Abstract
Even after appropriate treatment, a proportion of Lyme disease patients suffer from a constellation of symptoms, collectively called Post-Treatment Lyme Disease Syndrome (PTLDS). Brain PET scan of patients with PTLDS have demonstrated likely glial activation indicating persistent neuroinflammatory processes. It is possible that unresolved bacterial remnants can continue to cause neuroinflammation. In previous studies, we have shown that non-viable Borrelia burgdorferi can induce neuroinflammation and apoptosis in an oligodendrocyte cell line. In this follow-up study, we analyze the effect of sonicated remnants of B. burgdorferi on primary rhesus frontal cortex (FC) and dorsal root ganglion (DRG) explants. Five FC and three DRG tissue fragments from rhesus macaques were exposed to sonicated B. burgdorferi and analyzed for 26 inflammatory mediators. Live bacteria and medium alone served as positive and negative control, respectively. Tissues were also analyzed for cell types mediating inflammation and overall apoptotic changes. Non-viable B. burgdorferi induced significant levels of several inflammatory mediators in both FC and DRG, similar to live bacteria. However, the levels induced by non-viable B. burgdorferi was often (several fold) higher than those induced by live ones, especially for IL-6, CXCL8 and CCL2. This effect was also more profound in the FC than in the DRG. Although the levels often differed, both live and dead fragments induced the same mediators, with significant overlap between FC and DRG. In the FC, immunohistochemical staining for several inflammatory mediators showed the presence of multiple mediators in astrocytes, followed by microglia and oligodendrocytes, in response to bacterial remnants. Staining was also seen in endothelial cells. In the DRG, chemokine/cytokine staining was predominantly seen in S100 positive (glial) cells. B. burgdorferi remnants also induced significant levels of apoptosis in both the FC and DRG. Apoptosis was confined to S100 + cells in the DRG while distinct neuronal apoptosis was also detected in most FC tissues in response to sonicated bacteria. Non-viable B. burgdorferi can continue to be neuropathogenic to both CNS and PNS tissues with effects likely more profound in the former. Persistence of remnant-induced neuroinflammatory processes can lead to long term health consequences.
Highlights
Abbreviations blood brain barrier (BBB) Blood brain barrier BDNF Brain derived neurotrophic factor BLC B lymphocyte chemoattractant BSK Barbour–Stoenner–Kelly CNS Central nervous system CCL2 Chemokine (C–C) motif ligand 2 CCL3 Chemokine (C–C) motif ligand 3 cerebrospinal fluid (CSF)-3 Colony stimulating factor 3 CXCL8 Chemokine (C–X–C) motif ligand 8 CXCL10 (C–X–C) motif chemokine ligand CXCL11 (C–X–C) motif chemokine ligand CXCL13 (C–X–C) motif chemokine ligand 13 dorsal root ganglion (DRG) Dorsal root ganglion Enzyme-Linked Immuno-Sorbent assays (ELISAs) Enzyme linked immunosorbent assay EM Experimental medium fetal bovine serum (FBS) Fetal bovine serum frontal cortex (FC) Frontal cortex
As DRG plays a role in neuropathic pain[14], in this follow-up study, we analyzed the neuropathological effect of sonicated B. burgdorferi on primary rhesus frontal cortex tissues and primary rhesus dorsal root ganglion tissues, sourced from multiple animals
These results indicated that non-viable spirochetes are potent and consistent inducers of inflammatory mediators in primary rhesus frontal cortex tissues, and maybe more so than the live ones
Summary
Abbreviations BBB Blood brain barrier BDNF Brain derived neurotrophic factor BLC B lymphocyte chemoattractant BSK Barbour–Stoenner–Kelly CNS Central nervous system CCL2 Chemokine (C–C) motif ligand 2 CCL3 Chemokine (C–C) motif ligand 3 CSF-3 Colony stimulating factor 3 CXCL8 Chemokine (C–X–C) motif ligand 8 CXCL10 (C–X–C) motif chemokine ligand CXCL11 (C–X–C) motif chemokine ligand CXCL13 (C–X–C) motif chemokine ligand 13 DRG Dorsal root ganglion ELISA Enzyme linked immunosorbent assay EM Experimental medium FBS Fetal bovine serum FC Frontal cortex. Bacterial transmission occurs through tick bite, followed typically by appearance of a bulls-eye type rash called erythema migrans that appears at the site of tick attachment This early localized phase is generally accompanied by fever, headache, and malaise, followed by an early dissemination phase affecting multiorgan systems. Primary human microglia have been shown to induce chemokines and cytokines in response to antibiotic killed or sonicated B. burgdorferi[13] This suggested that in the subset of patients with persistent glial activation, residual antigens from antibiotic treatment, could be a source of this neuroinflammation. Since our and other in vitro studies used immortalized oligodendrocytes or primary microglia alone, it was not clear if such a phenotype can occur in primary brain tissues in response to non-viable B. burgdorferi It is possible, for instance, that this neuroinflammatory effect is masked in a tissue setting in the presence of other cells. The results show that like our in vitro study on the oligodendrocyte
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