC is essential only for the secretion of IL-1β/IL-18 but dispensable for caspase 1-mediated IL-6 and tumor necrosis factor alpha secretion and NF-κB and p38 activation (40). Thus, although both caspase 1^−/− mice and ASC^−/− mice have defects in the production of IL-1β/IL-18, caspase 1^−/− mice have additional defects in the activation of NF-κB.Several published reports have established that the inflammasome is important for immunity to intracellular bacteria and viruses, but much less is known about the contribution of the inflammasome to host defense against extracellular pathogens that elicit cytokines activated by caspase 1 (27, 29, 30, 32, 38, 42, 48). Thus, we sought to determine whether the inflammasome is also important during infection with the B. burgdorferi spirochete as representative of a subset of extracellular pathogens. We found that while B. burgdorferi can elicit IL-1β in a caspase 1-dependent fashion from mouse macrophages in vitro, the caspase 1-dependent inflammasome is not essential for the ultimate control of B. burgdorferi infection and disease.     

CCR2 receptor is essential to activate microbicidal mechanisms to control Toxoplasma gondii infection in the central nervous system

Chemokines comprise a structurally related family of cytokines that regulate leukocyte trafficking. Because infection with Toxoplasma gondii can induce an important inflammatory reaction that, if left uncontrolled, can lead to death, we investigated the role of the chemokine receptor CCR2 in T. gondii infection. We orally infected CCR2^−/− mice with five ME-49 T. gondii cysts and monitored morbidity, survival, and immune response thereafter. The CCR2^−/− mice displayed higher susceptibility to infection as all mice died on day 28 after infection. Despite similar Th1 responses, a more evident anti-inflammatory response was induced in the peripheral organs of CCR2^−/− mice compared with wild-type C57BL/6 mice. Additionally, CCR2^−/− mice presented greater parasitism and a milder inflammatory reaction in their peripheral organs with lesser CD4⁺ and MAC-1⁺ and greater CD8⁺ cell migration. The parasite load decreased in these organs in CCR2^−/− mice but remained uncontrolled in the central nervous system. Additionally, we observed down-regulated inducible nitric oxide synthase expression in peripheral organs from CCR2^−/− mice that was associated with a small nitric oxide production by spleen macrophages. In conclusion, in the absence of CCR2, another mechanism is activated to control tissue parasitism in peripheral organs. Nevertheless, CCR2 is essential for the activation of microbicidal mediators that control T. gondii replication in the central nervous system.     

Gamma Interferon Inhibits Production of Anti-OspA Borreliacidal Antibody In Vitro

The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-γ). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-γ was not affected by the time of treatment. In addition, treatment with rIFN-γ inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-γ marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-γ does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody.     

Matrix Metalloproteinase 9 Plays a Key Role in Lyme Arthritis but Not in Dissemination of Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme arthritis, does not produce any exported proteases capable of degrading extracellular matrix despite the fact that it is able to disseminate from a skin insertion site to infect multiple organs. Prior studies have shown that B. burgdorferi induces the host protease, matrix metalloproteinase 9 (MMP-9), and suggested that the induction of MMP-9 may allow the organism to disseminate and produce local tissue destruction. We examined the role of MMP-9 in dissemination of B. burgdorferi and pathogenesis of Lyme arthritis. In a MMP-9^−/− mouse model, MMP-9 was not required for the dissemination of the spirochete to distant sites. However, MMP-9^−/− exhibited significantly decreased arthritis compared to wild-type mice. The decrease in arthritis was not due to an inability to control infection since the spirochete numbers in the joints were identical. Levels of inflammatory chemokines and cytokines were also similar in MMP-9^−/− and wild-type mice. We examined whether decreased inflammation in MMP-9^−/− mice may be the result of decreased production of neoattractants by MMP-9-dependent cleavage of collagen. MMP-9 cleavage of type I collagen results in increased monocyte chemoattraction. MMP-9 plays an important role in regulating inflammation in Lyme arthritis, potentially through the cleavage of type I collagen.Borrelia burgdorferi, the causative agent of Lyme disease, causes a vast array of clinical symptoms in the human host including but not limited to erythema migrans skin lesions, meningitis, myocarditis, and arthritis (5, 12, 50). In order to establish infection, the spirochete migrates from the initial site of inoculation, spreads into the surrounding skin, and subsequently enters the bloodstream, where it can colonize more distal organs. While this dissemination almost certainly requires breakdown of cellular barriers and digestion of extracellular matrix (ECM) proteins, the sequenced B. burgdorferi genome does not encode for any spirochetal genes that share homology with known exported proteases that degrade the ECM. One class of host molecules that may aid in the dissemination of the spirochete during infection is the matrix metalloproteinases (MMPs). MMPs are zinc and calcium ion-dependent, secreted proteases that function in the degradation and remodeling of ECM (42).MMP-9 (also known as gelatinase B) is a 92-kDa enzyme whose substrates include collagen (types I, II, IV, V, and VII), elastin, and gelatin (18, 39, 48). MMP-9 is upregulated in erythema migrans in the skin of patients with acute Lyme disease and in in vitro models of skin infection (54). It has been proposed to play an important role in the dissemination of the spirochete from the initial site of infection (54). Supporting the notion that MMP-9 may promote the dissemination of the spirochete, activated MMP-9 released from human peripheral monocytes and human neutrophils has been shown to promote the translocation of B. burgdorferi through matrices of type I and type IV collagen and laminin (27). However, the role of MMP-9 in dissemination has not been confirmed in an animal or human model.In addition to a potential role in dissemination of B. burgdorferi, MMPs may play a role in the development of some of the signature manifestations of Lyme disease, including arthritis and carditis. MMPs have been shown to play a role in many different forms of arthritis and can be responsible for the degradation of bone and cartilage tissue. Increased amounts of MMP-9 have been found in the synovial fluids of some Lyme arthritis patients, as well as in cartilage explants; however, in vitro expression from chondrocytes were not seen after B. burgdorferi infection, suggesting that the source of MMP-9 may be from other infiltrating cells (7, 32). Using gene arrays of infected joints, which contain multiple cell types, Crandall et al. (19) found that MMP-9 was significantly increased at 2 weeks but not at 4 weeks postinfection in C3H mice. Changes in the balance of activated to inactive MMP-9 were not examined. B. burgdorferi has been shown to induce the MMP-9 activator, MMP-3, in the joint tissues of infected mice (7, 19).Activated MMP-9 may play a role in modulation of inflammation through its actions on inflammatory mediators. MMPs can have multiple effects on inflammation through their action in cleaving and activating or inactivating chemokines, cytokines, and their receptors. In addition, certain MMPs, including MMP-8, MMP-12, and MMP-14, have been shown to cleave ECM proteins to release peptides with chemoattractive and/or inflammatory properties (24, 31, 41). Because of the potential for opposing effects on inflammation, it is difficult to predict the inflammatory responses in MMP-9 deficient mice to various challenges (43). To date, different types of pathogens tested in MMP-9 deficient mice have shown different responses in terms of control of infection and level of inflammation with Staphylococcus aureus and Escherichia coli in an abdominal sepsis model causing greater inflammation, while Francisella tularensis, Mycobacterium tuberculosis, and Chlamydia muridarum infection showed decreased inflammation in MMP-9^−/− mice (13, 34, 40, 45, 51). These findings suggest that the effect of MMP-9 may be both pathogen and tissue specific.We were interested in determining the effects of MMP-9 deletion in an animal model of Lyme disease. We report here the impact of MMP-9 on the ability of the organism to disseminate to distant sites and on the development of murine arthritis and carditis.     

Distinct Characteristics of Resistance to Borrelia burgdorferi-Induced Arthritis in C57BL/6N Mice

Studies of mice infected with Borrelia burgdorferi have indicated that the severity of arthritis is influenced by the genetic composition of the host: the C3H mouse develops severe arthritis while BALB/c and C57BL/6 mice develop mild arthritis. In this study, the effects of increasing infectious dose on the severity of arthritis were determined in these three mouse strains. C3H/He mice developed severe arthritis at all infectious doses, with 100% infection requiring 200 spirochetes. In BALB/cAnN mice, arthritis severity was dependent on infectious dose; symptoms were mild with infection by 200 B. burgdorferi and progressively more severe with increasing infectious dose. Infection of BALB/cAnN mice with 2 × 10⁴ B. burgdorferi resulted in arthritis with severity identical to that in C3H/He mice. Spirochete levels in rear ankle joints of C3H/HeJ and C3H/HeN mice were relatively high, as detected by PCR, and did not increase with infectious dose. Spirochete levels in joints from BALB/cAnN mice increased with increasing infectious dose to levels found in severely arthritic C3H/He mice. Thus, resistance to severe arthritis in BALB/cAnN mice was conditional: it could be overcome by high infectious dose and the arthritis became severe when high levels of B. burgdorferi were present in joints. A unique response to increasing infectious dose was seen in C57BL/6N mice, which displayed mild to moderate arthritis at all doses of B. burgdorferi tested, up to 2 × 10⁵. At all infectious doses, the levels of spirochetes in ankle joints of C57BL/6N mice were high, equivalent to those found in the severely arthritic C3H/He mice. The arthritis observed in infected (C57BL/6N × C3H/HeN)F₁ mice was of severity intermediate between those of the two parental strains. The finding that resistance to severe arthritis in C57BL/6N mice could not be overcome by high infectious doses and was independent of spirochete levels in joints suggested that it was mediated by a distinct mechanism from that operating in BALB/cAnN mice.Lyme disease is caused by infection with the tick-transmitted spirochete Borrelia burgdorferi and is characterized by multisystem involvement (14, 16, 24). Many tissues may display disease involvement, and there is variability in the degree to which patients are affected. This variability could be due to host, microbial, or environmental factors. In fact, infection in Europe by related members of the B. burgdorferi sensu lato group is more frequently associated with chronic skin abnormalities and central nervous system involvement, while infection by B. burgdorferi sensu stricto in the United States is more commonly associated with arthritis (4, 38). Studies using the murine model of Lyme disease, developed by Barthold and colleagues, indicate host factors also influence disease outcome. Arthritis seen in this model is representative of human disease and is characterized by tendonitis, synovial hyperproliferation, and infiltration of neutrophils and other leukocytes (7). Interestingly, a spectrum of arthritis severity has been observed among inbred strains of mice in response to infection by B. burgdorferi. Infected C3H mice develop severe arthritis, whereas infected BALB/c and C57BL/6 mice develop only mild to moderate arthritis (8). Thus, inbred strains of mice provide opportunities to study host influences on disease severity.The results of several studies using the mouse model suggest the presence of inflammatory and/or anti-inflammatory cytokines can influence disease development and resolution. For example, manipulations of interleukin 12, interleukin 4, and gamma interferon levels by treating infected mice with neutralizing antibodies can influence disease severity and alter its resolution (2, 17, 21). The acquired defenses, particularly antibody production, are clearly involved in disease resolution (9, 30) but do not appear to be required for arthritis and carditis development. Not only does disease develop in scid mice, which lack mature T and B lymphocytes, but the relative differences in severity of arthritis in C3H/He and BALB/c mice is maintained in the presence of the scid mutation (12). Finally, studies with congenic mice expressing distinct major histocompatibility complex haplotypes on resistant or susceptible backgrounds suggest that the major histocompatibility complex itself had little influence on disease severity, but rather, that genes located at distinct chromosomal locations were important determinants of disease (41). These studies suggest that genes independent of acquired defenses play a large role in determining severity of disease in infected mice.In order to identify host genes that influence disease severity, the phenotypes of severe and mild arthritis must be well characterized. We previously compared B. burgdorferi levels in many tissues of C3H/HeJ and BALB/cJ mice, at several times following infection (42). Quantitative PCR demonstrated that the highest levels of spirochetes were found in the hearts and ankle joints at most time points. C3H/HeJ mice harbored 5- to 10-fold more B. burgdorferi in ankles and hearts than did BALB/cJ mice. This suggested that the severity of arthritis in C3H/HeJ mice was directly related to the high levels of spirochetes in tissues and that the relative resistance in BALB/cJ mice was associated with more restricted growth of the spirochetes.In this study we report that there are at least two different mechanisms for resistance to severe arthritis in mice. Resistance in BALB/cAnN mice could be overcome by increasing the infectious dose of B. burgdorferi and was associated with low levels of spirochetes in tissues. In contrast, resistance to severe arthritis in C57BL/6N mice was not overcome by increasing infectious dose and did not require the levels of spirochetes in joints to be low. F₁ mice from BALB/cAnN × C3H/HeJN mating developed severe arthritis upon infection, suggesting that resistance in BALB/cAnN mice could be masked by alleles from C3H/HeN mice (42). In contrast, infection of F₁ mice from a C57BL/6N × C3H/HeN cross resulted in arthritis of intermediate severity, suggesting more equal contribution by C57BL/6N and C3H/HeN genes.     

Neither Classical nor Alternative Macrophage Activation Is Required for Pneumocystis Clearance during Immune Reconstitution Inflammatory Syndrome

Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4⁺ T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2^−/− mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2^−/− mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR^−/− nor RAG/IL-4Rα^−/− mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR^−/− mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα^−/− mice. RAG/IFN-γR^−/− mice had elevated numbers of lung CD4⁺ T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8⁺ T suppressor cells. Impaired lung CD8⁺ T cell responses in RAG/IFN-γR^−/− mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8⁺ T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.     

Borrelia burgdorferi lacking DbpBA exhibits an early survival defect during experimental infection

Several Borrelia burgdorferi genes induced under mammalian host conditions have been purported to be important in Lyme disease pathogenesis based on their binding to host structures. These genes include the dbpBA locus, whose products bind host decorin and glycosoaminoglycans. Recently, the dbpBA genes were reported to be involved in borrelial infectivity. Here we extended the previous observations by using culture and quantitative PCR to evaluate low- and high-dose murine infection by a ΔdbpBA::Gent^r derivative of B. burgdorferi strain B31. The results indicate that the ΔdbpBA::Gent^r mutant is attenuated in the ability to initially colonize and then persist in multiple tissues. The mutant exhibited a colonization defect as early as 3 days postinfection, before the development of an adaptive immune response, and after low-dose infection of SCID mice, which are deficient in adaptive immunity. These findings suggest that the inability to adhere to host decorin may promote clearance of B. burgdorferi, presumably via innate immune mechanisms. In a high-dose infection, the mutant disseminated to several tissues, particularly joint tissue, but it was generally cleared from these tissues by 3 weeks postinfection. Finally, following high-dose infection of SCID mice, the dbpBA mutant exhibited only a mild colonization defect, suggesting that the adaptive response is involved in the clearance of the mutant in immunocompetent mice. Taken together, these results suggest that the DbpBA proteins facilitate the colonization of multiple tissues by B. burgdorferi and are required for optimal resistance to both innate and adaptive immune mechanisms following needle inoculation.     

Deficiency of the Complement Component 3 but Not Factor B Aggravates Staphylococcus aureus Septic Arthritis in Mice

The complement system plays an essential role in the innate immune response and protection against bacterial infections. However, detailed knowledge regarding the role of complement in Staphylococcus aureus septic arthritis is still largely missing. In this study, we elucidated the roles of selected complement proteins in S. aureus septic arthritis. Mice lacking the complement component 3 (C3^−/−), complement factor B (fB^−/−), and receptor for C3-derived anaphylatoxin C3a (C3aR^−/−) and wild-type (WT) control mice were intravenously or intra-articularly inoculated with S. aureus strain Newman. The clinical course of septic arthritis, as well as histopathological and radiological changes in joints, was assessed. After intravenous inoculation, arthritis severity and frequency were significantly higher in C3^−/− mice than in WT controls, whereas fB^−/− mice displayed intermediate arthritis severity and frequency. This was in accordance with both histopathological and radiological findings. C3, but not fB, deficiency was associated with greater weight loss, more frequent kidney abscesses, and higher bacterial burden in kidneys. S. aureus opsonized with C3^−/− sera displayed decreased uptake by mouse peritoneal macrophages compared with bacteria opsonized with WT or fB^−/− sera. C3aR deficiency had no effect on the course of hematogenous S. aureus septic arthritis. We conclude that C3 deficiency increases susceptibility to hematogenous S. aureus septic arthritis and impairs host bacterial clearance, conceivably due to diminished opsonization and phagocytosis of S. aureus.     

Borrelia burgdorferi and Interleukin-1 Promote the Transendothelial Migration of Monocytes In Vitro by Different Mechanisms

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% ± 3%, 65% ± 2%, or 25% ± 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold ± 2-fold increase in the migration of human CD4⁺ T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4⁺ T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.     

Detrimental role of CC chemokine receptor 4 in murine polymicrobial sepsis

CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are critically involved in different immune processes. In models of lipopolysaccharide-induced shock, CCR4-deficient (CCR4^−/−) mice showed improved survival rates associated with attenuated proinflammatory cytokine release. Using CCR4^−/− mice with a C57BL/6 background, this study describes for the first time the role of CCR4 in a murine model of polymicrobial abdominal sepsis, the colon ascendens stent peritonitis (CASP). CASP-induced sepsis led to a massive downregulation of CCR4 in lymphoid and nonlymphoid tissues, whereas the expression of CCL17 and CCL22 was independent of the presence of CCR4. After CASP, CCR4^−/− animals showed a strongly enhanced bacterial clearance in several organs but not in the peritoneal lavage fluid and the blood. In addition, significantly reduced levels of proinflammatory cytokines/chemokines were measured in organ supernatants as well as in the sera of CCR4^−/− mice. CCR4 deficiency consequently resulted in an attenuated severity of systemic sepsis and a strongly improved survival rate after CASP or CASP with intervention. Thus, our data provide clear evidence that CCR4 plays a strictly detrimental role in the course of polymicrobial sepsis.     

Association of CD4+ CD25+ T Cells with Prevention of Severe Destructive Arthritis in Borrelia burgdorferi-Vaccinated and Challenged Gamma Interferon-Deficient Mice Treated with Anti-Interleukin-17 Antibody

CD4⁺ CD25⁺ T cells are a population of regulatory T cells responsible for active suppression of autoimmunity. Specifically, CD4⁺ CD25⁺ T cells have been shown to prevent insulin-dependent diabetes mellitus, inflammatory bowel disease, and pancreatitis. Here, we present evidence that CD4⁺ CD25⁺ T cells also play a major role in controlling the severity of arthritis detected in Borrelia burgdorferi-vaccinated gamma interferon-deficient (IFN-γ°) C57BL/6 mice challenged with the Lyme spirochete. When B. burgdorferi-vaccinated and challenged IFN-γ° mice were treated with anti-interleukin-17 (IL-17) antibody, the number of CD4⁺ CD25⁺ T cells increased in the local lymph nodes. Furthermore, histopathologic examination showed the mice to be free of destructive arthritis. When these anti-IL-17-treated B. burgdorferi-vaccinated and challenged mice were also administered anti-CD25 antibody, the number of CD4⁺ CD25⁺ T cells in the local lymph nodes decreased. More importantly, severe destructive arthropathy was induced. In addition, delayed administration of anti-CD25 antibody decreased the severity of the arthritis. These results suggest that CD4⁺ CD25⁺ T cells are involved in regulation of a severe destructive arthritis induced with an experimental model of vaccination and challenge with B. burgdorferi.     

Activation of Natural Killer Cells in Arthritis-Susceptible but Not Arthritis-Resistant Mouse Strains following Borrelia burgdorferi Infection

Infection of susceptible mouse strains with Borrelia burgdorferi, the agent of Lyme disease, results in the development of arthritis. Components of the innate immune system may be important mediators of this pathology. To investigate the potential role of NK cells in development of experimental Lyme arthritis, we examined their activation in vivo in both resistant and susceptible mouse strains. Following inoculation of B. burgdorferi into the footpad, lymph node NK cells from susceptible C3H/HeJ (C3H) mice produced more gamma interferon than NK cells from resistant DBA/2J mice. Lymph node cells from susceptible C3H and AKR mice also had increased ability to lyse YAC-1 target cells 2 days following infection. Antibody depletion of NK cells from susceptible mice, however, did not alter the development of arthritis following B. burgdorferi challenge. In addition, NK cell depletion had little effect on spirochete burden. Thus, there is a marked activation of NK cells in susceptible mouse strains following infection. Although NK cells are not absolutely required for arthritis, events occurring prior to NK cell activation might be important in mediating pathology in experimental Lyme disease.     

Attenuated Coxiella burnetii phase II causes a febrile response in gamma interferon knockout and Toll-like receptor 2 knockout mice and protects against reinfection

Coxiella burnetii is a highly infectious obligate intracellular bacterium. The phase I form is responsible for Q fever, a febrile illness with flu-like symptoms that often goes undiagnosed. The attenuated C. burnetii phase II (having a truncated “O” chain of its lipopolysaccharide) does not cause disease in immunocompetent animals; however, phase II organisms remain infectious, and we questioned whether disease could be produced in immunodeficient mice. To study C. burnetii phase II infections, febrile responses in gamma interferon knockout (IFN-γ^−/−), BALB/c, Toll-like receptor 2 knockout (TLR2^−/−), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain of C. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-γ^−/− and TLR2^−/− mice, which showed elevated rectal temperatures that were sustained for ~15 days with transient increases in splenic weights. Reinfection of IFN-γ^−/− and TLR2^−/− mice with C. burnetii NMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection of Coxiella in TLR2^−/− mouse spleens was observed, greater colonization was evident in the IFN-γ^−/− mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-γ^−/− macrophages produced elevated levels of IL-6, IL-10, and IL-12, while TLR2^−/− macrophages produced GM-CSF, IL-12, and minimal IL-10. To distinguish immunity conferred by innate or adaptive systems, adoptive transfer studies were performed and showed that immune lymphocytes obtained from immunocompetent mice protected against a subsequent challenge with NMII, indicating that adaptive immunity mediates the observed protection. Thus, our data show that NMII is capable of eliciting disease in immunocompromised mice, which may help in evaluation of vaccine candidates as well as the study of host-pathogen interactions.     

Neutralization of Gamma Interferon Augments Borreliacidal Antibody Production and Severe Destructive Lyme Arthritis in C3H/HeJ Mice

Development of a high level of sustained borreliacidal antibody is paramount for maintaining protection against infection with Borrelia burgdorferi. We show that production of borreliacidal antibody can be enhanced by preventing the effects of gamma interferon (IFN-γ). When lymph node cells capable of producing borreliacidal antibody were cultured with anti-murine IFN-γ, an eightfold increase in borreliacidal antibody production was obtained. However, anti-IFN-γ treatment of these cells also enhanced their ability to adaptively induce arthritis. When anti-IFN-γ-treated lymph node cells producing borreliacidal antibody were infused into C3H/HeJ mice and the mice were then challenged with B. burgdorferi, the mice developed severe destructive Lyme arthritis. Additional studies are needed to delineate the immune response responsible for the induction of arthritis and production of borreliacidal antibody. These studies are needed to ensure an effective and safe vaccine against infection with B. burgdorferi.