The pathogenesis of Lyme disease neuropathies is poorly understood. B. burgdorferi infection may damage neural cells by the direct action of spirochetes or spirochetal products on glial and neuronal cells. It is also possible that spirochetes induce cytotoxic or inflammatory mediators locally in glial, neuronal or endothelial cells and, thus, cause indirect damage. Infiltrating immune cells and/or the presence of cross-reactive antibodies to self-antigens at the site of infection/inflammation may also be deleterious to neural cells.
Neuronal proteins, anti-myelin antibodies and cells secreting antibodies to MBP have been detected in the CSF of patients with LNB, indicating possible glial and neuronal damage [32, 33]. The antigenic determinants on the 41 kDa flagellar protein of B. burgdorferi are shared by several human tissue components such as Schwann cells from the PNS . Long-term murine intrathecal exposure to a lipoprotein of B. burgdorferi, outer-surface protein C, resulted in axonal damage. Intrathecal exposure to B. burgdorferi lipoproteins may be one of the causes of the neurologic manifestations of Lyme disease . The involvement of sensory ganglia in human LNB has also been previously documented [15, 36]. One rare peripheral nervous system manifestation of LNB is the Guillain-Barré-like syndrome, a prototype of immune-mediated peripheral neuropathies where neither the initial event nor the antigen that triggers the immune reaction is known. Autoimmune mechanisms similar to those suggested in MS have also been implied in the pathogenesis of central nervous system LNB .
Our earlier findings in the rhesus model of peripheral LNB of both the early disseminated and chronic phases in the PNS mirror several aspects of these forms of the disease in humans [38, 39]. The primary findings of axonal degeneration and regeneration, and multifocal nerve lesions showing perivascular inflammatory cellular infiltrates have been documented in almost all patients with Lyme-associated peripheral neuropathy [15, 16, 40–42]. The results of these studies suggest that immune mediated neuronal and glial cell damage could be involved in the neuropathy of LNB.
Cytokines and chemokines are key immune mediators that play an important role in promoting CNS injury in various kinds of inflammatory neurodegenerative diseases [43–47]. Importantly, various inflammatory cytokines and chemokines have also been reported in the CSF of patients with LNB [48–51]. The potential of B. burgdorferi to induce cytokines, chemokines and other inflammatory mediators in glial and neuronal cells as well as glial and neuronal apoptosis has been well documented [20, 23, 25, 52–56].
In this study we explored the potential of the Lyme disease bacteria to cause inflammation in tissue explants of rhesus DRG, as well as in primary cultures of rhesus DRG cells and human Schwann cells, as a representation of the Schwann cells that ensheath the dorsal roots. Primary cultures of DRG cells from rhesus monkeys may prove useful in understanding the mechanisms involved in Lyme peripheral neuropathy, as well as in other human peripheral neuropathies .
We documented the ability of B. burgdorferi to elicit the production of IL-6, IL-8, and CCL2 in cells of the DRG and to induce the death of sensory neurons in DRG cell cultures. The ability of sensory neurons of the DRG to express CCL2 has also been documented in pain models . Our in vitro DRG culture did not favor the growth of Schwann cells, which are known to ensheath the dorsal root axons in the DRG, possibly because the culture medium was tailored to supporting the neurons. Interestingly, the few satellite glial cells that were present in our DRG cultures did not undergo apoptosis in response to B. burgdorferi. We had previously observed the apoptosis of satellite glial cells in DRG of rhesus monkeys infected intrathecally with live B. burgdorferi. It is possible that there are additional regulatory factors that come into play in the in vivo environment that are absent in the in vitro culture system. Specifically, this difference may have been brought about by the absence of Schwann cells in the DRG cultures, especially considering their documented contribution to inflammation in response to B. burgdorferi.
Using rhesus DRG tissue explants we identified the phenotypes of the producer cells as satellite glial cells by the expression of glial markers such as S-100 [59, 60] and GFAP. Like other glial cells, the satellite glial cells are known to respond to nerve injury by up-regulating GFAP . We identified Schwann cells in rhesus DRG tissue explants by the expression of p75NTR and CNPase [29, 30, 62]. These cells, in addition to DRG satellite cells, produced IL-6, IL-8 and CCL2, while neurons, which were characterized by the expression of NeuN , produced IL-6 and CCL2 but not IL-8. The DRG cell cultures as well as the cultures of HSC stimulated with live B. burgdorferi produced IL-6, IL-8 and CCL2. Since we did not find IL-8 to be produced by neurons in rhesus DRG explants, it is likely that satellite glial cells contributed the IL-8 found in the DRG cell culture supernatants.
These results support our hypothesis and show that innate responses of neuronal and glial cells of the DRG to B. burgdorferi mediate inflammation and that neuronal apoptosis occurs in this context. In agreement with this notion we found that the anti-inflammatory drug dexamethasone reduced both the levels of inflammatory mediators and neuronal apoptosis as induced by B. burgdorferi, suggesting that the two phenomena may be causally related.
Cytokine/chemokine signaling and apoptosis are of key importance in the regulation of neuroinflammatory responses [43–47]. Since DRG axons project centrally into the spinal cord and peripherally into the spinal nerves, inflammation and cell death in the DRG elicited by the Lyme disease spirochete could affect neuronal survival and function both in the CNS and PNS. Further, as the sensory neurons of the DRG play a key role in the sensation of pain, inflammation in glial and neuronal cells and cell death in the DRG could also modulate the pain response . Neurogenic pain secondary to radiculitis or inflammation of the dorsal roots is often the earliest and sometimes only symptom in patients with LNB . It typically radiates from the spine into the extremities or trunk, and is described as ‘sharp or jabbing’ pain . The immune mediators IL-6, IL-8 and CCL2 that we found to be elevated in the DRG cultures exposed to live B. burgdorferi have been reported to play a role in modulating inflammation and the pain response [66–69]. IL-8 is known to induce expression of matrix metalloproteinases, cell cycle and pro-apoptotic proteins, and cell death in neurons . IL-6 and CCL2 have been reported to increase the sensitivity of sensory neurons to pain [71–73].
The expression of the chemokine CCL2 and its receptor (CCR2) is also up-regulated by DRG neurons in rodent models of neuropathic pain . Disruption of CCL2 signaling has been shown to block the development of neuropathic pain . CCL2 is also known to be involved in the signaling and upregulation of several genes and proteins that participate in the signal transduction of the pain response both in the DRG and in the spinal cord [72, 73]. This chemokine also functions as a neuromodulator in DRG neurons . We observed the levels of CCL2 to be the highest among the immune mediators elicited by B. burgdorferi in both DRG cell and HSC cultures. Similarly, we have reported high levels of CCL2 in the CSF of rhesus macaques infected with B. burgdorferi. It is possible that CCL2 is a major player in orchestrating inflammation as well as the pain response in LNB.
The local application of IL-6 to the DRG of rats has been shown to induce TNF-α and results in apoptosis of DRG cells . Earlier, we also reported the presence of IL-6 in the sensory neurons of the DRG in rhesus macaques that were inoculated intrathecally with B. burgdorferi. Patients with LNB sometimes have persistent symptoms such as fatigue, cognitive difficulties, depression and pain even after appropriate antibiotic treatment. Because of the subjective nature of these complaints, many of these patients are believed to have a primary psychiatric diagnosis, such as depression or somatization disorder. However, experiments such as those presented here raise the possibility that some of these complaints may be associated with inflammatory biochemical changes in the CNS. Elevated levels of IL-6 can cause symptoms of fatigue and malaise, common to many infectious conditions, as well as Lyme disease . Research in other animal models of peripheral neuropathy has demonstrated that peripheral inflammation alone can trigger the brain cytokine system via afferent neural pathways from the periphery to the CNS . Similar cascades of inflammation and apoptosis could also be involved in the DRG in LNB. Ongoing cytokine activation in the nervous system could contribute to the persistent symptoms of fatigue, pain and cognitive dysfunction that patients sometimes continue to experience despite having been treated for Lyme disease.
It is possible that B. burgdorferi, as well as the mediators elicited in cells of the DRG and Schwann cells that we report here, could contribute to mediating inflammatory and apoptotic signaling cascades in Schwann cells, the myelinating cells of the PNS. This, in turn, may result in axonal degeneration .
The potential of Schwann cells to initiate the process of Wallerian degeneration by releasing pro-inflammatory cytokines that are involved in leukocyte recruitment and differentiation (for example, IL-1β, CCL2, IL-8 and IL-6) has been documented . As we found the inflammatory mediators IL-6, IL-8 and CCL2 in our HSC culture supernatants stimulated with live B. burgdorferi, as well as in the Schwann cells in the dorsal roots of the DRG tissue explants incubated with live B. burgdorferi, it is possible that similar mechanisms of inflammatory mediated axonal damage could be contributing to the peripheral neuritis seen in LNB.
The high levels of CCL2 that we found to be elicited in DRG neurons and satellite glial cells, as well as in Schwann cells in response to B. burgdorferi, could trigger mechanisms of demyelination in the PNS similar to those thought to cause CNS demyelination in MS and experimental autoimmune encephalomyelitis (EAE) [81, 82]. CCR2, the receptor of CCL2, has been documented to be involved in macrophage recruitment to the injured PNS .
The ability of B. burgdorferi to activate the innate defense mechanisms of the host  and particularly in cells of the CNS is well documented [48, 51, 85–88]. It is possible that similar signaling cascades could be involved in triggering the immune mechanisms that result in the pathogenesis of peripheral LNB. We propose that inflammation of nerve roots and DRG and subsequent apoptosis in the DRG could be early events that contribute to peripheral neuropathy in Lyme neuroborreliosis.