Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis

Abstract

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PG^Bb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PG^Bb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PG^Bb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PG^Bb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PG^Bb In addition, systemic administration of PG^Bb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PG^Bb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PG^Bb fragments during growth suggests a potential role for PG^Bb in the immunopathogenesis of other Lyme disease manifestations.

Keywords: Borrelia burgdorferi; Lyme disease; arthritis; inflammation; peptidoglycan.

Fig. 1.

B. burgdorferi sheds muropeptides into its extracellular environment. (A, Top) Chromatogram of cellosyl-digested and reduced PG^Bb isolated from B. burgdorferi B31. Numbers correspond to the identified chemical species shown below. The asterisk indicates an unidentified species (SI Appendix, Table S1). Analysis performed on three separate preparations produced highly similar chromatograms. (A, Bottom) Chemical composition of muropeptides in peaks shown in the chromatogram. Muropeptide identification was accomplished by MS. MurNAc(r) and Anh indicate N-acetylmuramitol and 1,6-anhydro group, respectively. (B) Plot showing PG turnover over multiple generations in B. burgdorferi grown in vitro. PG^Bb was pulse-radiolabeled by incubating cells in medium containing 7.5 µCi/mL of ³H- or ¹⁴C-l-Orn for 48 h. Cells were then washed to remove unincorporated isotope, and outgrowth was tracked in complete BSK II medium lacking radioactive l-Orn. At each time point, the same volume of batch culture was removed, bacterial density was determined, and PG^Bb was purified for quantification of its radioactivity per volume equivalent. The retained radioactivity was then plotted as a percentage of total radioactivity in the PG at time 0 (i.e., start of outgrowth). (C) Muropeptide accumulation in the culture medium. Cultures of B. burgdorferi (5 × 10⁷ cells per milliliter) were diluted to a starting density of 10⁴ cells per milliliter and monitored for muropeptide release during growth in complete BSK II medium (lacking phenol red) using an hNOD2 reporter cell line in the presence or absence of the RIP2 inhibitor gefitinib. NF-κB activity (absorbance at 650 nm) provides a measure of NOD2-specific muropeptide levels present in the culture medium samples collected at the indicated time points. Shown are the mean and SD of NF-κB activation for two biological replicates at each time point.

Fig. 2.

Patients with LA develop an adaptive immune response specifically to PG^Bb. Purified PG from B. burgdorferi B31, E. coli K-12, S. aureus SA113, or B. subtilis 168 was immobilized, and synovial fluid from patients with different types of arthritis (groups 1–6 and 7) were blindly assayed for the presence of IgG by ELISA (****P < 0.0001, Kruskal–Wallis test followed by Dunn’s post hoc pairwise test for B and Mann–Whitney U test for A and D). Horizontal black lines indicate means and SDs. (A) Levels of IgG against different PG types. After the results were obtained, the patient sample type was decoded and organized based on the arthritis type and treatment stage. Results for a control joint fluid sample obtained from a patient with a torn ACL (group 8) were also included. Values were background-subtracted based on the IgG level measured for each individual sample in the absence of PG ligand. (Inset) All anti-PG^Bb IgG values are shown as a bee-swarm plot for LA samples and controls (all non-LA samples). (B) Specificity of IgGs from LA synovial fluid samples for PGs from different bacteria. (C) Specificity of IgGs from control synovial fluid samples for different PG types. (D) Comparison of IgG responses to PG^Bb for serum and synovial fluid samples from patients with LA relative to those for serum from healthy humans and synovial fluid samples from control (non-LA) patients. (E) Correlation analysis of anti-PG^Bb IgG responses between the serum and synovial fluid of patients with LA. The linear fit and Pearson correlation coefficient (r) for the LA synovial fluid samples are also shown.

Fig. 3.

Detection of PG^Bb in synovial fluid samples of patients with LA. (A) Competitive ELISA using rabbit antiserum raised against PG^Bb to quantify the concentration of PG (in picograms per milliliter) present in each sample. Horizontal black lines indicate means (****P < 0.0001, Kruskal–Wallis test followed by Dunn’s post hoc pairwise test). (B) Plot showing the PG^Bb concentration of each sample as a function of its anti-PG^Bb IgG level. The linear fit and the Pearson correlation coefficient (r) for the LA synovial fluid samples are also shown.

Fig. 4.

Cytokine profile in serum and synovial fluid samples from patients with LA or after in vitro stimulation of human PBMCs with PG^Bb. (A) Bee-swarm plots showing levels of indicated cytokines in LA patient samples. Horizontal black lines indicate geometric means (****P < 0.0001 and **0.001 < P < 0.01, Mann-Whitney U test). Pound signs indicate samples that yielded no signal but were included for completeness, as zero values cannot be displayed on log-scale axes. (B) Cytokine levels produced by control human PBMCs stimulated by PBS or 100 μg/mL polymeric PG (pPG) or mutanolysin-digested PG (dPG) for 72 h. The 18-h results are shown in SI Appendix, Fig. S7A. All stimulatory studies were performed on pooled, mixed donor samples assayed in duplicate (mean ± SD).

Fig. 5.

Systemic administration of PG^Bb induces acute arthritis in mice. (A) A BALB/c mouse 24 h after IV injection of 200 μg PG^Bb exhibits bilateral ankle edema not present in an uninjected control mouse. (B) Average composite arthritis score (i.e., average sum of individual scores for left and right hind limbs) within each mouse group 24, 48, 72, and 96 h after IV administration of PG^Bb or PBS. Error bars indicate SEMs; n = 12 mice per group at 24 and 48 h postinjection and n = 6 mice per group at all subsequent time points. (C) Arthritis prevalence as a function of time after injection with PG^Bb or PBS. Only mice with a composite arthritis score ≥1 were considered as having arthritis. (D) Sum of left and right ankle histopathological scores for individual mice at 48 or 96 h after IV injection of PG^Bb or PBS. Horizontal black lines indicate means and SEM (**P < 0.01 and *0.01 < P < 0.05, respectively, Mann–Whitney U test). (E) Representative light micrographs of hematoxylin-eosin–stained sections of mouse ankles collected 48 or 96 h after IV administration of PG^Bb show peritendon inflammation (single pound symbols) and synovial space edema (double pound symbols). PBS-injected control mice lack both histopathological features when examined at the same time points.