Janis J. Weis

Cutting Edge: Repurification of Lipopolysaccharide Eliminates Signaling Through Both Human and Murine Toll-Like Receptor 2

Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as “endotoxin protein,” suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.

Signaling by Toll-Like Receptor 2 and 4 Agonists Results in Differential Gene Expression in Murine Macrophages

ABSTRACT Lipopolysaccharide (LPS) derived from the periodontal pathogenPorphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.

Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi

Borrelia burgdorferi lipoproteins activate inflammatory cells through Toll-like receptor 2 (TLR2), suggesting that TLR2 could play a pivotal role in the host response to B. burgdorferi. TLR2 does play a critical role in host defense, as infected TLR2−/− mice harbored up to 100-fold more spirochetes in tissues than did TLR2+/+ littermates. Spirochetes persisted at extremely elevated levels in TLR2-deficient mice for at least 8 wk following infection. Infected TLR2−/− mice developed normal Borrelia-specific Ab responses, as measured by quantity of Borrelia-specific Ig isotypes, the kinetics of class switching to IgG, and the complexity of the Ags recognized. These findings indicate that the failure to control spirochete levels in tissues is not due to an impaired acquired immune response. While macrophages from TLR2−/− mice were not responsive to lipoproteins, they did respond to nonlipoprotein components of sonicated spirochetes. These TLR2-independent responses could play a role during the inflammatory response to B. burgdorferi, as infected TLR2−/− mice developed greater ankle swelling than wild-type littermates. Thus, while TLR2-dependent signaling pathways play a major role in the innate host defense to B. burgdorferi, both inflammatory responses and the development of the acquired humoral response can occur in the absence of TLR2.

MyD88 Plays a Unique Role in Host Defense but Not Arthritis Development in Lyme Disease

To assess the contribution of TLR signaling in the host response to Borrelia burgdorferi, mice deficient in the common TLR adaptor protein, myeloid differentiation factor 88 (MyD88), were infected with B. burgdorferi. MyD88-deficient mice harbored extremely high levels of B. burgdorferi in tissues when compared with wild-type littermates and greater amounts of spirochetes in tissues than TLR2-deficient mice. These findings suggest that, in addition to TLR2, other MyD88-dependent pathways play a significant role in the host defense to B. burgdorferi. MyD88−/− mice maintained the ability to produce Abs directed against B. burgdorferi. Partial clearance of spirochetes was evident in long term infection studies and immune sera from MyD88-deficient mice were able to protect naive mice from infection with B. burgdorferi. Thus, the acquired immune response appeared to be functional in MyD88−/− mice, and the inability to control spirochete numbers was due to a failure of cells involved in innate defenses. Although macrophages from MyD88−/− mice responded poorly to Borrelia sonicate in vitro, MyD88−/− mice still developed an inflammatory arthritis after infection with B. burgdorferi characterized by an influx of neutrophils and mononuclear cells. The findings presented here point to a dichotomy between the recruitment of inflammatory cells to tissue and an inability of these cells to kill localized spirochetes.

Resistance to Lyme disease in decorin-deficient mice

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Delineating the Requirement for the Borrelia burgdorferi Virulence Factor OspC in the Mammalian Host

ABSTRACT We previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferi is essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospC mutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospC mutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferi is prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

Host-pathogen interactions promoting inflammatory Lyme arthritis: use of mouse models for dissection of disease processes.

Recent studies have confirmed the infectious and inflammatory nature of arthritis induced by Borrelia burgdorferi, or Lyme arthritis. This arthritis is directed by the presence of the bacteria in joint tissue, and is mediated through activation of the Toll-like receptor 2 (TLR2) signaling pathways by borrelial lipoproteins. Several host genes regulate the severity of arthritis, possibly by regulating the balance of pro- and anti-inflammatory responses.

IMMUNITY TO LYME DISEASE : PROTECTION, PATHOLOGY AND PERSISTENCE

Persistence of the Lyme disease spirochete, Borrelia burgdorferi, in the presence of an active immune response has been well documented. Evidence from the past year indicates that modulation of surface antigens by the spirochete may be a major mechanism for evading the immune response.

Genetic control of susceptibility to experimental Lyme arthritis is polygenic and exhibits consistent linkage to multiple loci on chromosome 5 in four independent mouse crosses.

C3H/He mice infected with Borrelia burgdorferi develop severe arthritis and are high antibody responders, while infected C57BL/6 and BALB/c mice develop mild arthritis and less robust humoral responses. Genetic analysis using composite interval mapping (CIM) on reciprocal backcross populations derived from C3H/HeN and C57BL/6N or C3H/HeJ and BALB/cAnN mice identified 12 new quantitative trait loci (QTL) linked to 10 murine Lyme disease phenotypes. These QTL reside on chromosomes 1, 2, 4, 6, 7, 9, 10, 12, 14, 15, 16, and 17. A reanalysis of an F2 intercross between C57BL/6N and C3H/HeN mice using CIM identified two new QTL on chromosomes 4 and 15 and confirmed the location of seven previously identified loci. Two or more experimental crosses independently verified six QTL controlling phenotypes after B. burgdorferi infection. Additionally, Bb2 on chromosome 5 was reproduced in four experimental populations and was linked to the candidate locus Cora1. Evidence of four distinct QTL residing within the 30-cM region of chromosome 5 encompassing the previously mapped Bb2 and Bb3 loci was shown by CIM. Interestingly, some alleles contributing to susceptibility to Lyme arthritis were derived from C57BL/6N and BALB/cAnN mice, showing that disease-resistant strains harbor susceptibility alleles.

A critical role for type I IFN in arthritis development following Borrelia burgdorferi infection of mice.

Gene expression analysis previously revealed a robust IFN-responsive gene induction profile that was selectively up-regulated in Borrelia burgdorferi-infected C3H mice at 1 wk postinfection. This profile was correlated with arthritis development, as it was absent from infected, mildly arthritic C57BL/6 mice. In this report we now demonstrate that profile induction in infected C3H scid mice occurs independently of B or T lymphocyte infiltration in the joint tissue. Additionally, type I IFN receptor-blocking Abs, but not anti-IFN-γ Abs, dramatically reduced arthritis, revealing a critical but previously unappreciated role for type I IFN in Lyme arthritis development. Certain examined IFN-inducible transcripts were also significantly diminished within joint tissue of mice treated with anti-IFNAR1, whereas expression of other IFN-responsive genes was more markedly altered by anti-IFN-γ treatment. These data indicate that induction of the entire IFN profile is not necessary for arthritis development. These findings further tie early type I IFN induction to Lyme arthritis development, a connection not previously made. Bone marrow-derived macrophages readily induced IFN-responsive genes following B. burgdorferi stimulation, and this expression required a functional type I IFN receptor. Strikingly, induction of these genes was independent of TLRs 2,4, and 9 and of the adapter molecule MyD88. These data demonstrate that the extracellular pathogen B. burgdorferi uses a previously unidentified receptor and a pathway traditionally associated with viruses and intracellular bacteria to initiate transcription of type I IFN and IFN-responsive genes and to initiate arthritis development.