Cytotoxic mechanisms may play a role in the local immune response in the central nervous system in neuroborreliosis
Introduction
Lyme borreliosis (LB), caused by the spirochete Borrelia burgdorferi sensu lato (s.l) (Bb) is a complex infection that can affect various organs. In Sweden and Finland, neuroborreliosis (NB) is the most common manifestation of the disseminated stage. Bb infection can follow an asymptomatic course (Ekerfelt et al., 2001, Fahrer et al., 1998, Steere et al., 2003), or lead to a subacute disease that resolves after antibiotic treatment (Pfister and Rupprecht, 2006). However, despite adequate antibiotic therapy, some individuals have symptoms lasting more than six months (Asch et al., 1994, Kaiser, 1994, Nocton and Steere, 1995, Oschmann et al., 1998, Steere, 2001), here called NB with persisting symptoms post treatment (NBpspt). The mechanisms behind NBpspt are not fully known. Current hypotheses for explaining differences in clinical course include; a) an uncontrolled immune response, possibly leading to post infectious autoimmunity (Hemmer et al., 1999, Roessner et al., 1998, Sigal, 1997, Singh and Girschick, 2004) often associated with host specific characteristics as HLA-DRB*0401(Steere et al., 2006), impaired macrophage or lymphocyte activation (Iliopoulou et al., 2008) b) pathogen-specific genomic differences between subspecies of Bb (Wang et al., 2002) and c) long-time persistence of the spirochete despite antibiotic therapy (Frey et al., 1998, Oksi et al., 1999, Preac-Mursic et al., 1989). Regardless of suggested mechanisms, an optimal immune response is important to resolve the infection. This is supported by studies in patients with borreliosis which have shown predominating Bb-specific Th1-responses, also being most pronounced within the target organ (Ekerfelt et al., 1997, Ekerfelt et al., 1998, Grusell et al., 2002, Oksi et al., 1996b, Wang et al., 1995, Widhe et al., 2004). We agree with the hypothesis that a strong inflammatory Th-1 response is needed to eradicate the Bb-infection, which however, if excessive or persistent, may cause tissue injury, thereby contributing to NBpspt (Kang et al., 1997, Sjowall et al., 2005, Widhe et al., 2004).
We have previously shown that an early IFN-γ response with a subsequent up-regulation of IL-4 is associated with a benign disease course, whereas patients with NBpspt showed a persistent IFN-γ response and a lack of IL-4 (Widhe et al., 2004). Thus a switch to a type 2 response seems to be necessary to terminate the inflammatory type 1 response and hinder tissue injury (Ekerfelt et al., 1997, Jarefors et al., 2006, Widhe et al., 2004), which corroborates the findings in an experimental study in mice (Kang et al., 1997).
Involvement of cytotoxic cells in the immune response against Bb has been suggested based on in vitro studies (Beermann et al., 2000) and activated CD8+ T cells have been shown to accumulate in the central nervous system (CNS) during the early phase of NB (Jacobsen et al., 2003).We have previously reported indications of a cytolytic phenotype of IFN-γ producing cells from patients with NB (Ekerfelt et al., 2003) and decreased Bb-induced upregulation of the IL-12 receptor beta2 on blood CD8+ cytotoxic T cells (CTL:s) from patients with NBpspt compared to individuals with asymptomatic Bb infection (Jarefors et al., 2007). This may indicate a role for cytotoxic cells in NBpspt, which is intriguing since enrichment of CTL:s in the CNS has been reported in multiple sclerosis (MS) (Babbe et al., 2000, Jacobsen et al., 2002). NBpspt shares some features with MS (Garcia-Monco and Benach, 1995, Halperin, 1997, Oksi et al., 1996a) including autoimmune responses (Baig et al., 1986, Weigelt et al., 1992). Indeed, a role for aberrant immune responses to Bb in the pathogenesis of MS has been discussed (Frietzsche, 2005). A third T cell subset, Th17, was recently discovered (Harrington et al., 2005, Park et al., 2005) and the Th17 cells seems to produce e.g. IL-17A, IL-17F, TNF-α, IL-22, IL-26 and IFN-γ (Tesmer et al., 2008). IL-17 A (here called IL-17) is a pro-inflammatory cytokine that is secreted by cells of both the innate and the adaptive immune systems, suggesting a bridging function (Korn et al., 2009). Reports on the role of IL-17 in borreliosis are scarce, but interestingly Bb lipopeptides induce production of IL-17 in T-cells (Infante-Duarte et al., 2000) and IL-17 has been suggested as a major contributor to the pathogenesis of Bb arthritis in mice (Burchill et al., 2003, Nakae et al., 2003). Furthermore, increased levels of IL-17 have been reported to be secreted by in vitro stimulated synovial fluid T cells from patients with Lyme arthritis (Codolo et al., 2008). Chronic IL-17 expression induced by Bb lipopeptides could thus be an important mediator of infection-induced immunopathology. On the other hand, considering the coordinating activities of IL-17 in local inflammation, release of IL-17 early in infection may be crucial for effective eradication (Kolls and Linden, 2004).
An increased expression of IL-17 is also seen in MS lesions (Lock et al., 2002) and increased numbers of IL-17 mRNA expressing mononuclear cells (MNC) in blood and cerebrospinal fluid (CSF) were reported in patients with MS (Matusevicius et al., 1999). Recently, cells secreting both IFN-γ and IL-17 have been reported (Acosta-Rodriguez et al., 2007, Tesmer et al., 2008), occurring also in brain tissue of MS patients (Kebir et al., 2009), making investigation of IL-17 secretion also in the IFN-γ dominated disorder NB interesting (Ekerfelt et al., 1998, Forsberg et al., 1995, Oksi et al., 1996b).
The aim of this study was to investigate if cytokines associated with cytotoxicity (IL-2, IL-7, IL-10, IL-12p70, IL-15 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) and the Th 17-associated cytokine IL-17 are increased in CSF from patients with NB and if lymphocytes from patients with NBpspt kill Bb-infected target cells.
Section snippets
Patients
This prospective follow-up study was conducted with a total of 310 included patients referred to Åland Central Hospital with symptoms of suspected clinical NB, e.g. symptoms or signs compatible with meningitis, radiculitis, facial palsy, other cranial nerve affections, arthralgia, dementia or fatigue. Collection of EDTA plasma and lumbar puncture was done in all patients in the acute phase. Twenty patients were excluded from the study; eleven whose CSF/serum sample volumes lacked or were too
Cytokine levels in cerebrospinal fluid and plasma
Kruskal–Wallis test indicated significant differences in CSF levels of IL-2 (p < 0.0001), IL-7 (p = 0.0012), IL-10 (p < 0.0001), IL-12p70 (p = 0.0011), IL-17 (p < 0.0001) and GM-CSF (p < 0.0009), but not of IL-15 when comparing NB patients, patients with non-CNS Bb infections and non-Bb controls. No significant differences for any of the cytokines were found in plasma across the groups (data not shown).
CSF-data where Kruskal–Wallis test indicated differences between the groups were further analyzed using
Discussion
In this study six of the seven cytokines chosen, because of their association with cytotoxic responses, namely IL-2, IL-7, IL-10, IL-12p70, IL-17 and GM-CSF, were increased in CSF but not in plasma in patients with NB compared to patients with non-CNS Bb infection. Three of the cytokines, IL-2, IL-10 and GM-CSF were also increased in NB CSF compared to non-Bb controls, whereas IL-7 and IL-12p70 showed tendencies to be increased. Furthermore, in a pilot study, blood lymphocytes from all three
Acknowledgements
This study was supported by grants from The Swedish Research Council (Medicine), the Swedish Society of Medicine, the University Hospital in Linköping, ALF Grants, County Council of Östergötland, Wilhelm and Else Stockmann Foundation and the Foundation for Åland Medical Research of the Åland Culture Foundation. We are grateful to MSc Petra Cassel for help with the cytotoxic assay and Luminex assay. We also thank Susanne Olausson and Eivor Asplund, Åland Central hospital, Mariehamn for help with
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