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. Author manuscript; available in PMC: 2009 Dec 1.
Published in final edited form as: Arthritis Rheum. 2008 Dec;58(12):3892–3901. doi: 10.1002/art.24028

Borrelia burgdorferi infected HLA-DR4-positive CD28-negative mice have persistent arthritis post antibiotic treatment

Bettina Panagiota Iliopoulou 1, Joseph Alroy 2, Brigitte T Huber 1,*
PMCID: PMC2775539  NIHMSID: NIHMS68864  PMID: 19035513

Abstract

Objective:

The immunological events that lead to persistent joint inflammation in certain Lyme arthritis patients post antibiotic treatment have been elusive so far. The prevalence of this condition is highest in individuals with RA-associated HLA.DR alleles. The aim of this study was to generate a murine model that presents persistent arthritis post antibiotic treatment.

Methods:

We have previously show that CD28−/− mice develop intermittent monoarticular Lyme arthritis that is antibiotic responsive. Since there seems to be a link between persistent arthritic manifestations post antibiotic treatment in human patients and HLA-DR4 allele, we generated DR4+/+CD28−/−MHC-II−/− mice, infected them with Bb and subsequently treat them with antibiotics.

Results:

We now show that 38% of Bb-infected DR4+/+CD28−/−MHC-II−/− mice, but none of the Bb-infected CD28−/−MHC-II−/− mice, remained arthritic post antibiotic treatment. A significant fraction (36%) of these mice, but none of the mice that resolved arthritis, had serum antibodies to OspA of Bb. After abrogation of active Bb infection, the inflammatory reaction in mice with persistent joint inflammation is restricted to the joints, since their draining lymph nodes were no longer enlarged. Increased CD20 and IFN-γ mRNA expression in the inflamed joints of these mice suggest a possible role for B cells and inflammatory cytokines in the pathogenesis of persistent arthritis post antibiotic treatment.

Conclusion:

The establishment of this murine model allows, for the first time, the elucidation of the immunological events that lead to persistent Lyme arthritis post antibiotic therapy in genetically susceptible individuals.

INTRODUCTION

Lyme disease, caused by the tick-borne spirochete Borrelia burgdorferi (Bb), is the most common vector-borne illness in the United States. After inoculation into the skin, Bb quickly disperses in the mammalian host by binding to components of the extracellular matrix (1). Three clinical stages of Lyme disease have been described in humans. Early infection consists of localized erythema migrans, followed within days or weeks by disseminated infection that affects the nervous system, heart or joints, and subsequently by late or persistent infection (2). While the spirochetes can be eliminated from Lyme patients by antibiotic treatment, chronic arthritis may persist, mainly in patients with RA-associated HLA-DR alleles, such as HLA-DRB1*0401 (DR4) and HLA-DRB1*0101 (3, 4). Two basic hypotheses have been proposed to explain this phenomenon: persistent infection and infection induced autoimmunity. The latter hypothesis is supported by the fact that the arthritic manifestations persevere despite the absence of Bb DNA, documented by PCR analysis of the synovial fluid (5-7). Interestingly, 70% of the patients who continue to experience arthritis after antibiotic treatment mounted an antibody (Ab) response to outer surface protein A (OspA) of Bb during periods of maximal arthritis that seems to parallel the severity and duration of arthritis (8, 9). In addition, an anti-OspA Th1 response has been documented in the synovial fluid of these patients (10-12).

Murine systems have been developed to analyze the immunological events occurring upon Bb infection in humans. It is well established that the arthritic manifestations depend upon the administered dose of Bb, the age and the genetic background of the mice (13-15). It has also been suggested that T cells, more specifically the CD4+ Th1 subset, as well as the pro-inflammatory cytokine IFN-γ, are responsible for arthritis exacerbation upon Bb infection (16-20). Murine Lyme arthritis peaks within the first 2 weeks post infection and then resolves spontaneously and only very few individual mice continue to present chronic arthritis (21, 22). These manifestations are reminiscent of the acute phase of Lyme arthritis in humans (15). Thus, a detailed study of the chronic phase of Lyme arthritis has been elusive thus far.

By studying the development of Lyme arthritis in different inbred mouse strains, it has become apparent that arthritis severity depends upon a fine balance between pro-inflammatory factors and immunoregulatory mechanisms. We hypothesized that by interfering with this balance, such as in the CD28−/− mouse, we could increase the incidence of chronic Lyme arthritis. We have recently demonstrated that CD28−/− mice, but not wild type C57BL/6J (B6) mice, develop chronic Lyme arthritis upon Bb infection. The persistent arthritic attacks observed in these mice last for over 6 months, but were sensitive to antibiotic treatment (23). Since a prerequisite for the development of persistent arthritis post antibiotic treatment in humans is the presence of HLA.DR4 or related alleles, we introduced the CD28−/− genotype onto the DR4+/+MHC-II−/− background. Here we show that a significant fraction of Bb-infected DR4+/+CD28−/−MHC-II−/− mice continue to manifest arthritis after antibiotic therapy. A third of these mice, but none of the mice that resolve arthritis, maintain an OspA Ab titer after antibiotic treatment. Furthermore, we show that CD20 and IFN-γ expression are increased in the joints of the DR4+/+CD28−/−MHC-II−/− mice suggesting that B cells and inflammatory cytokines may be involved in the perpetuation of inflammation. This animal system will allow, for the first time, the direct examination of the inflammatory events that lead to persistent Lyme arthritis, after antibiotic treatment.

MATERIALS AND METHODS

Mice

CD28−/− mice (B6.129S2-Cd28tm1Mak/J) were bred at the Tufts University Division of Laboratory Animal Medicine from breeding pairs that were initially obtained from The Jackson Laboratories (Stock number: 002666) (Bar Harbor Maine). Jackson Laboratories confirmed that these mice have been backcrossed for at least 10 generations to the B6 background. DR4+/+MHC-II−/− transgenic mice were a gift of T. Forsthuber (Case Western Reserve University, Cleveland) and were bred in our facility. These mice were generated with HLA-DRA-IEα and HLA-DRB1*0401-IEβ chimeric genes and are also on a B6 background (24). DR4+/+CD28−/−MHC-II−/− mice were generated by breeding the CD28−/− genotype onto DR4+/+MHC-II−/− mice. The F1 DR4+/−CD28+/−MHC-II+/− mice were backcrossed to the CD28−/− mice, the DR4+/−CD28−/−MHC-II+/− mice were selected by FACS and then intercrossed to generate DR4+/+CD28−/−MHC-II−/− mice. All offspring were screened by FACS analysis for surface expression of DR4, CD28 and I-Ab molecules (all Abs were purchased by Pharmingen, San Diego, CA). All animal experiments were approved by IACUC, the institutional animal review board at Tufts/NEMC.

Bb

Low-passage (passage 2) of the infectious Bb N40 clone D10E9A1-E (kind gift of Jenifer Coburn) (25, 26) was used for all infections. Bb were cultured in complete Barbour-Stoenner-Kelly medium (Sigma, St. Louis, MO) at 34° C until mid-log phase (5×107 Bb/ml) and were counted by darkfield microscopy.

Development of murine model for persistent Lyme arthritis post antibiotic treatment arthritis

Four to five weeks old, sex-matched DR4+/+CD28−/−MHC-II−/− , CD28−/− and DR4+/+MHC-II−/− mice were infected intradermally in the skin of the femoral area of both hind limbs with a total dose of 2×104 Bb per mouse (1×104 Bb in 50 μl per hind limb). This protocol was used for all the infections. No difference in arthritis development was observed between male and female mice; therefore both male and female mice were included in the experiments. For each experiment however, great care was taken that all the mice in the experimental group (DR4+/+CD28−/−MHC-II−/− mice) were sex- and aged-matched with those of the control groups (CD28−/− and DR4+/+MHC-II−/− mice). Arthritis by means of edema formation was assessed in a blinded fashion two to three times a week by measuring the anterior-posterior tibiotarsal joint thickness, using a gauge caliper (Mahr Federal, Providence, RI). Upon establishment of chronic arthritis (within approximately 3 months post Bb infection), ceftriaxone (50 mg/Kg/dose, for 5 days) was administered i.p., a treatment that has been reported to be 100% effective in eradicating Bb (27, 28), and the presence of arthritis was monitored over a period of two to five months after the antibiotic therapy. Since we have established already that when mice on the CD28−/− background are not treated with antibiotics they continue to experience monoarticular intermittent arthritic attacks for over at least five months (which is as long as they have been tested (23), we did not include a group of mice that were not treated with antibiotics in this set of data. In addition, when DR4+/+CD28−/−MHC-II−/− mice were infected with Bb, but not treated with antibiotics, they developed chronic joint inflammation similar to the CD28−/− mice. At the end of the experiment, mice were sacrificed by CO2 asphyxiation. Hearts and ankles were harvested and processed for histology. We also followed the development of anti-OspA Ab throughout the whole course of the experiment. Mice were bled weekly, and the serum was assayed in an ELISA for the presence of anti-OspA Abs. In order to assess the effectiveness of the antibiotic therapy, DNA was extracted from ear punch tissue before antibiotics and at the end of the experiment. The experiments have been repeated four times.

Histopathology

Ankles were harvested and decalcified overnight in Decalcifier I Solution (Surgipath, Richmond, IL). The hearts were cut in half through bisection across the atria and ventricles, and kept in formalin. Both ankles and hearts were processed for histology as described in (23).

Anti-OspA ELISA

Flat bottom Immulon 2HB plates (Fisher Scientific, Pittsburgh, PA) were coated overnight with 5 μg/ml of recombinant OspA or OspA fragments in binding buffer (0.1M Na2HPO4, pH 9). ELISA was performed as described in (23). The cut-off to calculate the endpoint titer was set as the lowest serum concentration, in which the ODU signal was twice that of the background wells (all reagents included, expect the serum) for each isotype.

In vitro restimulation and IL-17 ELISA

Popliteal as well as inguinal lymph nodes cells were stimulated with anti-CD3 (145.2C11, 1:150 dilution) for 48 hrs in vitro, and IL-17 cytokine production was assayed by ELISA. IL-17 ELISA was performed as described in (23), with the following modifications: Plates were coated overnight with 3 μg/ml of capture anti-mouse IL-17 Ab (R & D systems, Minneapolis, MN) in 1× PBS. Coated plates were blocked with 2% BSA, 5% sucrose in 1×PBS at RT for 1hr. Recombinant mouse IL-17 (standard curve) and the supernatant from the in vitro restimulation were added in duplicates to the ELISA plates and incubated for 45 min at 37° C. Plates were washed and incubated with biotinylated anti-mouse IL-17 (R & D systems) for 1 hr at 37° C, followed by another wash and incubation with neutrAvidin-AP for 30 min at RT. Plates were then developed as described in (23).

Bb burden

DNA was extracted from ear punch tissue and the Bb burden was determined by real time qPCR, as described in (23).

mIL-17, mIFN-γ and mCD20 real-time reverse transcription-PCR

Ankles were harvested from the mice and were immediately frozen in liquid nitrogen. Frozen tissue was pulverized using a mortar and pestle pre-cooled in liquid nitrogen. RNA from pulverized ankles, popliteal as well as inguinal lymph nodes cells was extracted using the RNAeasy Mini kit (Qiagen, Valencia, CA) per the manufacturer instructions. cDNA synthesis and removal of genomic DNA was performed using the Quanti Tect reverse transcription kit (Qiagen). The c-DNA was then diluted 1:20 and was used as template in 20 μl reaction containing primers and probes specific for mIL-17, mIFN-γ, mCD20 and 18S. Primers and FAM-labeled probes specific for mIL-17 (Mm00439619-m1) , mIFN-γ (Mm00801778-m1) and mCD20 (Mm00545909-m1), as well as VIC-labeled probes specific for murine 18S, were purchased (Applied Biosystems, Foster City, CA) and used as loading control. The PCR reaction was carried out using iTaq SuperMix with Rox (Bio-Rad, Hercules, CA) under the same cycling parameters described above. The amount of template DNA was first normalized by the signal of the 18S housekeeping gene.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (San Diego, CA). All data were tested for Gaussian distribution, using the Shapiro-Will normality test. Quantitative differences between groups were assessed by a two-tailed Student's t test, if 2 groups were compared, and by analysis of variance, if more than two groups were compared, for normally distributed data. For skewed data, on the other hand, the Mann-Whitney test was used, if 2 groups were compared, and the Kruskal-Wallis test was used, if more than two groups were compared. Statistical differences in the proportion of mice with persistent joint inflammation post antibiotic treatment as well as in the proportion of mice with anti-OspA Abs were also determined by a two-tailed Fisher's exact probability test. The correlation between two variables was assessed by calculating the Pearson correlation coefficient for normally distributed data, or the Spearman correlation coefficient for skewed data. The significance level of each correlation was addressed by the P value. Significance was declared at a two-sided 0.05 level for all statistical analyses.

RESULTS

DR4+/+CD28−/−MHC-II−/− mice present persistent Lyme arthritis post antibiotic treatment

DR4+/+CD28−/−MHC-II−/− mice were generated (Figure 1) and subsequently infected with Bb, while DR4+/+MHC-II−/− and CD28−/− mice served as controls in our murine model of persistent Lyme arthritis post antibiotic treatment. Upon establishment of chronic arthritis in these mice, ceftriaxone was administered intraperitoneally, a treatment that has been reported to be 100% effective in eradicating Bb (27, 28), and the presence of arthritis was monitored over a period of two to five months after the antibiotic therapy. While the joint inflammation was completely eradicated after antibiotic treatment in the CD28−/− mice, a significant fraction of DR4+/+CD28−/−MHC-II−/− mice (38%) remained arthritic (Figures 2A and 2B). In our hands, the effectiveness of antibiotic therapy in all groups is shown by the fact that virtually none of the control groups (0/25 of the CD28−/− mice, and 1/31 of the DR4+/+MHC-II−/− mice) continue to present joint inflammation after antibiotics, as opposed to the DR4+/+CD28−/−MHC-II−/− group. In case of persistent spirochetes that would elicit an inflammatory reaction in the joints of these mice, the percentage of edema formation after antibiotic treatment should have been the same in all groups. The elimination of Bb in these mice systemically was also confirmed by real time qPCR (Figure 2A), indicating that chronic arthritis may be propagated in DR4+/+CD28−/−MHC-II−/− mice in the absence of Bb. However, the possibility of persistent infection due a small number of spirochetes that survived in the microenvironment of the joint cannot be excluded at this point. In accordance with the observations made in human Lyme patients with persistent arthritis after antibiotic therapy, these mice continued to manifest mostly persistent and in some cases intermittent arthritic attacks after antibiotic therapy, which was in some mice monoarticular (Figure 3A). More specifically, 8/11 mice presented prolonged and persistent joint inflammation (ankle width 2.9mm-3.1mm) that did not resolved with time. In addition, in the remaining three mice, we never observed a remission phase where inflammation was completely gone. We rather observed a fluctuation of the ankle width that ranged from 2.7mm-3.1mm. These results imply that the presence of the HLA.DR4, or a related RA-associated DR allele, is indispensable for the development of a murine model with persistent Lyme arthritis post antibiotic treatment.

Figure 1.

Figure 1

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Phenotype of the DR4+/+CD28−/−MHC-II−/− mice. HLA.DR4 and I-Ab surface expression on B220+ gated DR4+/+CD28−/−MHC-II−/− lymphocytes was determined by FACS analysis. For the CD28 staining the B220+ lymphocytes were gated out.

Figure 2.

Figure 2

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DR4+/+CD28−/−MHC-II−/− mice present persistent Lyme arthritis post antibiotic treatment. DR4+/+CD28−/−MHC-II−/−, CD28−/− and DR4+/+MHC-II−/− mice were infected with Bb, and arthritis was assessed by measuring the ankles using a caliper. On day 55 post infection, mice were treated with antibiotics, and the arthritis was monitored over the following two months. (A) A significant fraction of DR4+/+CD28−/−MHC-II−/− mice (11/29) remained arthritic post antibiotic treatment, compared to CD28−/− (0/25) and DR4+/+MHC-II−/−(1/31). All mice were negative for Bb's RecA gene post antibiotic treatment. The Bb burden among mice in the three groups, pre-antibiotic treatment is not statistically significant (P=0.6, by Kruskal-Wallis test). Data are expressed as median (25th-75th percentile). Asterisks indicate statistical significance; *P=0.004, **P=0.0004; two-tailed Fisher exact probability test. Data include the pooled results of four independent experiments. (B) Ankle widths of three representative mice per group are shown over a 120 day time course. Asterisks indicate significant difference in ankle swelling between experimental and mock-infected mice *P<0.001; Student's two-tailed t test. Error bars represent SEM

Figure 3.

Figure 3

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Antibiotic treated DR4+/+CD28−/−MHC-II−/− mice develop either persistent or intermittent arthritic attacks, which is in some cases monoarticular. (A) Left (L) and right (R) ankles of DR4+/+CD28−/−MHC-II−/−, CD28−/−, and DR4+/+MHC-II−/− mice (three representative mice/group, each shown in different color) pre-, as well as, post-, antibiotic treatment. Grey shaded areas represent the mock-infected mice. (B) Hematoxylin and eosin-stained joint sections, representative for CD28−/− and DR4+/+MHC-II−/− (a & b) and DR4+/+CD28−/−MHC-II−/− mice (c & d) post-antibiotic treatment are shown at a magnification of 100× (a & c) and 400× (b & d). The mice were sacrificed four months after antibiotic therapy. Heavy infiltration, mostly of neutrophils, occurred in the joints of DR4+/+CD28−/−MHC-II−/− mice.

Joint inflammation over the course of arthritic disease in mice is routinely measured by caliper, which allows monitoring of the edema formation. To examine whether cellular infiltration occurred in the inflammatory process that was established in these arthritic mice after antibiotic treatment, we performed histological analyses of their ankles at the termination of the experiment, four months after antibiotic therapy. Out of eleven mice with persistent joint inflammation post antibiotic treatment, six (54%) showed cellular infiltration, mostly of neutrophils, as opposed to the synovial infiltrate in human patients that contains mainly lymphocyes and macrophages and very few neutrophils (2) (Figure 3B). This observation is in accordance with the data obtained using the caliper, since not all the mice had continuous joint inflammation; instead, some exhibited recurring ankle swelling over time that can be attributed only to edema formation. Besides cellular infiltration, we did not detect any significant changes in the synovial tissue of these mice. Another manifestation of Lyme disease in Bb-infected mice is carditis, an inflammatory response characterized by cellular infiltration of macrophages (30). To exclude the possibility that the edema in the joints of these mice was due to obstruction of lower limb circulation caused by carditis, we performed histological analyses of their hearts. None of the mice examined showed cellular infiltration in the heart, suggesting that the edema formation was due to perpetuated inflammation in the joints, even after elimination of Bb with antibiotics (results not shown).

Incidence of anti-OspA Abs in DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation post antibiotic treatment

As stated in the introduction, 70% of chronic Lyme arthritis patients mount an anti-OspA immune response that seems to correlates with periods of maximal arthritis (8, 9). Thus, we monitored the OspA serum Ab titers in our murine model pre-, as well as post-, antibiotic treatment. Anti-OspA Abs were observed in the majority of Bb-infected DR4+/+CD28−/−MHC-II−/− mice before antibiotic therapy. Following antibiotic treatment, a significant proportion of the mice with persistent joint inflammation (36%) retained anti-OspA titers, as opposed to none of the mice that cleared inflammation in the joints (Figure 4). In addition, none of the CD28−/− nor the DR4+/+MHC-II−/−mice had an antibody response to OspA post antibiotic treatment (results not shown). No significant differences were observed between mice that had anti-OspA Abs and those that did not.

Figure 4.

Figure 4

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Incidence of anti-OspA Abs in DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation post antibiotic treatment. Anti-OspA Ab levels were determined by ELISA in DR4+/+CD28−/−MHC-II−/− mice pre-, as well as post-, antibiotic treatment. The open squares represent the anti-OspA Ab titers pre-antibiotic treatment, and the closed squares represent the anti-OspA Ab titers post-antibiotic treatment. The horizontal lines represent the median. The difference in the anti-OspA Ab levels pre- and post- antibiotic therapy in the mice that eventually develop persistent arthritis is statistically significant (**P=0.003, Mann-Whitney test). The absolute number of DR4+/+CD28−/−MHC-II−/− mice that developed anti-OspA Abs pre-, as well as post-, antibiotics is also shown in parenthesis. The proportion of anti-OspA Abs in arthritic mice post-antibiotic treatment is significantly higher than those in mice that cleared arthritis (*P=0.014; two-tailed Fisher exact probability test).

Since mice with persistent joint inflammation were weekly bled, they were further analyzed post antibiotic therapy, to determine whether there is a correlation between arthritis development and anti-OspA Ab titers. In accordance with the observations made in chronic Lyme patients, there was a significant correlation over time between anti-OspA Abs and development of arthritis post antibiotic treatment in these mice (Spearman correlation coefficient=0.53, p<0.0001) (results not shown).

In addition, we determined the isotype of the OspA-specific Abs. We observed IgM and IgG3, as well as Th1-dependent IgG2c (results not shown). Based on the notion that IgG3 class switching can also occur in the presence of IFN-γ (31), we could speculate that the inflammatory milieu is biased towards a Th1 response. The complete absence of Th2-dependent IgG1 Abs from all four mice may also be indicative of a Th1/Th2 imbalance.

Absence of active immune response in the draining lymph nodes of mice with persistent joint inflammation post antibiotic treatment

To investigate the cellular mechanisms that contribute to the persistent inflammation post antibiotic treatment, we assessed the dynamics and the cytokine profile of the draining popliteal, as well as the inguinal, lymph node cells in Bb-infected antibiotic treated DR4+/+CD28−/−MHC-II−/− mice, while Bb-infected CD28−/− mice served as positive control. The size and absolute number of popliteal lymph node cells was much larger in the Bb-infected CD28−/− mice with active arthritis compared to the Bb-infected CD28−/− mice that cleared the inflammation in the joints. On the contrary, the absolute number of the popliteal lymph node cells in the antibiotic treated DR4+/+CD28−/−MHC-II−/− mice with persistent arthritis was identical to that of the DR4+/+CD28−/−MHC-II−/− mice which cleared arthritis (Figure 5A). A similar pattern was also observed in the inguinal lymph nodes, providing an independent confirmation that arthritis persists in these mice in the absence of systemic Bb infection (data not shown). The presence of IL-17-producing T cells has been associated with development of inflammation in a wide range of infectious, as well as autoimmune diseases (32-34). In our arthritic model, IL-17 mRNA expression was observed only in the lymph nodes of the Bb-infected CD28−/− mice (Figure 5B, left panel), even though an IL-17 memory T cell response was detectable in the draining popliteal lymph nodes of antibiotic treated DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation (Figure 5C, right panel).

Figure 5.

Figure 5

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Absence of active immune response in the draining lymph nodes of mice with persistent joint inflammation post antibiotic treatment. (A) Absolute number of popliteal lymph node cells in antibiotic treated DR4+/+CD28−/− MHC-II−/− mice with persistent joint inflammation and in actively Bb-infected CD28−/− mice. Data include the pooled results of three independent experiments. (B) IL-17 mRNA expression in draining popliteal, as well as in inguinal, lymph node cells in antibiotic treated DR4+/+CD28−/−MHC-II−/− mice (right), and in Bb-infected CD28−/− mice (left), as assessed by real time RT-PCR. Data were normalized by the signal of the 18S housekeeping gene. These results are representative of three independent experiments. (C) IL-17 cytokine expression in draining popliteal, as well as in inguinal, lymph node cells in DR4+/+CD28−/−MHC-II−/− mice (right) and in Bb-infected CD28−/− mice (left), upon anti-CD3 stimulation for 48 hrs, as assessed by IL-17 ELISA. Data include the pooled results of three independent experiments. Asterisks indicate statistical significance; *P<0.05, **P<0.001; two-tailed Student's t test. Data are shown as mean ± SEM.

Increased CD20 and IFN-γ mRNA expression in arthritic joints of DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation

The presence of B cells, as well as of the IFN-γ cytokine, has been associated with persistent arthritic manifestations in humans (10, 11, 35, 36), providing a mechanism for the perpetuation of inflammation in the absence of Bb. However, it has not been possible thus far to confirm this observation in an in vivo animal model. To assess the CD20 and IFN-γ mRNA expression in antibiotic treated DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation, we isolated the arthritic and non-arthritic joints of each mouse and perfromed RT-PCR. There was a significant increase in both the CD20 and IFN-γ mRNA expression in the inflammed joints, compared to the non-inflammed ones, within each examined DR4+/+CD28−/−MHC-II−/− mouse (Figure 6). This observation is indicative of a possible role of B cells, as well as inflammatory cytokines, in the manifestation of persistent inflammation after antibiotic therapy. No difference in IL-17 mRNA expression was observed in the joints of DR4+/+CD28−/−MHC-II−/− mice with persistent arthritis and those isolated from mice that cleared arthritis (results not show).

Figure 6.

Figure 6

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Increased CD20 and IFN-γ mRNA expression in arthritic joints of DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation. CD20 (top panel) and IFN- γ (bottom panel) mRNA expression in the joints of DR4+/+CD28−/−MHC-II−/− mice, as assessed by real time RT-PCR. Data were normalized by the signal of the 18S housekeeping gene. The arthritic (black bars) and non-arthritic (white bars) joints of three representative antibiotic treated DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation are shown. Statistical significance between the arthritic and non-arthritic joints was assessed by a two-tailed Student's t test. Data are shown as mean ± SEM.

DISCUSSION

The immunological mechanisms that trigger persistent joint inflammation after elimination Bb infection have been elusive thus far due to the absence of an animal model for this human inflammatory disease (37). Here we describe the development of a murine system, in which we observe self-perpetuating arthritis upon Bb eradication systemically, as assessed by PCR analysis. Based on the clinical data, persistent arthritis is manifested in a small percentage of antibiotic treated Lyme arthritis patients that have been experiencing intermittent inflammation in the joints before treatment. These patients had a preponderance of RA-associated HLA-DR alleles, such as HLA-DR4 (1, 3, 4).

The intriguing phenomenon of persistent arthritis in antibiotic treated Lyme arthritis patients has been extensively studied. Two main hypotheses have been formulated to explain its pathogenesis, namely, persistent Bb infection after antibiotic treatment or infection-induced autoimmunity (1). The fact, however, that spirochetal DNA is no longer detectable in the inflamed joints after antibiotic treatment seems to rule out the first hypothesis (5-7). On the other hand, the strong HLA.DR restriction observed in patients with persistent arthritis manifestation post antibiotic treatment favors the autoimmune-based hypothesis. The importance of appreciating the mechanism(s) that triggers this chronic arthritic disease is two-fold. It contributes to the general understanding of chronic inflammatory arthritic manifestations, and it may provide a model for other infection-induced autoimmune diseases.

There seems to be a well defined linkage between certain HLA.DR haplotypes and various autoimmune diseases. HLA alleles are accountable for the presentation of autoantigens, as well as the positive and negative selection of autoreactive T cells in the thymus (38, 39). Treatment refractory Lyme arthritis patients have a high frequency of HLA-DR4-related alleles compared to the ones that are treatment responsive (1, 3, 4). Interestingly, the same HLA-DR alleles have also been associated with susceptibility to RA (40, 41). Our murine studies indicate that the presence of HLA-DR4 or a related allele accentuates persistent Lyme arthritis post antibiotic treatment, since a significant fraction of antibiotic treated DR4+/+CD28−/−MHC-II−/− mice, but none of the CD28−/− mice, showed persistent joint inflammation after antibiotic treatment. Interestingly, it has been shown that there is an increased frequency of CD4+CD28−/− T cells in RA patients, which correlates with the severity of the disease (42, 43). This observation validates the use of DR4+/+CD28−/−MHC-II−/− mice in order to get a murine model for persistent Lyme arthritis post antibiotic treatment.

The humoral response against Bb antigens in antibiotic treated Lyme arthritis patients with persistent joint inflammation is of particular interest. Clinical studies have shown that antibodies against OspA mark the initiation of prolonged arthritic episodes, as opposed to other Bb proteins that are associated more with brief and mild arthritis (3, 9). While the majority of Bb-infected DR4+/+CD28−/−MHC-II−/− mice with arthritic symptoms developed anti-OspA Abs prior to antibiotic treatment, the anti-OspA humoral response was detectable in some, but not all, antibiotic treated mice with mice with persistent joint inflammation. These results provide evidence that in our murine model of persistent joint inflammation post antibiotic treatment anti-OspA Abs play an important, but not necessarily exclusive, role in the pathogenesis of the disease. However, there was a significant correlation between anti-OspA Ab titers and development of arthritis in the mice that developed an anti-OspA humoral immune response post antibiotic treatment. This is in accordance with clinical data, where the levels of anti-OspA Abs seem to parallel the severity and duration of arthritis (3, 9).

Bb has been shown to induce IL-17 production in murine T cells in vitro, as well as in synovial fluid T cells isolated from patients with Lyme arthritis (44, 45). In addition, Bb-induced arthritis was ameliorated by anti-IL-17 administration (46). On the other hand, it has not yet been demonstrated whether Th17 cells participate in the pathogenesis of persistent joint inflammation post antibiotic therapy. However, in our mouse model we could not detect any IL-17 mRNA expression in the inflamed joints, although the Bb-infected mice showed increased IL-17 mRNA production in the inguinal and popliteal lymph nodes before antibiotic treatment, in accordance with previous reports. It is possible that the number of infiltrated Th17 cells is small, rendering the IL-17 mRNA negligible in the context of a whole joint. Alternatively, IL-17 does not play an active role in the pathogenesis of persistent inflammation upon antibiotic treatment. A memory IL-17 response, however, was detectable in the lymph nodes of the treatment refractory DR4+/+CD28−/−MHC-II−/− mice, indicating that Th17 cells were induced during the Bb infection phase in these mice, prior to antibiotic treatment.

Increased CD20 mRNA expression was found in the joints of antibiotic treated DR4+/+CD28−/−MHC-II−/− mice with persistent joint inflammation, suggesting that inflammation is locally perpetuated in the microenvironment of the joint. These data, in association with the presence of anti-OspA Abs in the periphery of these mice, are indicative of a possible role of B cells in our murine system, consistent with previous clinical studies (3, 9). In addition to the presence of a humoral response in the inflammatory milieu of the joints, a critical role of T cells, in particular of Th1 cells, has been documented in human Lyme patients with persistent arthritis after antibiotic therapy. IFN-γ production has been associated with increased Lyme arthritis severity in mice (20), and IFN-γ producing cells were identified in the synovial tissue of human Lyme patients with persistent arthritis post antibiotic treatment (10, 11, 47, 48). In this regard, it is of special interest that we observed increased IFN-γ mRNA expression in the joints of antibiotic treated DR4+/+CD28−/−MHC-II mice with persistent joint inflammation, suggesting that IFN-γ plays a pathogenic role in the our murine model. Interestingly, the presence of IFN-γ in the inflamed joint may justify the absence of IL-17 transcription, since IL-17 production is negatively regulated by IFN-γ in vitro (49, 50).

In conclusion, we have described, a mouse model that presents persistent joint inflammation post antibiotic treatment. Although the possibility of residual infection due to the presence of a small number of spirochetes in the joints cannot be excluded, it is apparent from this model that the expression of the HLA-DR4 allele accentuates chronic inflammation in the Bb-infected CD28−/− mice after antibiotic therapy. The persistent joint inflammation observed in these mice may be due to an autoimmune response that is linked to the presence of HLA-DR4 allele. Therefore, our murine system will allow, for the first time, the precise definition of the mechanism involved in the development of persistent Lyme arthritis post antibiotic treatment. Unraveling its manifestation represents a unique opportunity, because it provides insights into other chronic inflammatory arthritides with a possible autoimmune etiology, such as RA, where the infecting agent is unknown.

ACKNOWLEDGEMENTS

We are grateful to Dr. Jenifer Coburn for the gift of B. burgdorferi and to Dr. Jeffrey Bluestone for guiding us to the CD28−/− system. We would like to thank members of the Huber laboratory for critical reading of the manuscript and Lin Miao and Francesca Chang for technical help. The authors have no conflicting financial interests.

This work was funded by NIHRO1 grant AR45386, GRASP center NIH/NIDDK grant DK34924

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