Pathogenesis

To get from the vector to the host, the Borrelia have to penetrate several barriers and survive the vector and host immune system. In unfed ticks, B. burgdorferi is mainly located in the mid-gut, attached to the gut by the interaction of the outer surface protein (Osp) A with the corresponding receptor of the tick, TROSPA.^[18,19] While human blood streams into the gut during the feeding process, B. burgdorferi has to defend itself already against various components of the host immune system (e.g., the complement system or the leucocytes). To this end, B. burgdorferi possesses several mechanisms of defense. For movement from the gut to the salivary glands, they possess so-called endoflagellae, which are located between the outer and inner membrane and enable longitudinal axis rotation. Binding of host plasminogen by OspA and OspC probably enables penetration of barriers (e.g., gut wall and salivary glands).

Antigenic Variation

The surface proteins of B. burgdorferi are very immunogenic^[20] and can induce several proinflammatory cytokines.^[21] To prevent detection by the host immune surveillance system, B. burgdorferi is able to regulate the expression of several surface proteins. During the feeding process, OspA expression ceases, while OspC – most probably due to the rise of temperature and the different pH of the inflowing blood – is upregulated.^[19,22–25] OspC appears to play an important role in this early phase of infection. It can bind to the protein Salp15, which inhibits CD4⁺ T-cell activation,^[26] protects the Borrelia from antibody-mediated killing^[27] and prevents the deposition of complement factors on the borrelial surface.^[28] Accordingly, the surface protein OspC is necessary for the survival of B. burgdorferi, as OspC-negative Borrelia are unable to survive in the mammalian host.^[29] An example of true antigenic variation can be found in the surface protein VlsE. It has been shown that infection of mice induces sequence changes and thus alters the antigenic properties of VlsE, which consecutively leads to immune evasion.^[30]

Inactivation of the Host Immune System

As depicted previously, B. burgdorferi is able to bind to tick salivary proteins with protective properties on its own surface. Besides Salp15, the salivary proteins ISAC and IRAC are also capable of inactivating the mammalian complement system.^[31–33] In addition, Borrelia possess their own anticomplement proteins, the complement regulator-acquiring surface proteins CRASP 1–5, the factor H-binding OspE paralog or the complement-regulating protein CD59, which is also known as ‘protectin’.^[34–36] The CRASPs appear to play a very important role for complement resistance and might be responsible for the higher complement sensitivity of B. garinii compared with B. afzelii.^[37,38] Furthermore, B. burgdorferi induces the production of the anti-inflammatory cytokine IL-10.^[39] Accordingly, IL-10-deficient mice are able to eradicate the Borrelia much more efficiently than wild-type mice and the bacterial load is ten-times lower.^[40] Finally, B. burgdorferi produces soluble antigens that can bind and thus inactivate B. burgdorferi-specific antibodies in immune complexes.^[41–43]

Hiding in a Protective Niche

Another way to escape from the host immune system is hiding in less accessible compartments, (e.g., the extracellular matrix), also known as immunologically privileged sites.^[44,45] B. burgdorferi possesses the ability to bind via OspA plasminogen on its surface.^[46] Plasminogen can be activated by plasmin, which in turn leads to the degradation of the extracellular matrix as a prerequisite for invasion.^[47–49] However, it must be remembered that OspA is known to be downregulated during the early phase of infection.^[50,51] Therefore, this mechanism might only play a role for intrathecal pathogenesis, as there are several indications that OspA is upregulated in the CSF of patients with LNB. In addition, there are proteins with degenerating capacities, such as matrix metalloproteinases (MMPs). MMP-9 is upregulated in both erythema migrans skin lesions and the CSF in patients with LNB.^[52,53] On the other hand, studies in MMP-9-deficient mice have not shown an impaired dissemination of B. burgdorferi.^[54] Taken together, while increased levels of MMP-9 have been shown, the functional role of MMP-9 in Lyme disease remains unclear.

After invasion of the extracellular matrix, the Borrelia are able to attach to the matrix proteins with their decorin (DbpA und DbpB)- and fibronectin (BBK32)-binding proteins.^[55,56] Decorin, for example, is necessary not only for dissemination but also survival in the extracellular matrix.^[57] Another potential protective niche could be the intracellular location of B. burgdorferi, as is known to be used by other bacteria, such as Chlamydia or Mycoplasma. However, it must be remembered that these bacteria are approximately 50-times smaller than Borrelia and are adapted to intracellular survival. Nevertheless, Borrelia have been found in both endothelial, synovial, neuronal and glial cells in vitro,^[58–60] and can be cultivated again once extracted from the mammalian cells.^[60] It is tempting to speculate that this might be a location that is protected from the immune system and antibiotic therapy, and thus explaining the etiology of chronic disease. Nonetheless, these findings have not yet been reproduced in vivo and, therefore, these findings have to be interpreted very cautiously.

Invasion of the CNS

The most frequent clinical manifestation of Lyme disease is erythema migrans, which, as described previously, accounts for approximately 90% of cases.^[9] The expansion of this rash is caused by centrifugal migration of the Borrelia from the site of the tick bite.^[61] How and especially why the Borrelia disseminate to other organs is not well understood. It was recently suggested that in North America, the Borrelia disseminate predominantly via the blood, while in Europe, they appear to prefer a migration along other structures (e.g., the peripheral nerves) directly to the nerve roots.^[62] This suggestion was based on the higher prevalence of Borrelia in the blood and there being more patients with multiple erythema migrans in North America than in Europe.^[63–68] In addition, the clinical picture of LNB hints at a different mode of invasion into the nervous system. In Europe, meningopolyradiculitis, with the maximal intensity of the pain close to the site of the tick bite (Bannwarth’s syndrome), predominates, but the presentation of CNS disease in North America is more diffuse, with mostly meningitis or encephalopathy.^[69,70] A reason for this could be the different borrelial species: while B. burgdorferi sensu stricto is the only species found in North America, B. garinii and especially the recently distinguished species B. bavariensis (formerly defined as B. garinii OspA type 4^[2]) are typically found in patients with Bannwarth’s syndrome. In a European study, 65% of the patients with B. garinii found in the CSF suffered from typical meningoradiculitis, in contrast to none of the ten patients with B. afzelii.^[71] In the future, analyzing the borrelial genome in more detail might aid in further elucidating the respective species-specific pathogenetic principles.^[72]

Inflammatory Reaction of the CNS

Once inside the CNS, B. burgdorferi induces an inflammatory reaction, presenting as a lymphomonocytic pleocytosis in the CSF. Physiologically, the CNS is considered to be an immunoprivileged site, as there are only very few immune cells to be found.^[73] While a fairly low number of dendritic and monocytic cells are responsible for the immune surveillance in the CSF/CNS compartment compared with the systemic circulation, neutrophils and components of the complement system are only rarely found, if at all.^[74] In addition, the CNS lacks a well-formed lymphatic system, as there are no lymphatic drainage vessels to be found.^[75] Therefore, once bacteria have crossed the BBB, the CSF and the CNS are virtually defenseless, as shown by the impressive example of pneumococcal meningitis with high lethality despite antibiotic treatment.^[76] By contrast, neuroborreliosis is not such a fatal disease, most probably due to the comparably long division time of B. burgdorferi and the lack of classic endotoxins.^[77] Therefore, the host organism has enough time to react to the borrelial invasion. B. burgdorferi inside the CSF are first encountered by microglial cells,^[78] perivascular cells,^[79,80] dendritic cells^[81] or astrocytes.^[82] In particular, the Toll-like receptors (TLRs) of the innate immune system appear to play an important role in the recognition process. The lipoproteins of B. burgdorferi are detected by TLR2^[83–85] and, as recently noted in transfected cell lines, TLR7 and 9.^[86] In addition, it has been observed that astrocyte and microglial TLR1, 2 and 5 are involved in the in vivo response of primate glial cells to B. burgdorferi.^[87] Furthermore, studies have shown that TLRs have an essential role in the control of B. burgdorferi burden, because the respective knockout mice have up to 250-fold more spirochetes than wild-type controls.^[88,89] For example, TLR2 engagement results in NF-κB nuclear translocation, which not only induces the generation of bactericidal nitric oxide (NO) and superoxide, but also the production and release of cytokines (e.g., IL-1, IL-6, IL-12 or TNF-α) and chemokines.^[85,90] The chemokines, in turn, attract further immune cells from the systemic circulation, thus leading to the aforementioned CSF pleocytosis.^[91]

Using a protein-array as a screening test, the upregulation of four chemokines, the GRO family (equivalent to CXCL1–3 according to the new nomenclature), CXCL8, CXCL10 and CXCL13 has been found in the CSF of patients suffering from LNB.^[92] Of particular interest is the high expression of the B-cell-attracting chemokine CXCL13 in LNB, which was not detectable at all or at least in much lower concentrations in most other inflammatory CNS diseases (e.g., pneumococcal or viral meningitis or multiple sclerosis).^[92] Local monocytic cells appear to be the source of this chemokine, as the production of CXCL13 could be induced by cultivating human monocytes with B. burgdorferiin vitro.^[85] Using the only reliable animal model for LNB – the nonhuman primate – dendritic cells, microglia, endothelial cells and T cells were identified as other potential sources of CXCL13.^[93,94] In one of these studies, the expression of CXCL13 correlated with the spirochetal load.^[93] In addition, a recent study has shown that CXCL13 plays a key role for the immigration of B cells into the CSF in LNB.^[91] Taken together, these results fit very well into the observation that B cells are one of the characteristic cells of LNB, as their proportion in the CSF is much higher than in other inflammatory CNS diseases.^[95,96] In addition, it explains why an elevated concentration of CXCL13 can be measured days before the intrathecal production of B. burgdorferi-specific antibodies (Figure 2).^[94,97,98]

Figure 2.

Enlarge

The Inflammatory B-cell Response in the Cerebrospinal Fluid in Response to the CNS Infection. Borrelia are recognized by monocytic cells (A), which produce the B-cell-attracting chemokine CXCL13 (B). B cells immigrate into the CSF (C) and mature into plasma cells (D). These plasma cells can produce Borrelia burgdorferi-specific antibodies (E) that can eventually destroy the invading spirochetes (F).
CSF: Cerebrospinal fluid; Osp: Outer surface protein; TLR: Toll-like receptor.
Adapted with permission from ⁶².

Besides B lymphocytes and plasma cells, there is also a clonal accumulation of activated CD8⁺ T cells in the CSF during early LNB.^[99] This lymphocyte subtype could be attracted by the local production of chemokines such as CCL2, CCL4, CCL5, CXCL10 or CXCL11,^[94,100] as increased levels of these chemokines have either been found in the rhesus monkey model of LNB or in the CSF of LNB patients.^{[94,101–103]} However, a functional role for the immigration of T lymphocytes, has only been reported for CXCL11 so far.^[103]

Neuronal Dysfunction

Unfortunately, little is known about the pathogenesis of LNB itself (e.g., the neuronal dysfunction evoked by B.b). Principally, there are four mechanisms to be discussed: a direct cytotoxic effect of the Borrelia; secretion and/or release of cytotoxic mediators by B. burgdorferi as, for example, lipoproteins; a result of the host inflammatory reaction – a so-called ‘bystander effect’; and autoimmunity through molecular mimicry.

There are several arguments for a direct cytotoxic effect of B. burgdorferi. Borellia are known to adhere to different murine neuronal or glial cell lines^[104,105] and also to primary rat brain cultures.^[104] Probably the most relevant observation for European LNB was the adherence of B. garinii to dorsal root ganglia cells, as this reflects the presumed pathogenesis of meningoradiculitis (Bannwarth’s syndrome) with lancinating, radicular pain.^[106] This adherence process appears to be mediated by the borrelial OspA and the proteoglycans^[106] or the galactocerebrosides.^[104] The adherent Borrelia can be cytotoxic for the neural cells,^[105] and OspA induces apoptosis and astrogliosis.^[107] Besides adherence, one study also observed the invasion of B. burgdorferi into neuroglial and cortical brain cells, where they were found to be viable without a cytotoxic effect. As this in vitro observation has not yet been confirmed in vivo, these findings have to be interpreted cautiously and their relevance remains unclear.^[60]

There are only two studies that have shown proteins that are similar to lipopolysaccharide in B. burgdorferi with pyrogenic, cytotoxic and IL-1, IL-6 and TNF-α-inducing effects,^[108,109] while a classical endotoxin has not been identified so far.^[77] Therefore, there is only limited evidence for a pathogenic effect of mediators secreted and/or released by the spirochetes. Instead, a bystander effect appears more probable. For example, Schwann cells produce NO in the rhesus monkey model of LNB,^[110] and the incubation of glial-enriched primary cultures of neonatal rat brain cells with B. burgdorferi leads to the release of NO into the culture medium.^[111] Macrophages incubated with B. burgdorferiin vitro produce quinolonic acid. This agonist of N-methyl-D-aspartic synaptic function can be neurotoxic in higher concentrations.^[112] Recent experiments with microglia incubated with either B. burgdorferi or lipidated OspA in vitro found both an inflammatory reaction with the production of IL-6, IL-8, TNF-α and CCL2–5 and apoptosis of cocultured neuronal cells. The authors concluded that the neurotoxic surroundings generated by the microglial cells might have contributed to the neuronal cell damage.^[113]

Finally, autoimmunity through molecular mimicry is another potential mechanism of neuronal dysfunction in Lyme disease. Antibodies against two homologous OspA peptides generated in rabbits were found to react with neurons in the human brain, spinal cord and dorsal root ganglia by immunohistochemistry,^[114] and immunization of Lewis rats with B. burgdorferi induces ganglioside antibodies.^[115] In addition, antibodies against the flagellin of B. burgdorferi bind to a human axonal protein.^[116] Finally, the serum of patients with Lyme disease contains IgM antibodies to B. burgdorferi that crossreact with neuronal antigens,^[117] and antibodies found in the CSF in LNB patients might not only be directed against B. burgdorferi, but also against the CNS parenchyma.^[118] In accordance with the concept of molecular mimicry, it has been shown that a patient with an autoimmune neuropathy following acute LNB improved after treatment with intravenous immunoglobulins.^[119] Whether autoimmune processes could also be responsible for the frequently debated and not clearly defined ‘post-Lyme disease’ (PLD) is tempting to speculate. Of interest, a very recent study has found antineuronal antibodies (directed against cortical cells and dorsal root ganglia) in 49.4% of patients with PLD, in contrast to only 18.5% of healthy patients after subsided Lyme disease.^[120]