Association of a Toll-like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis.

OBJECTIVE: Single-nucleotide polymorphisms (SNPs) that alter immune function, inflammatory responses, and disease susceptibility have been identified in several genes encoding Toll-like receptors (TLRs). The TLR SNPs with the best evidence of an effect on immune function are those in TLR1 (1805GG), TLR2 (2258GA), and TLR5 (1174CT). This study was undertaken to assess the frequency and functional outcomes of these polymorphisms in patients with Lyme disease. METHODS: SNP frequencies and functional outcomes were assessed in 248 patients with Lyme disease. Cytokine and chemokine levels were determined using multiplex assays in the serum of patients with erythema migrans (EM), joint fluid of patients with Lyme arthritis, and supernatants of Borrelia burgdorferi-stimulated peripheral blood mononuclear cells (PBMCs) from patients with Lyme arthritis. RESULTS: The frequency of the TLR1-1805GG polymorphism was greater in patients with antibiotic-refractory arthritis compared with patients with EM or those with antibiotic-responsive arthritis. Early in the illness, patients with EM carrying 1805GG, primarily those infected with B burgdorferi 16S-23S ribosomal spacer RNA intergenic type 1 (RST1) strains, had higher serum levels of interferon-γ (IFNγ), CXCL9, and CXCL10 and had more severe infection than EM patients carrying the 1805TG/TT polymorphism. These inflammatory responses were amplified in patients with Lyme arthritis, and the highest responses were observed in patients with 1805GG in the antibiotic-refractory group who had been infected with RST1 strains. When PBMCs from patients with Lyme arthritis were stimulated with a B burgdorferi RST1 strain, the 1805GG group had a significantly larger fold increase in the levels of IFNγ, CCL2, CXCL9, and CXCL10 compared to the 1805TG/TT group. In contrast, the TLR2 and TLR5 polymorphisms did not vary in frequency or function among the groups. CONCLUSION: The TLR1-1805GG polymorphism in B burgdorferi RST1-infected patients was associated with stronger Th1-like inflammatory responses, an environment that may set the stage for antibiotic-refractory arthritis.

Relationship between immunity to Borrelia burgdorferi outer-surface protein A (OspA) and Lyme arthritis.

Antibiotic-refractory Lyme arthritis may result from Borrelia burgdorferi-induced autoimmunity in affected joints. Such patients usually have certain HLA-DRB1 molecules that bind an epitope of B. burgdorferi outer-surface protein A (OspA163₋175), and cellular and humoral immune responses to OspA are greater in patients with antibiotic-refractory arthritis than in those with antibiotic-responsive arthritis. Recent work in a mouse model suggests that, during B. burgdorferi infection, OspA in genetically susceptible individuals stimulates a particularly strong T(H)1 response, which may be one of several factors that can help set the stage for a putative autoimmune response in affected joints. However, vaccination with OspA did not induce arthritis in this mouse model, and case and control comparisons in human vaccine trials did not show an increased frequency of arthritis among OspA-vaccinated individuals. Thus, a vaccine-induced immune response to OspA does not replicate the sequence of events needed in the natural infection to induce antibiotic-refractory Lyme arthritis.

Borrelia burgdorferi stimulation of chemokine secretion by cells of monocyte lineage in patients with Lyme arthritis.

INTRODUCTION: Joint fluid in patients with Lyme arthritis often contains high levels of CCL4 and CCL2, which are chemoattractants for monocytes and some T cells, and CXCL9 and CXCL10, which are chemoattractants for CD4+ and CD8+ T effector cells. These chemokines are produced primarily by cells of monocyte lineage in TH1-type immune responses. Our goal was to begin to learn how infection with Borrelia burgdorferi leads to the secretion of these chemokines, using patient cell samples. We hypothesized that B. burgdorferi stimulates chemokine secretion from monocytes/macrophages in multiple ways, thereby linking innate and adaptive immune responses. METHODS: Peripheral blood mononuclear cells (PBMC) from 24 Lyme arthritis patients were stimulated with B. burgdorferi, interferon (IFN)-γ, or both, and the levels of CCL4, CCL2, CXCL9 and CXCL10 were measured in culture supernatants. CD14+ monocytes/macrophages from PBMC and synovial fluid mononuclear cells (SFMC) were stimulated in the same way, using available samples. CXCR3, the receptor for CXCL9 and CXCL10, and CCR5, the receptor for CCL4, were assessed on T cells from PBMC and SFMC. RESULTS: In patients with Lyme arthritis, B. burgdorferi but not IFN-γ induced PBMC to secrete CCL4 and CCL2, and B. burgdorferi and IFN-γ each stimulated the production of CXCL9 and CXCL10. However, with the CD14+ cell fraction, B. burgdorferi alone stimulated the secretion of CCL4; B. burgdorferi and IFN-γ together induced CCL2 secretion, and IFN-γ alone stimulated the secretion of CXCL9 and CXCL10. The percentage of T cells expressing CXCR3 or CCR5 was significantly greater in SFMC than PBMC, confirming that TH1 effector cells were recruited to inflamed joints. However, when stimulated with B. burgdorferi or IFN-γ, SFMC and PBMC responded similarly. CONCLUSIONS: B. burgdorferi stimulates PBMC or CD14+ monocytes/macrophages directly to secrete CCL4, but spirochetal stimulation of other intermediate cells, which are present in PBMC, is required to induce CD14+ cells to secrete CCL2, CXCL9 and CXCL10. We conclude that B. burgdorferi stimulates monocytes/macrophages directly and indirectly to guide innate and adaptive immune responses in patients with Lyme arthritis.

Treg cell numbers and function in patients with antibiotic-refractory or antibiotic-responsive Lyme arthritis.

OBJECTIVE: In a murine model of antibiotic-refractory Lyme arthritis, the numbers of Treg cells are dramatically reduced. The aim of this study was to examine Treg cell numbers and function in patients with antibiotic-refractory Lyme arthritis. METHODS: CD4+ T cell subsets were enumerated in the peripheral blood (PB) and synovial fluid (SF) of 12 patients with antibiotic-refractory arthritis and 6 patients with antibiotic-responsive arthritis. Treg cell function was examined using Borrelia-specific and nonspecific Treg cell proliferation assays. RESULTS: In both patient groups, interferon-gamma-positive Th1 cells in SF were abundant and enriched (approximately 50% of CD4+ T cells). In patients with antibiotic-refractory arthritis, the median percentages of FoxP3-positive Treg cells were significantly higher in SF than in PB (12% versus 6%; P = 0.03) or in SF from patients with antibiotic-responsive arthritis (12% versus 5%; P = 0.04). Moreover, in the antibiotic-refractory group, a higher percentage of Treg cells in SF correlated with a shorter duration until resolution of arthritis (r = -0.74, P = 0.006). In contrast, patients with fewer Treg cells had suboptimal responses to disease-modifying antirheumatic drugs and a longer duration of arthritis after antibiotic treatment, and they often required synovectomies for arthritis resolution. In each group, Treg cells in SF dampened Borrelia burgdorferi-specific proliferative responses, and in 2 patients with antibiotic-refractory arthritis, Treg cells were functional in nonspecific suppression assays. CONCLUSION: Treg cells were functional in patients with antibiotic-refractory arthritis, and in some patients, higher numbers of these cells in SF appeared to participate in arthritis resolution. However, as in the murine model, patients with antibiotic-refractory arthritis and lower numbers of Treg cells seemed unable to achieve resolution of synovial inflammation.

Analysis of Borrelia burgdorferi genotypes in patients with Lyme arthritis: High frequency of ribosomal RNA intergenic spacer type 1 strains in antibiotic-refractory arthritis.

OBJECTIVE: Most of the Borrelia burgdorferi genotypes have been isolated from erythema migrans (EM) skin lesions in patients with Lyme disease. OspC type K strains, which are 16S-23S ribosomal RNA intergenic spacer type 2 (RST2) strains, are most commonly recovered, but a higher percentage of OspC type A strains (RST1), the next most commonly recovered type, is detectable in blood. The goal of this study was to determine the B burgdorferi genotypes in the joints of patients with Lyme arthritis. METHODS: Joint fluid samples from 124 patients seen over a 30-year period were analyzed for OspC types by semi-nested polymerase chain reaction (PCR) and sequencing, and for RSTs by nested PCR and restriction fragment length polymorphism analysis. These results were correlated with clinical outcome. RESULTS: OspC and RST genotypes were identified in 49 of the 124 joint fluid samples (40%). In these 49 samples, OspC type K strains (RST2) were identified in 21 samples (43%), OspC type A strains (RST1) were identified in 11 samples (22%), and 8 other OspC types and all 3 RSTs were identified among the remaining 17 samples (35%). However, among the 17 patients who had been treated with antibiotics according to current guidelines, all 7 patients who were infected with RST1 strains had antibiotic-refractory arthritis, compared with 4 of 6 patients infected with RST2 strains and only 1 of 4 infected with RST3 strains (P = 0.03). CONCLUSION: Most of the B burgdorferi genotypes, particularly OspC type K (RST2), were identified in the joint fluid of patients with Lyme arthritis, and the genotype frequencies found in joints reflected those in EM skin lesions. However, RST1 strains were most frequent in patients with antibiotic-refractory arthritis. Our results help to further the understanding of the differential pathogenicity of strains of B burgdorferi.

Strong IgG antibody responses to Borrelia burgdorferi glycolipids in patients with Lyme arthritis, a late manifestation of the infection.

In this study, the membrane lipids of B. burgdorferi were separated into 16 fractions; the components in each fraction were identified, and the immunogenicity of each fraction was determined by ELISA using sera from Lyme disease patients. Only the 2 glycolipids, acylated cholesteryl galactoside (ACG, BbGL-I) and monogalactosyl diacylglycerol (MgalD, BbGL-II), were immunogenic. Early in the infection, 24 of 84 patients (29%) who were convalescent from erythema migrans and 19 of the 35 patients (54%) with neuroborreliosis had weak IgG responses to purified MgalD, and a smaller percentage of patients had early responses to synthetic ACG. However, almost all of 75 patients with Lyme arthritis, a late disease manifestation, had strong IgG reactivity with both glycolipids. Thus, almost all patients with Lyme arthritis have strong IgG antibody responses to B. burgdorferi glycolipid antigens.

Arthritogenicity of Borrelia burgdorferi and Borrelia garinii: comparison of infection in mice.

Arthritogenicity, as determined by joint swelling and synovial histology, was compared between or within two Borrelia genospecies that cause Lyme arthritis in humans. The spirochete burden in bladder tissue (a site of spirochete persistence) was documented by quantitative polymerase chain reaction, and immune responses were analyzed. In C3H/HeJ mice, three B. burgdorferi isolates and two of the three B. garinii isolates induced severe arthritis and swelling. Previous designation as invasive or noninvasive B. garinii, or RNA spacer type of B. burgdorferi did not determine arthritis severity induced by isolates. Compared with the other five isolates, the B. garinii PBi isolate induced significantly less arthritis, a lower humoral immune response, and persisted at a much lower level in bladder tissue. However, B. garinii PBi isolates induced similar Borrelia antigen-specific inflammatory T cell responses from the local draining lymph node. Thus, diverse B. burgdorferi and B. garinii isolates were highly arthritogenic in immune competent mice.

Higher mRNA levels of chemokines and cytokines associated with macrophage activation in erythema migrans skin lesions in patients from the United States than in patients from Austria with Lyme borreliosis.

BACKGROUND: Erythema migrans (EM) is caused primarily by Borrelia afzelii in Europe and solely by Borrelia burgdorferi in the United States. B. burgdorferi infection in the United States has previously been associated with faster expansion of EM lesions and with more associated symptoms, compared with B. afzelii infection in Europe. However, reasons for these differences are not yet known. METHODS: We determined the Borrelia species infecting 67 US or Austrian patients with EM. The clinical pictures and chemokine and cytokine mRNA levels in lesional skin were then compared in the 19 B. burgdorferi-infected US patients and the 37 B. afzelii-infected Austrian patients, the 2 largest groups. RESULTS: The 19 B. burgdorferi-infected US patients had faster-expanding EM lesions and a median of 4 associated signs and symptoms, whereas the 37 B. afzelii-infected Austrian patients had slower-expanding lesions and usually did not experience associated symptoms. Compared with the EM lesions of B. afzelii-infected Austrian patients, those of B. burgdorferi-infected US patients had significantly higher mRNA levels of chemokines associated with activation of macrophages, including chemoattractants for neutrophils (CXCL1), macrophages (CCL3 and CCL4), and T helper 1 cells (CXCL9, CXCL10, and CXCL11). In addition, compared with the EM lesions of Austrian patients, the EM lesions of US patients tended to have higher mRNA levels of the macrophage-associated proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha, and they had significantly higher mRNA expression of the antiinflammatory cytokines interleukin 10 and transforming growth factor beta. CONCLUSIONS: The EM lesions of B. burgdorferi-infected US patients expanded faster, were associated with more symptoms, and had higher mRNA levels of macrophage-associated chemokines and cytokines than did the EM lesions of B. afzelii-infected Austrian patients.

Antibody responses to Borrelia burgdorferi in patients with antibiotic-refractory, antibiotic-responsive, or non-antibiotic-treated Lyme arthritis.

OBJECTIVE: To compare the pattern of antibody responses to Borrelia burgdorferi in patients with antibiotic-refractory, antibiotic-responsive, or non-antibiotic-treated Lyme arthritis as an indirect measure of spirochetal persistence or eradication. METHODS: At least 3 serial serum samples from 41 patients with antibiotic-refractory arthritis and 23 patients with antibiotic-responsive arthritis, and samples from 10 non-antibiotic-treated, historical control patients were tested for IgG reactivity with B burgdorferi sonicate and 4 differentially expressed outer surface lipoproteins of the spirochete, by enzyme-linked immunosorbent assay. RESULTS: Among non-antibiotic-treated patients, antibody titers to B burgdorferi antigens remained high throughout a 2-5-year period of arthritis. In contrast, in patients with antibiotic-responsive arthritis, in whom joint swelling usually resolved during a 1-month course of oral antibiotic therapy, the median antibody titers to most of the spirochetal antigens remained steady or decreased during the first 1-3 months after starting antibiotic therapy. In patients with antibiotic-refractory arthritis, who had persistent joint swelling for a median duration of 10 months despite 2-3 months of oral or intravenous antibiotics, the median titers to most antigens increased slightly during the first 1-3 months. However, by 4-6 months after starting antibiotic therapy, reactivity with all antigens declined similarly in both antibiotic-treated groups. CONCLUSION: Whereas the antibody titers to B burgdorferi remained high in non-antibiotic-treated patients, the titers declined similarly 4-6 months after starting therapy in patients with antibiotic-responsive or antibiotic-refractory arthritis, suggesting that synovial inflammation persisted in patients with antibiotic-refractory arthritis after the period of infection.

Chemokine signatures in the skin disorders of Lyme borreliosis in Europe: predominance of CXCL9 and CXCL10 in erythema migrans and acrodermatitis and CXCL13 in lymphocytoma.

The three skin disorders of Lyme borreliosis in Europe include erythema migrans, an acute, self-limited lesion; borrelial lymphocytoma, a subacute lesion; and acrodermatitis chronica atrophicans, a chronic lesion. Using quantitative reverse transcription-PCR, we determined mRNA expression of selected chemokines, cytokines, and leukocyte markers in skin samples from 100 patients with erythema migrans, borrelial lymphocytoma, or acrodermatitis chronica atrophicans and from 25 control subjects. Chemokine patterns in lesional skin in each of the three skin disorders included low but significant mRNA levels of the neutrophil chemoattractant CXCL1 and the dendritic cell chemoattractant CCL20 and intermediate levels of the macrophage chemoattractant CCL2. Erythema migrans and particularly acrodermatitis lesions had high mRNA expression of the T-cell-active chemokines CXCL9 and CXCL10 and low levels of the B-cell-active chemokine CXCL13, whereas lymphocytoma lesions had high levels of CXCL13 and lower levels of CXCL9 and CXCL10. This pattern of chemokine expression was consistent with leukocyte marker mRNA in lesional skin. Moreover, using immunohistologic methods, CD3(+) T cells and CXCL9 were visualized in erythema migrans and acrodermatitis lesions, and CD20(+) B cells and CXCL13 were seen in lymphocytoma lesions. Thus, erythema migrans and acrodermatitis chronica atrophicans have high levels of the T-cell-active chemokines CXCL9 and CXCL10, whereas borrelial lymphocytoma has high levels of the B-cell-active chemokine CXCL13.

High levels of inflammatory chemokines and cytokines in joint fluid and synovial tissue throughout the course of antibiotic-refractory lyme arthritis.

OBJECTIVE: To investigate the possible role of chemokines and cytokines in the pathogenesis of Lyme arthritis. METHODS: Using cytometric bead array and flow cytometry techniques, chemokine and cytokine levels were determined in 65 synovial fluid (SF) samples and 7 synovial tissue (ST) samples from 17 patients with antibiotic-responsive Lyme arthritis and 35 patients with antibiotic-refractory Lyme arthritis seen during the past 18 years. In the ST samples, expression of chemokine receptors was measured using immunohistochemistry. RESULTS: Before or during antibiotic therapy, when the majority of patients had positive polymerase chain reaction (PCR) results for Borrelia burgdorferi DNA, SF from patients with antibiotic-refractory arthritis contained exceptionally high levels of Th1 chemoattractants and cytokines, particularly CXCL9 and interferon-gamma (IFNgamma). Compared with the patients whose arthritis was responsive to antibiotic treatment, those with antibiotic-refractory arthritis had significantly higher levels of CXCL9 and CXCL10 (both P

Short report: Association of macrophage inflammatory response and cell death after in vitro Borrelia burgdorferi infection with arthritis resistance.

Susceptibility to Borrelia burgdorferi infection and subsequent arthritis is genetically determined in mice and determined by innate immunity. Accordingly, macrophage responses to B. burgdorferi challenge may differ between mouse strains. Bone marrow-derived macrophages were infected ex vivo with clonal B. burgdorferi strain N40. Interleukin-12 and tumor necrosis factor-alpha (TNF-alpha) production were higher in macrophages from resistant C57Bl/6 mice than in macrophages from susceptible C3H/HeJ mice. However, TNF-alpha production was observed in lower concentrations in C3H/HeJ (toll-like receptor-4(-/-)) macrophages than in C3H/FeJ (TLR4(+/+)) macrophages, suggesting that TLR4 might contribute to the response to B. burgdorferi. A higher cytokine response to B. burgdorferi was associated with cell death in macrophages from resistant C57Bl/6 mice. Understanding variability in the response of macrophages to B. burgdorferi may contribute to understanding Lyme arthritis.

Borrelia burgdorferi genetic markers and disseminated disease in patients with early Lyme disease.

Three genetic markers of Borrelia burgdorferi have been associated with disseminated disease: the OspC type, the 16S-23S rRNA intergenic spacer type (RST), and vlsE. Here, we modified previous methods so as to identify the three markers by PCR and restriction fragment length polymorphism in parallel, analyzed B. burgdorferi isolates from erythema migrans (EM) skin lesions in 91 patients, and correlated the results with evidence of dissemination. OspC type A was found approximately twice as frequently in patients with disseminated disease, whereas type K was identified approximately twice as often in those without evidence of dissemination, but these trends were not statistically significant. The remaining seven types identified were found nearly equally in patients with or without evidence of dissemination. RST 1 strains were significantly associated with dissemination (P=0.03), whereas RST 2 and RST 3 strains tended to have an inverse association with this outcome. The vlsE gene was identified in all 91 cases, using primer sets specific for an N-terminal sequence of B. burgdorferi strain B31 (vlsEB31) or strain 297 (vlsE297), but neither marker was associated with dissemination. Specific combinations of the three genetic markers usually occurred together. OspC type A was always found with RST 1 and vlsEB31, type K was always identified with RST 2 and more often with vlsE297, and types E and I were almost always found with RST 3 and equally often with vlsEB31 and vlsE297. We conclude that B. burgdorferi strains vary in their capacity to disseminate, but almost all strains isolated from EM lesions sometimes caused disseminated disease.

Molecular characterization of the OspA(161-175) T cell epitope associated with treatment-resistant Lyme arthritis: differences among the three pathogenic species of Borrelia burgdorferi sensu lato.

Treatment-resistant Lyme arthritis, which may result from infection-induced autoimmunity, is associated with reactivity to a T cell epitope of outer-surface protein A (OspA(161-175)) of Borrelia burgdorferi sensu stricto (Bb). This syndrome has been noted primarily in the United States where only Bb is present, and rarely in Europe where Borrelia garinii (Bg) and Borrelia afzelii (Ba) predominate. To gain a better understanding of this epitope, we identified its species-specific polymorphisms, determined their immunogenicity, and characterized the contribution of individual amino acids. Based on published sequences the Bb peptide differed from the Ba peptide in six of the nine core residues (amino acids 165-173), whereas the Bg peptide usually differed in three of the nine residues. Lymphocytes from seven patients with treatment-resistant Lyme arthritis proliferated in response to the Bb peptide, but not to the Ba or Bg peptide. Substitution analysis showed that valine166 and threonine172 were critical for the immunogenicity of the Bb peptide. Thus, consistent with the geographic distribution of the illness, the European causative agents of Lyme borreliosis usually lack the putative pathogenic OspA epitope. These observations are consistent with the hypothesis that T cell recognition of this epitope is important in the induction of autoimmunity in treatment-resistant Lyme arthritis.