Borrelia burgdorferi is a spirochetal bacterium responsible for human Lyme disease, a multisystem illness transmitted by ticks (1). Lyme disease usually begins with the appearance of erythema migrans, a characteristic expanding skin lesion (2). Within days or weeks after the onset of local infection, bacteria may spread through the bloodstream to different sites, causing meningitis, cranial or peripheral neuritis, carditis, or musculoskeletal pain. After several months, severe and chronic symptoms that mainly affect joints may develop in untreated or inadequately treated patients (3); this condition, defined as Lyme arthritis (LA), is characterized by joint swelling, synovial hypertrophy, vascular proliferation, and infiltration of inflammatory cells (4). The presence of B burgdorferi in connective tissue of the joints of infected individuals likely plays an important role in establishing the course of LA (5).
The recruitment of neutrophils (polymorphonuclear cells [PMNs]) to the site of infection is a critical first step in the immune response to pathogens. In experimental LA, but also in patients with LA, PMNs are the primary cell type present during the acute inflammatory phase, and they appear to be critical for development of the disease (6, 7). Accordingly, not only are PMNs likely to be responsible for the tissue damage that may occur upon extracellular release of granule content but, more importantly, they may also play an active role in driving an immune response that, instead of being beneficial, contributes to the induction of LA (7). However, the mechanism by which PMNs contribute to LA has not yet been clarified.
The inflammation characterizing LA has traditionally been defined as a Th1 cell–mediated response; however, recent studies have shown that LA is induced by cytokines other than interferon-γ (IFNγ), because experimental LA can occur and propagate even in IFNγ-deficient mice (8, 9). The disease paradigm based solely on a Th1 cell–mediated inflammatory response has been extended to include Th17 cells, a new subset of helper T cells (7); indeed, inhibition of interleukin-17 (IL-17) prevents the development of arthritis in vaccinated mice challenged with B burgdorferi (10). Furthermore, T cells from the synovial fluid of patients with LA produce IL-17 in response to neutrophil-activating protein A (NapA), a major antigen produced by B burgdorferi (11). Consistent with the fact that 2 major types of T cells are involved in the pathogenesis of LA, there is a predominance of some chemokines crucial for their recruitment into the synovial fluid of patients, such as CXCL10, which is specific for Th1 cells, and CCL2, which is specific for both Th1 and Th17 cells (12). Moreover, although it has not yet been evaluated in LA, CCL20-mediated recruitment of Th17 cells into the inflamed joints of patients with rheumatoid arthritis (RA) has also been described (13).
The contribution of macrophages to the creation of a milieu rich in chemoattractants for T cells in the joint fluid of patients with LA has been established (11, 12, 14). However, the possibility exists that PMNs are also involved; these versatile cells, either spontaneously or following appropriate stimulation, have been shown to express and/or produce numerous cytokines and chemokines (15).
We previously demonstrated that the immune modulatory properties of the antigen NapA are relevant for the differentiation of T cells toward the Th17 phenotype (11). However, we did not elucidate whether NapA accumulates in the joint fluid of patients with LA. Therefore, the potential participation of NapA in orchestrating the entire process of joint inflammation also remained unexplored.
In this study, we demonstrate that the protein NapA, which accumulates in the joint cavity of patients with LA, recruits PMNs in the early stage of disease and subsequently recruits T lymphocytes. We show that NapA recruits T cells via the contribution of chemokines released by PMNs. Recruited T cells include both Th1 and Th17 cells, as well as a subset producing both IFNγ and IL-17. Taken together, our results show that NapA is a major bacterial factor driving the generation of a proinflammatory T cell response responsible for clinical onset and histopathologic changes in LA.
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Arthritis is a well-documented complication that follows infection with the tick-borne spirochete B burgdorferi. The severity of arthritis can range from mild to moderate inflammation of the joints that develops months after infection, to a chronic, debilitating osteoarthropathy with destruction of cartilage and erosion of bone that develops in a subset of patients within a few years. After dissemination of the spirochete from the skin to the joint, macrophages, T cells, B cells, and plasma cells infiltrate the synovial tissue, while joint fluid contains large numbers of PMNs.
The recruitment of PMNs into the infected joint appears to be a prerequisite for the development of LA, and these cells have been proposed to play a nonphagocytic immunoregulatory role (7, 36). However, this issue remains quite controversial, because some studies showed that arthritis develops independently of the presence of PMNs (36, 37). Another missing piece of the puzzle concerns the bacterial factor(s) that are responsible for the recruitment of PMNs.
Once B burgdorferi is disseminated from the site of inoculation, it establishes residence in the joint tissue; however, whether the bacterium survives for a long time in the synovial fluid is a matter of debate. Indeed, although almost all patients with LA have positive PCR results for detection of B burgdorferi DNA in synovial fluid (5, 38), only 2 cases have been reported in which the bacterium itself was recovered from this site, and in neither case was it possible to subculture the isolate (39, 40). Therefore, the idea that emerges is that in the synovial fluid of patients with LA, these bacteria are either moribund or dead (41), and the release of bacterial antigens in this context is absolutely expected. Interestingly, it has been recently proposed that retained spirochetal antigens might perpetuate synovial inflammation even after eradication of the spirochete from the joint (through antibiotic therapy), giving rise to chronic synovitis (6).
In a previous study, we demonstrated that patients with LA had a humoral immune response to the neutrophil-activating protein NapA, while patients with earlier manifestations of the disorder (erythema migrans and facial palsy) had minimal or no reactivity with this antigen (11). Moreover, we showed that NapA is endowed with immune modulatory properties that reflect the activation of PMNs and monocytes, which are induced to release cytokines that are crucial for the induction of Th17 cell responses. It is noteworthy that NapA-specific T cells were mainly confined to the synovial fluid, because IL-17 production could not be detected after stimulation of peripheral blood T cells from the same patients with LA. These latter data, suggesting that NapA could be one of the spirochetal antigens that accumulate in the joint, have been confirmed in the present study. Indeed, we demonstrated that NapA is retained in the synovial fluid of patients with LA.
In addition, using a rat model of arthritis, we observed that intraarticular injection of NapA triggered the accumulation of inflammatory and immune cells: PMNs accumulated as early as 2 hours after injection, and lymphocytes/monocytes accumulated 2 days after injection. In addition to monocyte/macrophages, the latter population included IFNγ- and IL-17–producing cells, as revealed by relevant staining of the 2 cytokines on the synovial membrane of NapA-injected rats.
Notably, we also observed that NapA per se exerted chemotactic activity for PMNs and T lymphocytes without involving chemokines of endothelial origin. Moreover, despite the high concentration of anti-NapA antibodies in synovial fluid from patients with LA, these antibodies did not impair the NapA-induced recruitment of PMNs (data not shown).
The role of chemokines released by Borrelia-stimulated macrophages in the homing of T cells to infected joints is well established (14). We therefore investigated whether NapA could stimulate recruited PMNs to release chemoattractants that would participate in guiding the adaptive immune response in patients with LA. We observed that NapA-stimulated PMNs released CCL2, CCL20, and CXCL10. Together with CCL2, CCL20 is involved in the recruitment of Th17 lymphocytes, while CXCL10, together with CCL2, induces migration of CXCR3-expressing Th1 effector cells (34). Therefore, it is expected that NapA, via the involvement of PMNs, recruits both Th1 and Th17 cells; this is consistent with the synovial staining we observed in NapA-injected joints. Alternatively, the fact that these chemokines were crucial in recruiting IFNγ- and IL-17–producing cells was confirmed by evidence showing that the application of anti-CCL2, anti-CCL20, and anti-CXCL10 blocking antibodies significantly reduced T cell recruitment promoted by NapA-conditioned supernatants of PMNs. In accordance with these data, we observed the accumulation of not only IFNγ and IL-17 in synovial fluid of patients with LA, but also the accumulation of the 3 chemokines that we demonstrated to be produced by PMNs exposed to NapA. Finally, we report that some T cells recruited by NapA-stimulated PMNs belong to the subset that produces both IFNγ and IL-17, and notably, such a subset was detectable in patients with LA (42–44).
Collectively, our data highlight the role of the NapA antigen in orchestrating the innate and adaptive immune response during the development of LA. Moreover, our data support the notion that other than monocyte/macrophages (14), PMNs (whose role in LA pathogenesis had remained unclear until now) display the potential to guide the adaptive immune response during arthritis onset.
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- PATIENTS AND METHODS
- 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. de Bernard 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 conception and design. Codolo, Cassatella, M. D'Elios, de Bernard.
Acquisition of data. Codolo, Bossi, Durigutto, Della Bella, Fischetti, Amedei, Tedesco, S. D'Elios, Cimmino, Micheletti.
Analysis and interpretation of data. Codolo, Bossi, Durigutto, Della Bella, Fischetti, Amedei, Tedesco, S. D'Elios, Cimmino, Micheletti, Cassatella, M. D'Elios, de Bernard.