Interactions Between Pathogenic Burkholderia and the Complement System: A Review of Potential Immune Evasion Mechanisms.

The genus Burkholderia contains over 80 different Gram-negative species including both plant and human pathogens, the latter of which can be classified into one of two groups: the Burkholderia pseudomallei complex (Bpc) or the Burkholderia cepacia complex (Bcc). Bpc pathogens Burkholderia pseudomallei and Burkholderia mallei are highly virulent, and both have considerable potential for use as Tier 1 bioterrorism agents; thus there is great interest in the development of novel vaccines and therapeutics for the prevention and treatment of these infections. While Bcc pathogens Burkholderia cenocepacia, Burkholderia multivorans, and Burkholderia cepacia are not considered bioterror threats, the incredible impact these infections have on the cystic fibrosis community inspires a similar demand for vaccines and therapeutics for the prevention and treatment of these infections as well. Understanding how these pathogens interact with and evade the host immune system will help uncover novel therapeutic targets within these organisms. Given the important role of the complement system in the clearance of bacterial pathogens, this arm of the immune response must be efficiently evaded for successful infection to occur. In this review, we will introduce the Burkholderia species to be discussed, followed by a summary of the complement system and known mechanisms by which pathogens interact with this critical system to evade clearance within the host. We will conclude with a review of literature relating to the interactions between the herein discussed Burkholderia species and the host complement system, with the goal of highlighting areas in this field that warrant further investigation.

Microcystin-LR (MC-LR) Triggers Inflammatory Responses in Macrophages.

We were the first to previously report that microcystin-LR (MC-LR) has limited effects within the colons of healthy mice but has toxic effects within colons of mice with pre-existing inflammatory bowel disease. In the current investigation, we aimed to elucidate the mechanism by which MC-LR exacerbates colitis and to identify effective therapeutic targets. Through our current investigation, we report that there is a significantly greater recruitment of macrophages into colonic tissue with pre-existing colitis in the presence of MC-LR than in the absence of MC-LR. This is seen quantitatively through IHC staining and the enumeration of F4/80-positive macrophages and through gene expression analysis for Cd68, Cd11b, and Cd163. Exposure of isolated macrophages to MC-LR was found to directly upregulate macrophage activation markers Tnf and Il1b. Through a high-throughput, unbiased kinase activity profiling strategy, MC-LR-induced phosphorylation events were compared with potential inhibitors, and doramapimod was found to effectively prevent MC-LR-induced inflammatory responses in macrophages.

Immune Response to Borrelia: Lessons from Lyme Disease Spirochetes.

The mammalian host responds to infection with Borrelia spirochetes through a highly orchestrated immune defense involving innate and adaptive effector functions aimed toward limiting pathogen burdens, minimizing tissue injury, and preventing subsequent reinfection. The evolutionary adaptation of Borrelia spirochetes to their reservoir mammalian hosts may allow for its persistence despite this immune defense. This review summarizes our current understanding of the host immune response to B. burgdorferi sensu lato, the most widely studied Borrelia spp. and etiologic agent of Lyme borreliosis. Pertinent literature will be reviewed with emphasis on in vitro, ex vivo and animal studies that influenced our understanding of both the earliest responses to B. burgdorferi as it enters the mammalian host and those that evolve as spirochetes disseminate and establish infection in multiple tissues. Our focus is on the immune response of inbred mice, the most commonly studied animal model of B. burgdorferi infection and surrogate for one of this pathogen's principle natural reservoir hosts, the white-footed deer mouse. Comparison will be made to the immune responses of humans with Lyme borreliosis. Our goal is to provide an understanding of the dynamics of the mammalian immune response during infection with B. burgdorferi and its relation to the outcomes in reservoir (mouse) and non-reservoir (human) hosts.

Borrelia burgdorferi CheY2 Is Dispensable for Chemotaxis or Motility but Crucial for the Infectious Life Cycle of the Spirochete.

The requirements for bacterial chemotaxis and motility range from dispensable to crucial for host colonization. Even though more than 50% of all sequenced prokaryotic genomes possess at least one chemotaxis signaling system, many of those genomes contain multiple copies of a chemotaxis gene. However, the functions of most of those additional genes are unknown. Most motile bacteria possess at least one CheY response regulator that is typically dedicated to the control of motility and which is usually essential for virulence. Borrelia burgdorferi appears to be notably different, in that it has three cheY genes, and our current studies on cheY2 suggests that it has varied effects on different aspects of the natural infection cycle. Mutants deficient in this protein exhibit normal motility and chemotaxis in vitro but show reduced virulence in mice. Specifically, the cheY2 mutants were severely attenuated in murine infection and dissemination to distant tissues after needle inoculation. Moreover, while ΔcheY2 spirochetes are able to survive normally in the Ixodes ticks, mice fed upon by the ΔcheY2-infected ticks did not develop a persistent infection in the murine host. Our data suggest that CheY2, despite resembling a typical response regulator, functions distinctively from most other chemotaxis CheY proteins. We propose that CheY2 serves as a regulator for a B. burgdorferi virulence determinant that is required for productive infection within vertebrate, but not tick, hosts.

The Borrelia burgdorferi CheY3 response regulator is essential for chemotaxis and completion of its natural infection cycle.

Borrelia burgdorferi possesses a sophisticated and complex chemotaxis system, but how the organism utilizes this system in its natural enzootic life cycle is poorly understood. Of the three CheY chemotaxis response regulators in B. burgdorferi, we found that only deletion of cheY3 resulted in an altered motility and significantly reduced chemotaxis phenotype. Although ΔcheY3 maintained normal densities in unfed ticks, their numbers were significantly reduced in fed ticks compared with the parental or cheY3-complemented spirochetes. Importantly, mice fed upon by the ΔcheY3-infected ticks did not develop a persistent infection. Intravital confocal microscopy analyses discovered that the ΔcheY3 spirochetes were motile within skin, but appeared unable to reverse direction and perform the characteristic backward-forward motility displayed by the parental strain. Subsequently, the ΔcheY3 became 'trapped' in the skin matrix within days of inoculation, were cleared from the skin needle-inoculation site within 96 h post-injection and did not disseminate to distant tissues. Interestingly, although ΔcheY3 cells were cleared within 96 h post-injection, this attenuated infection elicited significant levels of B. burgdorferi-specific IgM and IgG. Taken together, these data demonstrate that cheY3-mediated chemotaxis is crucial for motility, dissemination and viability of the spirochete both within and between mice and ticks.

Spirochetal motility and chemotaxis in the natural enzootic cycle and development of Lyme disease.

Two-thirds of all bacterial genomes sequenced to-date possess an organelle for locomotion, referred to as flagella, periplasmic flagella or type IV pili. These genomes may also contain a chemotaxis-signaling system which governs flagellar rotation, thus leading a coordinated function for motility. Motility and chemotaxis are often crucial for infection or disease process caused by pathogenic bacteria. Although motility-associated genes are well-characterized in some organisms, the highly orchestrated synthesis, regulation, and assembly of periplasmic flagella in spirochetes are just being delineated. Recent advances were fostered by development of unique genetic manipulations in spirochetes coupled with cutting-edge imaging techniques. These contemporary advances in understanding the role of spirochetal motility and chemotaxis in host persistence and disease development are highlighted in this review.

Motor rotation is essential for the formation of the periplasmic flagellar ribbon, cellular morphology, and Borrelia burgdorferi persistence within Ixodes scapularis tick and murine hosts.

Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts.

Delineating the importance of serum opsonins and the bacterial capsule in affecting the uptake and killing of Burkholderia pseudomallei by murine neutrophils and macrophages.

Infection of susceptible hosts by the encapsulated Gram-negative bacterium Burkholderia pseudomallei (Bp) causes melioidosis, with septic patients attaining mortality rates ≥ 40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt). Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS)-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis.

Borrelia burgdorferi elicited-IL-10 suppresses the production of inflammatory mediators, phagocytosis, and expression of co-stimulatory receptors by murine macrophages and/or dendritic cells.

Borrelia burgdorferi (Bb) is a tick-borne spirochete that is the causative agent for Lyme disease. Our previous studies indicate that virulent Bb can potently enhance IL-10 production by macrophages (MØs) and that blocking IL-10 production significantly enhances bacterial clearance. We hypothesize that skin-associated APC types, such as MØs and dendritic cells (DCs) are potent producers of IL-10 in response to Bb, which may act in autocrine fashion to suppress APC responses critical for efficient Bb clearance. Our goal is to delineate which APC immune functions are dysregulated by Bb-elicited IL-10 using a murine model of Lyme disease. Our in vitro studies indicated that both APCs rapidly produce IL-10 upon exposure to Bb, that these levels inversely correlate with the production of many Lyme-relevant proinflammatory cytokines and chemokines, and that APCs derived from IL-10(-/-) mice produced greater amounts of these proinflammatory mediators than wild-type APCs. Phagocytosis assays determined that Bb-elicited IL-10 levels can diminish Bb uptake and trafficking by MØs, suppresses ROS production, but does not affect NO production; Bb-elicited IL-10 had little effect on phagocytosis, ROS, and NO production by DCs. In general, Bb exposure caused little-to-no upregulation of several critical surface co-stimulatory markers by MØs and DCs, however eliminating Bb-elicited IL-10 allowed a significant upregulation in many of these co-stimulatory receptors. These data indicate that IL-10 elicited from Bb-stimulated MØs and DCs results in decreased production of proinflammatory mediators and co-stimulatory molecules, and suppress phagocytosis-associated events that are important for mediating both innate and adaptive immune responses by APCs.

Secretion of growth factors from macrophages when cultured with microparticles.

The aim of this study is to investigate the influence of macrophages on osteoblast (OB) performance and differentiation. In this regard, we studied the secretion of growth factors including bone morphogenetic proteins (BMPs) from before and after activation of macrophages. We also evaluated osteogenic markers in the co-culture of macrophages and OBs. The macrophages were seeded on microparticles (MPs) based on chitosan (CS). Two types of MPs were fabricated including CS MPs and 10% calcium phosphate (CaHPO4 )-incorporated CS MPs. Macrophage seeded on MPs was activated using lipopolysaccharide (LPS). The expression of BMP-2, BMP-6, BMP-7, and transforming growth factor beta (TGF-ß) from macrophages seeded and cultured on hybrid MPs before and after activation of LPS at predetermined times was quantified using a quantitative reverse transcription-polymerase chain reaction (RT-PCR). All of the above growth factors were expressed from MP-macrophage cultures before LPS activation. Osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and collagen I (COL-I) in the cultures of MP-OB-macrophage were quantified using a quantitative RT-PCR at days 2, 4, and 7. We found an elevation of gene expression of ALP and COL-1 in the co-cultures of OB-macrophage on MPs compared to OB on MP cultures. These data suggest that macrophages enhance expression of osteogenic markers in OBs, and demonstrate the importance of the role of macrophages in bone regeneration.

Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils.

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD(50) via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

Human platelets efficiently kill IgG-opsonized E. coli.

Platelets are known contributors of hemostasis but have recently been shown to be important in inflammation and infectious diseases. Moreover, thrombocytopenia is often observed in patients with sepsis. We previously reported that platelets actively phagocytosed IgG-coated latex beads. In this study, the capacity of human platelets to participate in host defense against bacterial infections was determined by assessing their ability to kill Escherichia coli. Washed human platelets were incubated with unopsonized or IgG-opsonized E. coli and evaluated for binding and killing of E. coli. We found that although both unopsonized and IgG-opsonized E. coli were associated with platelets, only IgG-opsonized E. coli were efficiently killed unless platelets were activated by a potent agonist. The bactericidal activity was dependent on FcγRIIA, was sensitive to cytochalasin D, but was not due to reactive oxygen metabolites. These data suggest that platelets may play an important role in protection against infection.

ELISA-based measurement of antibody responses and PCR-based detection profiles can distinguish between active infection and early clearance of Borrelia burgdorferi.

Borrelia burgdorferi is a spirochetal bacterium that causes Lyme disease. These studies address whether current research methods using either ELISA to detect seroconversion to B. burgdorferi antigens or PCR quantification of bacterial DNA within tissues can accurately distinguish between a productive infection versus a B. burgdorferi exposure that is rapidly cleared by the innate responses. Mice receiving even minimal doses of live B. burgdorferi produced significantly more B. burgdorferi-specific IgM and IgG than groups receiving large inocula of heat-killed bacteria. Additionally, sera from mice injected with varied doses of killed B. burgdorferi recognized unique borrelial antigens compared to mice infected with live B. burgdorferi. Intradermal injection of killed B. burgdorferi resulted in rapid DNA clearance from skin, whereas DNA was consistently detected in skin inoculated with viable B. burgdorferi. These data indicate that both ELISA-based serological analyses and PCR-based methods of assessing B. burgdorferi infection clearly distinguish between an established infection with live bacteria and exposure to large numbers of bacteria that are promptly cleared by the innate responses.

Roles for phagocytic cells and complement in controlling relapsing fever infection.

Relapsing fever spirochetes, such as Borrelia hermsii, proliferate to high levels in their hosts' bloodstream until production of IgM against borrelial surface proteins promotes bacterial clearance. The mechanisms by which B. hermsii survives in host blood, as well as the immune mediators that control this infection, remain largely unknown. It has been hypothesized that B. hermsii is naturally resistant to killing by the alternative pathway of complement activation as a result of its ability to bind factor H, a host complement regulator. However, we found that Cfh(-/-) mice were infected to levels identical to those seen in wild-type mice. Moreover, only a small minority of B. hermsii in the blood of wild-type mice had detectable levels of factor H adhered to their outer surfaces. In vitro, complement was found to play a statistically significant role in antibody-mediated inactivation of B. hermsii, although in vivo studies indicated that complement is not essential for host control of B. hermsii. Depletion of mphi and DC from mice had significant impacts on B. hermsii infection, and depleted mice were unable to control bloodstream infections, leading to death. Infection studies using muMT indicated a significant antibody-independent role for mphi and/or DC in host control of relapsing fever infection. Together, these findings indicate mphi and/or DC play a critical role in the production of B. hermsii-specific IgM and for antibody-independent control of spirochete levels.

Viable Borrelia burgdorferi enhances interleukin-10 production and suppresses activation of murine macrophages.

Although it is capable of eliciting strong innate and adaptive immune responses, Borrelia burgdorferi often evades immune clearance through largely unknown mechanisms. Our previous studies determined that infected interlukin-10-/- (IL-10-/-) mice show significantly lower B. burgdorferi levels than wild-type (B6) mice and that IL-10 inhibits innate immune responses critical for controlling B. burgdorferi infection. To determine whether virulent B. burgdorferi preferentially enhances IL-10 production, we developed an in vitro coculture medium (RPMI.B) in which both B. burgdorferi and primary macrophages (Mphis) remain viable. B. burgdorferi grew at similar rates and was able to regulate expression of immunoreactive proteins with similar kinetics in RPMI.B and in traditional BSK medium; in contrast, B. burgdorferi cultured in conventional tissue culture medium (RPMI) rapidly lost viability. Coculture of viable B. burgdorferi in RPMI.B with Mphis resulted in more rapid and significant increases in IL-10 transcripts and secreted proteins than coculture with nonviable B. burgdorferi in RPMI, which corresponded with decreased production of proinflammatory cytokines. Addition of live B. burgdorferi to Mphis in RPMI.B also elicited substantially higher IL-10 levels than heat-killed bacteria elicited, confirming that increased IL-10 production was not inherent to coculture in RPMI.B. Transfer of supernatants from B. burgdorferi-stimulated Mphis into naïve Mphi cultures resulted in suppressed activation upon subsequent stimulation with different bacterial agonists, and this suppression was obviated by IL-10-specific antibody. In vivo analyses determined that murine skin samples exhibited substantial upregulation of IL-10 within 24 h of injection of B. burgdorferi. Together, these results suggest that viable B. burgdorferi can suppress early Mphi responses during infection by causing increased release of IL-10.

Borrelia burgdorferi binding of host complement regulator factor H is not required for efficient mammalian infection.

The causative agent of Lyme disease, Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development of B. burgdorferi infection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways in B. burgdorferi survival during mammalian infection. While it was predicted that B. burgdorferi should be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis of B. burgdorferi acquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection by B. burgdorferi and indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing.

The Moraxella catarrhalis autotransporter McaP is a conserved surface protein that mediates adherence to human epithelial cells through its N-terminal passenger domain.

The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded beta-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.

IL-10 deficiency promotes increased Borrelia burgdorferi clearance predominantly through enhanced innate immune responses.

Borrelia burgdorferi is capable of persistently infecting a variety of hosts despite eliciting potent innate and adaptive immune responses. Preliminary studies indicated that IL-10-deficient (IL-10(-/-)) mice exhibit up to 10-fold greater clearance of B. burgdorferi from target tissues compared with wild-type mice, establishing IL-10 as the only cytokine currently known to have such a significant effect on spirochetal clearance. To further delineate these IL-10-mediated immune effects, kinetic studies indicated that spirochete dissemination to target tissues is similar in both wild-type and IL-10(-/-) mouse strains, and that enhanced clearance of B. burgdorferi in IL-10(-/-) mice is correlated with increased B. burgdorferi-specific Ab as early as 2 wk postinfection. Immunoblot analysis indicated that Abs produced by infected IL-10(-/-) and wild-type mice recognize similar ranges of spirochetal Ags. Immune sera from IL-10(-/-) and wild-type mice also exhibited similar bactericidal activity in vitro, and passive transfer of these immune sera into B. burgdorferi-infected SCID mice caused similar reductions of bacterial numbers in target tissues. Infectious dose studies indicated that 8-fold more B. burgdorferi were needed to efficiently infect naive IL-10(-/-) mice, suggesting these animals possess higher innate barriers to infection. Moreover, macrophages derived from IL-10(-/-) mice exhibit enhanced proinflammatory responses to B. burgdorferi stimulation compared with wild-type controls, and these responses are not significantly affected by the presence of immune serum. These findings confirm that B. burgdorferi clearance by innate immune responses is more efficient in the absence of IL-10, and these activities are not directly related to increased levels of B. burgdorferi-specific Ab.

Gene expression profiling reveals unique pathways associated with differential severity of lyme arthritis.

The murine model of Lyme disease provides a unique opportunity to study the localized host response to similar stimulus, Borrelia burgdorferi, in the joints of mice destined to develop severe arthritis (C3H) or mild disease (C57BL/6). Pathways associated with the response to infection and the development of Lyme arthritis were identified by global gene expression patterns using oligonucleotide microarrays. A robust induction of IFN-responsive genes was observed in severely arthritic C3H mice at 1 wk of infection, which was absent from mildly arthritic C57BL/6 mice. In contrast, infected C57BL/6 mice displayed a novel expression profile characterized by genes involved in epidermal differentiation and wound repair, which were decreased in the joints of C3H mice. These expression patterns were associated with disease state rather than inherent differences between C3H and C57BL/6 mice, because C57BL/6-IL-10(-/-) mice infected with B. burgdorferi develop more severe arthritis than C57BL/6 mice and displayed an early gene expression profile similar to C3H mice. Gene expression profiles at 2 and 4 wk postinfection revealed a common response of all strains that was likely to be important for the host defense to B. burgdorferi and mediated by NF-kappaB-dependent signaling. The gene expression profiles identified in this study add to the current understanding of the host response to B. burgdorferi and identify two novel pathways that may be involved in regulating the severity of Lyme arthritis.

Effects of vlsE complementation on the infectivity of Borrelia burgdorferi lacking the linear plasmid lp28-1.

The loss of linear plasmid lp28-1, which contains the vls antigenic variation locus, is associated with reduced infectivity of Borrelia burgdorferi in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable B. burgdorferi lacking lp25 and lp56, was used to determine the effect of trans expression of vlsE on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and vlsE. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured B. burgdorferi, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact vls antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the vlsE shuttle plasmid and was examined for vlsE sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion. Lack of vlsE sequence variation on the shuttle plasmid thus did not result in clearance of the trans-complemented strain in immunocompetent mice under the conditions tested.