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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Arthritis Rheum. Author manuscript; available in PMC 2014 January 1.
Published in final edited form as:
PMCID: PMC3535550
NIHMSID: NIHMS411425

A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease

Abstract

Objective

Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but uncovering disease-relevant autoantigens has been a difficult challenge. Our goal was to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, which is thought to result from infection-induced autoimmunity.

Methods

Using tandem mass spectrometry, naturally presented HLA-DR self-peptides from a patient’s synovium were identified, synthesized and reacted with his peripheral blood mononuclear cells (PBMC). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patients’ cells or sera.

Results

Of 120 HLA-DR-presented self-peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMC to proliferate. We then found that T and B cell responses to ECGF occurred systemically in about 10–30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. In non-antibiotic-treated historic patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis.

Conclusion

T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence for autoimmune T and B cell responses in this illness.

Presentation of autoantigens by HLA-DR molecules to CD4+ T cells is thought to be a central component of many autoimmune diseases (1), but in most instances, disease-relevant autoantigens have remained elusive. The problem is compounded by the fact that human autoimmune diseases are generally thought to be multifactorial, involving both genetic and environmental factors such as infection (2). Furthermore, in autoimmune diseases such as rheumatoid arthritis (RA) or lupus, multiple autoantigens are thought to be involved, and autoantibodies are often present months or years before the onset of clinical disease (3, 4), suggesting that additional critical factors are required to trigger tissue pathology (3). Even so, recognition of self-antigens is an essential component in the development of disease pathology.

Lyme arthritis, a late manifestation of infection with the tick-borne spirochete, Borrelia burgdorferi (Bb) (5, 6), provides an important human model to study questions surrounding infection-induced autoimmunity. Lyme arthritis can usually be treated successfully with 1–2 months of oral or intravenous (IV) antibiotics, called antibiotic-responsive arthritis (7). However, in a small percentage of patients, proliferative synovitis persists for months or several years after apparent spirochetal killing with ≥3 months of oral and IV antibiotics, referred to as antibiotic-refractory arthritis (8).

This disease course has been postulated to result from either persistent infection, retained spirochetal antigens, or infection-induced autoimmunity (9, 10). Against the persistent infection hypothesis, PCR and culture results of synovectomy specimens obtained in the post-antibiotic period have been uniformly negative (11), and relapse of infection has not been observed with the use of disease modifying anti-rheumatic drugs (DMARDs) after antibiotic therapy (8). Contrary to what might be expected with retained spirochetal antigens, T and B cell responses to Bb decline similarly in patients with refractory or responsive arthritis (12, 13), whereas inflammatory mediators in synovial fluid (SF), particularly IFN-γ, remain high or even increase in the refractory group during the post-antibiotic period (14). In support of the autoimmunity hypothesis, specific HLA-DR alleles, particularly the DRB1*0101 or 0401 alleles, are the greatest known genetic risk factor for antibiotic-refractory arthritis (15). As in other chronic inflammatory arthritides, HLA-DR molecules in antibiotic-refractory Lyme arthritis are intensely expressed in inflamed synovium (16).

In a search for molecular mimicry between spirochetal and host proteins, partial sequence homology was found between the human peptides, LFA-1αL332-340 (17) and MAWD-BP280-288 (18), and an epitope of Bb outer-surface protein A (OspA163-175) (19), which binds refractory arthritis-associated HLA-DR molecules (15). However, only a minority of patients had low-level T cell reactivity with these self peptides, and none had autoantibody responses to these self proteins (18, 20). Later, Ghosh et al., identified human cytokeratin 10 as a cross-reactive target ligand recognized by anti-OspA antibodies in a small group of patients with refractory arthritis (3 of 15), but not in those with responsive arthritis (0 of 5) (21). Finally, several neural proteins have been reported to induce T or B cell responses in patients with neuroborreliosis (2224) or post-Lyme syndrome (25). However, responses against neural proteins would be unlikely to explain antibiotic-refractory arthritis.

In this study, we utilized discovery-based proteomics and translational research in an effort to identify autoantigens in synovial tissue, the target tissue of the immune attack in antibiotic-refractory Lyme arthritis. Based on this approach, we report here the identification of a novel autoantigen, endothelial cell growth factor (ECGF), which is a target of T and B cell responses in a subset of patients with Lyme disease, thereby providing the first direct evidence for autoimmune T and B cell responses in this illness.

PATIENTS AND METHODS

Patients

All Lyme disease patients met the CDC criteria (26) and those with RA met the ACR/EULAR criteria (27). Studies from 1975–1987 were approved by the Human Investigations Committee at Yale University School of Medicine, those from 1988–2002 were approved by Tufts Medical Center, and those after 2002 were approved at Massachusetts General Hospital.

In patients with erythema migrans (EM), who were seen from 1998–2001 in a study of early Lyme disease, all available PBMC and serum samples were tested from culture-positive patients. In antibiotic-treated patients with Lyme arthritis, who were seen from 1988–2010 in a study called “Immunity in Lyme arthritis”, the first available sample was used. For comparison, serial serum samples were tested from non-antibiotic-treated patients who were seen in the late 1970s. In patients with RA, who were evaluated from 2008–2010 in a study of biomarkers for early disease, PBMC, serum and SF samples were obtained from patients during the first year of the disease. All RA patients had positive test results for rheumatoid factor or anti-CCP antibodies. PBMC were stored in liquid nitrogen, and serum samples were stored at − 80°C.

Isolation and identification of synovial HLA-DR-presented peptides

A detailed description of the isolation and identification of in vivo HLA-DR presented peptides from patients’ synovial tissue are found in our previous publication (28).

T cell Proliferation Assay

T cell proliferation assays were performed as previously described (29). Briefly, the patient’s PBMC were stimulated for 5 days with 2 uM of each peptide after which 3H-thymidine was added. All non-redundant HLA-DR presented peptides were synthesized by Mimotopes, (Victoria, Australia).

ELISpot

Assays were performed using ELISpotplus for human IFN-γ kits (MabTech). ECGF peptides were synthesized and HPLC-purified at the Tufts University Core Facility (Boston, MA). The peptide sequences were as follows with the predicted promiscuous HLA-DR binding peptides shown in bold: (A52DIRGFVAAVVNGSAQGAQI71; D123KVSLVLAPALAACG137; S220KKLVEGLSALVVDV234; K253TLVGVGASLGLRVAAALTAMD274; L340GRFERMLAAQGVDPG355; L302RDLVTTLGGALLWL316 and G387TVELVRALPLALVLH401. All peptides (1 μM) were tested in duplicate wells, as were positive (PHA) and negative (no antigen) controls. After 5 days, cells were transferred to ELISpot plates coated with IFN-γ antibody and incubated overnight. Images of wells were captured using ImmunoSpot series 3B analyzer, and spots were counted using ImmunoSpot software. Based upon 18 healthy control subjects (Figure 3), a positive T cell response was defined as a stimulation index (SI) that was 3 standard deviations (SD) above the mean of healthy subjects, which characteristically gave >40 spot forming units (SFU)/106 PBMC and a stimulation index of >8.

Figure 3
Testing of PBMC from healthy control subjects, or patients with RA, EM, or antibiotic-responsive or antibiotic-refractory Lyme arthritis for T cell recognition of ECGF peptides. PBMC were incubated with individual ECGF peptides (1 μM), PHA (positive ...

ELISA for serum IgG anti-ECGF antibodies

ELISA plates were coated with 0.5 μg/ml carrier free, recombinant human PD-ECGF (R&D Systems) dissolved in PBS and incubated overnight at 4°C. All subsequent steps were performed at room temperature with plates on a platform shaker set at 200 rpm. After washing with PBST (PBS and 0.05% Tween-20), the plates were incubated with blocking buffer (5% nonfat dry milk in PBST), followed by patient or control serum samples (100 μl, 1:100). Subsequently, goat anti-human IgG conjugated to horseradish peroxidase was added, followed by TMB substrate. For inter-plate standardization, 4 patient and 8 control samples were included on each plate.

Immunoblotting

Human recombinant PD-ECGF was electrophoresed through acrylamide gels and transferred to nitrocellulose membranes. Membranes were cut into strips and incubated for 1 h in blocking buffer (5% nonfat dry milk dissolved in 0.1% Tween-20 in 20 mM Tris, 500 mM NaCl; pH 7.5). The strips were incubated with patient or control sera (1:100), followed by goat anti-human IgG antibody conjugated to alkaline phosphatase, then visualized with NBT/BCIP substrate. Densitometric analysis of scanned blots was performed using IMAGEJ. To correct for external variation between experiments, the data were standardized to a positive control included in each blot.

ELISA for synovial fluid ECGF

ELISA plates were coated with goat anti-human PD-ECGF (5 ug/ml) and incubated overnight at 4°C. The next day, plates were incubated with blocking buffer (5% nonfat dry milk in PBST), followed by each patient’s SF sample (100 μl, 1:10). To generate a standard curve, serial dilutions of PD-ECGF were added to each plate. The plates were then incubated with mouse anti-human PD-ECGF antibody, followed by goat anti-mouse IgG conjugated to horse radish peroxidase, and then TMB substrate.

Immunohistochemistry

Synovial tissue samples were cut and fixed in cold acetone. Endogenous peroxidases were blocked with hydrogen peroxide in methanol, followed by 1-time power block solution containing 10% normal donkey serum. The sections were then incubated with anti-rabbit polyclonal ECGF (3μg/ml) at 4°C overnight. For negative controls, non-specific rabbit IgG was used. The following day, the sections were incubated with biotinylated anti-rabbit secondary antibody, peroxidase-streptavidin, and then diaminobenzidine substrate. The sections were counterstained with Mayer’s hemotoxylin and mounted with glycerol. Microscopic images were obtained with a Nikon eclipse ME6000 microscope. Each slide was read blindly, and intensity of ECGF staining on each tissue region was graded on a semi-quantitative scale of 0–3: 0 = no ECGF-positive cells; 1 = few (~50 positive cells); 2 = many (~50 to 100 positive cells); and 3= most (>100 positive cells).

Statistical analysis

Categorical data were analyzed using either the chi-square test or the Fisher’s exact test, and quantitative data were analyzed by the Mann-Whitney test. All analyses were performed using SigmaStat 3.0.

RESULTS

Identification of HLA-DR-peptides in synovial tissue

Based upon the hypothesis that HLA-DR molecules in inflamed synovial tissue, the target of the immune response in antibiotic-refractory Lyme arthritis, present disease-related autoantigens, we utilized a broadly applicable, unbiased approach for the identification of autoantigens in this tissue. The protocol had 3 steps: 1) a proteomics approach utilizing tandem mass spectrometry (MS/MS) for the identification of HLA-DR-presented peptides in an individual patient’s synovial tissue, 2) synthesis and testing of all non-redundant peptides identified for autoreactivity with the matching patient’s PBMC, and 3) determining whether any autoreactive peptides and their source proteins identified in a single patient also induced T and B cell responses in large numbers of patients with Lyme disease.

In step 1, we initially analyzed synovial tissue from 4 patients, 2 with antibiotic-refractory Lyme arthritis, and for comparison, 2 with RA (28). The approach is shown in Figure 1. Altogether, we identified 1,427 synovial HLA-DR in vivo presented peptides (220 to 464 per patient), which were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. These source proteins were substantially different from those identified from EBV cell lines (30), demonstrating the necessity of using patients’ target tissues or cells for identifying naturally presented HLA-DR epitopes. The complete list of peptides, their spectra, and their source proteins for each of the 4 patients is given in our previous publication (28).

Figure 1
An overview of the isolation and identification of in vivo HLA-DR presented peptides from patients’ synovial tissue. Antibiotic-refractory Lyme arthritis usually manifests as one swollen knee (A). In those cases in which therapeutic arthroscopic ...

Screening HLA-DR-presented peptides for autoantigenicity

In step 2, we first tested peptides identified from a youth who had a synovectomy for the treatment of antibiotic-refractory Lyme arthritis (LA1). He had one of the refractory arthritis-associated HLA-DR alleles (DRB1*0101). Of the 2,237 MS/MS spectra generated from his tissue sample, 464 had a consensus match with 2 or more mass spectrometry search programs (Mascot, OMSSA or X!Tandem), of which 104 were non-redundant. Since we wanted to test as many candidate peptides as possible, we also manually inspected the 53 peptides identified by only 1 of 3 search programs, of which 16 could be verified. Altogether, we tested 120 non-redundant peptides for autoreactivity with the patient’s PBMC in T cell proliferation assays. Because of limited cell numbers, peptides were pooled (3 per well) for testing.

Only two peptide sets (sets 33 and 40) induced proliferative responses that were >2 times background (Figure 2). When we retested the 6 peptides from these 2 sets, only one peptide from set 40 induced a proliferative response that was >2 times background, which was substantially higher than any other peptide. This peptide’s mass spectrum (L340GRFERMLAAQGVDPG355) is shown in Figure 1E. This peptide was 1 of the 16 peptides identified by only 1 of the 3 search programs and originated from the source protein platelet-derived endothelial cell growth factor (ECGF), also called thymidine phosphorylase or gliostatin. ECGF is a chemotactic factor, it has a proliferative effect on endothelial cells, and it induces angiogenesis (31). Moreover, it is not known to be a relevant autoantigen in any other autoimmune disease.

Figure 2
Screening of in vivo presented HLA-DR-peptides identified from the synovial tissue of one patient for T cell autoreactivity using the patient’s own PBMC. All 120 non-redundant HLA-DR-presented peptides identified were synthesized and tested in ...

T cell responses to ECGF

In step 3, all available PBMC and serum samples collected over a 25-year period were tested from patients with EM (erythema migrans), the initial skin lesion of early Lyme disease, and from those with Lyme arthritis, for T and B cell reactivity with ECGF. For comparison, samples from healthy control subjects and from patients with RA were tested. Although HLA-DR typing was performed only in patients with Lyme arthritis, it is likely that patients with RA would be enriched for the HLA-DRB1*0101 and 0401 alleles, as in patients with antibiotic-refractory Lyme arthritis (15).

Initially, patients’ PBMC were tested for T cell autoreactivity using commercially available recombinant ECGF. However, we found, as had others, that ECGF inhibited the read-out of the 3H-thymidine assay (32), and non-specifically induced PBMC to secrete IFN-γ. Therefore, using 3 HLA-DR T cell epitope prediction algorithms (33, 34), 7 T cell peptide epitopes of ECGF were identified and synthesized, including the peptide initially isolated in the patient’s synovial tissue sample (ECGF340-355). Five of the 7 peptides, including ECGF340-355, were predicted to be promiscuous HLA-DR binders (predicted to bind ≥19 HLA-DR molecules). Rather than proliferation assays, IFN-γ ELISpot assays were used due to their increased sensitivity.

Healthy control subjects and RA patients had only minimal responses to a few of the 7 ECGF peptides tested (Figure 3). Of the patients with EM, an early manifestation of Lyme disease, 16% had low-level T cell responses. In contrast, 30% of patients with antibiotic-responsive arthritis and 38% of those with antibiotic-refractory arthritis had robust responses, often to multiple ECGF peptides. Overall, Lyme arthritis patients had substantially greater T cell responses to ECGF peptides than those in the other groups; their cells recognized all 7 peptides tested; and 10 patients had responses to 2-to-4 ECGF peptides, suggestive of epitope spreading.

Previously, we showed that patients with antibiotic-refractory arthritis more often had HLA-DRB1*0101, 0102, 0401, 0402, 0404, or 1501 alleles (15). Of the 21 patients tested here with refractory or responsive arthritis who had T cell reactivity with ECGF peptides, 20 (95%) had known refractory arthritis-associated alleles. Therefore, T cell responses to ECGF appear to occur primarily in patients with antibiotic-refractory risk alleles.

B cell responses to ECGF

ECGF-reactive CD4+ T cells likely contribute to pathogenicity by providing help to B cells to produce anti-ECGF autoantibodies. Therefore, we tested patients’ serum samples for IgG anti-ECGF antibodies using two methods, ELISA and immunoblotting. When ECGF antibody responses were determined by ELISA, none of the 74 healthy control subjects had a positive response (defined as >3 SD above the mean value of healthy subjects) (Figure 4A). In comparison, 15% of patients with EM (P=0.001), 8% of patients with responsive arthritis (P=0.04), and 17% patients with refractory arthritis (P<0.0001) had positive responses. In addition, patients with antibiotic-refractory arthritis tended to have ECGF autoantibodies more frequently than those with antibiotic-responsive arthritis (17% vs. 8%; P=0.09). In contrast, none of the 33 patients with RA had a positive response. When these sera were tested by immunoblotting, similar results were obtained (Figure 4B), though immunoblotting was not done in RA patients as not enough serum remained. Moreover, the results by ELISA and immunoblotting were highly concordant in patients with refractory arthritis (P<0.0001), but not in those with EM or responsive arthritis. These 2 methods may not assess all of the same epitopes, and therefore, concordance may occur only with recognition of multiple epitopes.

Figure 4
IgG anti-ECGF autoantibody responses in the sera of healthy control subjects, or patients with EM, antibiotic-responsive or antibiotic-refractory Lyme arthritis, or RA as determined by ELISA and immunoblotting. For the ELISA, plates were coated with recombinant ...

When concordance was assessed between T cell (ELISpot) and B cell (ELISA) assays, 13 of 22 patients (59%) with responsive arthritis and 23 of 37 patients (62%) with refractory arthritis had concordant results, and similar results were obtained when the comparison was done with immunoblotting data. Although the overall frequencies of T and B cell immune responses in patients with EM were similar, concordance was difficult to show due to the small number of patients with positive T cell responses (N=3). T or B cell reactivity to ECGF did not correlate with the duration of arthritis or how long the sample had been frozen prior to testing. Taken together, T and B cell responses to ECGF occurred in patients with early or late manifestations of Lyme disease, most frequently in those with antibiotic-refractory arthritis.

Antibody responses to ECGF in non-antibiotic-treated patients

In a study of the natural history of Lyme disease carried out in the late 1970s prior to knowledge of the etiology of the illness, we followed 55 non-antibiotic-treated patients with EM longitudinally for a median duration of 6 years (6). Of the 55 patients, 21 (38%) had no subsequent manifestations or only brief joint pain, whereas 34 (62%) subsequently developed intermittent attacks or persistent arthritis, lasting from 2 weeks to 4 years. Serial serum samples were still available in 42 of the 55 patients. Of the 15 patients who did not develop arthritis, 2 (13%) had ECGF antibody responses 2 to 3 weeks after onset of EM, whereas 7 of the 27 patients (26%) who later had arthritis had positive ECGF antibody responses, a non-significant difference. In 6 of the 7 arthritis patients who had ECGF responses, reactivity developed weeks to months after disease onset, prior to joint inflammation. When patients’ attacks of arthritis were added together, the duration of active arthritis was significantly longer in the 7 patients who had ECGF responses than in the 20 patients who did not (median value, 67 versus 17 weeks, respectively, P=0.004). Correlation of disease activity and ECGF antibody levels are shown for the patient who had the most prolonged arthritis (Figure 5). Thus, in untreated patients, ECGF antibody responses usually developed early in the illness and in those who subsequently developed arthritis, this response was associated with significantly longer joint inflammation.

Figure 5
Correlation of clinical course with IgG anti-ECGF antibody responses as determined by ELISA in a non-antibiotic-treated historic patient followed for 6 years throughout his entire disease course. He initially had EM followed several months later by a ...

ECGF in joint fluid and synovial tissue

For ECGF to have pathogenic relevance as an autoantigen in antibiotic-refractory Lyme arthritis, one would predict that this protein would be present in high concentrations in patients’ inflamed SF and tissue. Although SF was available in patients with antibiotic-responsive arthritis, synovial tissue was not as therapeutic synovectomies are never necessary in this patient group. As determined by sandwich ELISA, patients with antibiotic-refractory arthritis often had very high concentrations of ECGF in SF (mean value = 448 ng/ml) (Figure 6A), which were significantly greater than those in patients with antibiotic-responsive arthritis (154 ng/ml) (P<0.0001). RA patients also often had high levels of ECGF (313 ng/ml), which is consistent with the findings of other investigators (32). They also showed that patients with osteoarthritis (OA), a minimally inflammatory form of arthritis, had much lower levels (8.7 ng/ml).

Figure 6
Detection of ECGF protein in joint fluid and synovial tissue. ECGF concentrations in joint fluid of patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis, or RA were measured by sandwich ELISA (A). Representative serial synovial ...

Synovial tissue from 16 patients with antibiotic-refractory arthritis and 5 patients with RA was examined for the presence of ECGF. Of the 16 patients with antibiotic-refractory arthritis, 10 (63%) had moderate-to-intense staining for ECGF in the lining and sublining of the synovial tissue, 4 (25%) had mild staining, and 2 (12%) had no staining in these areas. In comparison, the 5 RA patients had staining for ECGF primarily in the lining area, with little seen in sublining region. Representative examples from one patient each with antibiotic-refractory arthritis or RA are shown in Figure 6B. In the synovial tissue of the patients with antibiotic-refractory arthritis, ECGF staining was clearly evident in the sublining area around blood vessels (green circle) and in large cells, most likely synoviocytes (red arrow). In contrast, in the RA patient, staining was not seen in the sublining region. Thus, the majority of patients with antibiotic-refractory arthritis had large amounts of ECGF in SF and intense staining in synovial tissue where the protein could act as an autoantigen.

DISCUSSION

In this study, we utilized discovery-based proteomics to identify HLA-DR-presented self peptides in a patient’s synovium and determined the immunoreactivity of the peptides by testing them with his PBMC. In this way, one peptide derived from endothelial cell growth factor (ECGF) was shown to be autoreactive. We then found that about 10–30% of patients with early or late manifestations of Lyme disease had T or B cell responses to ECGF, and they were more common in patients with antibiotic-refractory arthritis. Analogous to the situation in RA or lupus (3, 4), autoimmune responses to ECGF in Lyme disease often began before the onset of arthritis. Moreover, in non-antibiotic-treated historic patients, ECGF antibody responses often developed early in the illness and were associated with more persistent arthritis. Previously, naturally presented HLA-DR peptides had been identified from other tissues and fluids by MS/MS (3539), but until our recent report (28), not from synovial tissue, and no previous studies had systematically tested the autoreactivity of each peptide identified using the matching patient’s PBMC, the key step here in autoantigen identification.

ECGF itself was found in high levels in SF and synovial tissue in patients with antibiotic-refractory Lyme arthritis and in those with RA. However, the protein appeared to be immunoreactive primarily in patients with Lyme disease. The reasons why Bb infection may lead to ECGF reactivity are not yet known. T cell epitope mimicry between a spirochetal and host protein is often the first hypothesis considered. In a search of the Bb proteome, the Bb peptide with the greatest sequence homology with ECGF340-355 was derived from BB_0580 (F78ERMLA83), a Bb protein that is not known to be immunoreactive (40). This Bb peptide has sequence homology with 6 of 9 ECGF340-355 core residues, but in our experience (18), this is insufficient to induce cross-reactivity. Moreover, contrary to what one would expect with a molecular mimicry mechanism, no single ECGF epitope was recognized by all, or even the majority of, ECGF-reactive patients.

Alternatively, ECGF antibody responses could reflect a cross-reactive antibody response to Bb. However, the fact that patients had both T and B cell responses to ECGF, including responses to multiple T cell epitopes, argues against this possibility. Another option is that excessive joint inflammation may lead to bystander activation of ECGF-specific T cells. However, the lack of robust ECGF reactivity in RA patients, despite high levels of the protein in joints, argues against this type of non-specific mechanism. One additional possibility is that Bb, which is known to bind several host proteins (41, 42), may also bind ECGF allowing Bb to act simultaneously as a conduit for enhanced presentation by antigen-presenting cells and as an adjuvant.

We postulate a three-step process in the pathogenesis of antibiotic-refractory Lyme arthritis. In the first step, autoimmune responses to ECGF develop systemically in about 10–20% of individuals with Lyme disease, often early in the illness. Both spirochetal and host genetic factors are probably important in the generation of this autoimmune response. For example, Bb RST1 strains, which account for ~30–50% of the infections in the northeastern U.S., are more inflammatory than other Bb strains (43), and more frequently cause antibiotic-refractory arthritis (44). Important host factors likely include specific HLA genotypes, such as DRB1*0101 or 0401 (15), which are also found more often in patients who develop antibiotic-refractory arthritis.

In the second step, we propose that this rather common systemic autoimmune response becomes pathogenic in only a small percentage of patients who have marked ECGF antigen accumulation, excessive inflammation, and immune dysregulation in joints. Patients with refractory arthritis had elevated concentrations of ECGF in their SF where it could be taken up by local APC and presented at high concentrations to T cells leading to their activation. Moreover, intense staining for ECGF was detected in synovia of patients with refractory arthritis where it could initiate immune complex deposition and tissue damage through excessive complement activation. However, more than one autoantigen or certain spirochetal antigens may be important in antibiotic-refractory arthritis, since a considerable number of patients with refractory arthritis do not have anti-ECGF antibodies. Moreover, in untreated patients, infection and autoimmunity may occur together, though the arthritis seems to be more persistent when the autoimmune component is present. It is only with antibiotic treatment that one may observe the autoimmune component independently.

Even though more than one triggering antigen may be involved, patients with antibiotic-refractory arthritis, particularly those with a TLR1 polymorphism (1805GG) (43), have exceptionally high levels of pro-inflammatory mediators such as TNF-α, IL1-β, and IFN-γ in SF and synovial tissue compared to patients with responsive arthritis (14). Although initially important for eradication of the spirochete, the inability of these individuals to down-regulate expression of these inflammatory mediators likely contributes to immune dysregulation (45, 46). Finally, in patients with antibiotic-refractory arthritis, CD4+ T effector cells in SF resist suppression by CD4+ Treg cells and lower numbers of CD4+ Treg cells correlate with longer durations of arthritis (47).

In the third step, synovitis in most patients resolves within months to several years after antibiotic therapy, assisted by DMARDs such as methotrexate, which are thought to inhibit T cell activation (48). In these patients, we postulate that the innate immune “danger” signals provided by live spirochetes are no longer present, and without these signals, the adaptive immune response to autoantigens eventually regains homeostasis. Similarly, in patients requiring synovectomies, the arthritis does not usually recur because innate immune signals associated with active infection are missing.

In summary, we have shown definitively that T and B cell responses to ECGF occur in a subset of patients with Lyme disease, thereby identifying the first autoantigen that is a target of autoimmune T and B cell responses in this illness. Moreover, the approach used here for the identification of novel autoreactive HLA-DR-presented peptides in synovia of patients with antibiotic-refractory Lyme arthritis should be valuable for the determination of immune targets in other forms of chronic inflammatory arthritis, including RA.

Acknowledgments

Supported by National Institutes of Health grants AR-20358 (to A.C.S.), P41 GM104603/RR10888, S10 RR15942, S10 RR20946, and contracts N01 HV28178 and NO1 HV00239 (to C.E.C.), the Dana Foundation (to A.C.S. and C.E.C.), and the Mathers Foundation, the English, Bonter, Mitchell Foundation, the Eshe Fund, and the Lyme/Arthritis Research Fund at Massachusetts General Hospital (to A.C.S.). Klemen Strle was the recipient of post-doctoral fellowships from the Arthritis Foundation and the Walter J. and Lille A. Berbecker Foundation, and Kianoosh Katchar received a scholarship for the study of Lyme disease from the Lillian B. Davey Foundation.

The authors thank Drs. Nitin Damle and Vijay Sikand for the collection of samples from patients with EM, Drs. Elena Massarotti and Robert Kalish for help in obtaining samples from patients with Lyme arthritis and Dr. Lisa Glickstein for helpful discussion about the manuscript.

Footnotes

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content and all authors approved the final version to be published. Dr. Drouin had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design. Drouin, Costello and Steere.

Acquisition of data. Drouin, Seward, Strle, McHugh, Katchar, Londono and Yao.

Analysis and interpretation of data. Drouin, Costello and Steere.

Contributor Information

Elise E. Drouin, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

Robert J. Seward, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

Klemen Strle, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

Gail McHugh, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

Kianoosh Katchar, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

Diana Londoño, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

Chunxiang Yao, Boston University School of Medicine, Boston, Massachusetts.

Catherine E. Costello, Boston University School of Medicine, Boston, Massachusetts.

Allen C. Steere, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.

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