Toll-like receptors: insights into their possible role in the pathogenesis of lyme neuroborreliosis

Abstract

Lyme neuroborreliosis is likely caused by inflammatory effects of the tick-borne spirochete Borrelia burgdorferi on the nervous system. Microglia, the resident macrophage cells within the central nervous system (CNS), are important in initiating an immune response to microbial products. In addition, astrocytes, the major CNS glial cell type, also can contribute to brain inflammation. TLRs (Toll-like receptors) are used by glial cells to recognize pathogen-associated molecular patterns (PAMPs), mediate innate responses, and initiate an acquired immune response. Here we hypothesize that because of their PAMP specificities, TLR1, -2, -5, and -9 may be involved in the pathogenesis of Lyme neuroborreliosis. Previous reports have shown that the rhesus monkey is the only animal model to exhibit signs of Lyme neuroborreliosis. Therefore, we used primary cultures of rhesus astrocytes and microglia to determine the role of TLRs in mediating proinflammatory responses to B. burgdorferi. The results indicate that microglia and astrocytes respond to B. burgdorferi through TLR1/2 and TLR5. In addition, we observed that phagocytosis of B. burgdorferi by microglia enhances not only the expression of TLR1, -2, and -5, but also that of TLR4. Taken together, our data provide proof of the concept that astrocyte and microglial TLR1, -2, and -5 are involved in the in vivo response of primate glial cells to B. burgdorferi. The proinflammatory molecules elicited by these TLR-mediated responses could be a significant factor in the pathogenesis of Lyme neuroborreliosis.

FIG. 1.

Relative expression of IL-6 and TLR1, -2 and -5 transcripts in microglia and astrocytes in response to specific TLR ligands and B. burgdorferi. Microglia (A) and astrocytes (B) were treated with L-OspA, live and sonicated B. burgdorferi cells (MOI of 10), and FliC for 8 h. The expression of each TLR transcript was determined using qRT-PCR, and each transcript was normalized with respect to the expression of GAPDH. Presented are the mean values obtained from triplicate specimens ± standard deviations. Asterisks indicate a significant difference between unstimulated and stimulated cells: * and **, P < 0.05 and P < 0.005, respectively. Similar results were obtained with microglial RNA obtained from three additional rhesus monkeys and astrocyte RNA from four additional animals.

FIG. 2.

(A) Western blot analysis of TLR1, TLR2, and TLR5 protein expression in microglia and astrocytes. Microglia (left panels) and astrocytes (right panels) either were left unstimulated or were stimulated with L-OspA (0.25 μg/ml), FliC (100 ng/ml), or live B. burgdorferi cells (MOI of 10) for 24 h. (B) Extracellular expression of TLR2 on activated microglia in response to L-OspA and live B. burgdorferi cells. Cells were stimulated with L-OspA (0.25 μg/ml) and B. burgdorferi (MOI of 10) for 4 and 24 h and evaluated by flow cytometry. The MFI values showed upregulation of TLR2 expression on cells stimulated with L-OspA at 4 and 24 h. An increase of TLR2 expression was observed at 4 h of stimulation with B. burgdorferi, followed by a decrease of expression at 24 h. Data are expressed as means ± standard deviations of three independent experiments (different animals). (C) Surface and intracellular expression of TLR1, TLR2, and TLR5 in microglia. Multilabel images show surface expression of TLR1 (Alexa 568, red [yellow arrowhead]) and intracellular expression of TLR2 (Alexa 633, blue [yellow arrows]) (panel 1); intracellular expression of TLR1 (blue [yellow arrows]), with TLR5 also shown, in red (panel 2); and intracellular expression of TLR5 (red [yellow arrows]) and surface expression of both TLR5 (yellow arrowhead) and TLR2 (blue, orange arrowhead) during phagocytosis of live B. burgdorferi cells (labeled with FITC, green) (panel 3). Cell nuclei were labeled with BoPro1 (gray). The images show a pseudo-three-dimensional representation, with the XY plane in the center, and the XZ and YZ planes on the sides. Similar results were obtained with cells from two additional rhesus monkeys.

FIG. 3.

Expression of TLR1, -2, and -5 by astrocytes. Confocal microscopy shows that astrocytes constitutively express TLR1 (A), TLR2 (B), and TLR5 (C). Antibody to GFAP was labeled with Alexa 568 (red); TLR1, -2, and -5 were labeled with Alexa 488 (green); and cell nuclei were labeled with ToPro3 (blue). Similar results were obtained with astrocytes isolated from two additional rhesus monkeys.

FIG. 4.

Secretion of proinflammatory mediators by microglia and astrocytes. Concentrations of IL-6, IL-8, CCL3, and CCL4 were determined after 24-h incubations of microglia (A) and astrocytes (B) with no stimulant, 0.25 μg/ml L-OspA, 0.25 μg/ml U-OspA, 12.5 ng/ml Pam3Cys, 1.0 μM CpG ODN M362, 10 or 100 ng/ml FliC, and live and sonicated B. burgdorferi cells (MOI of 10). Shown are the mean values ± standard deviations obtained from triplicate specimens. Asterisks (**, P < 0.005) indicate significant differences between unstimulated and stimulated cells. (C) Multilabel confocal microscopy showing increased cytoplasmic expression of IL-6 in phagocytic microglia after a 12-h stimulation with live B. burgdorferi cells, followed by 2.5 h of incubation with brefeldin A. Antibody to B. burgdorferi (Bb) was labeled with FITC (green), antibody to IL-6 was labeled with Alexa 568 (red), antibody to IBA1 was labeled with Alexa 633 (blue), and cell nuclei were labeled with BoPro1 (gray). Similar results were obtained with supernatants obtained from cells of three additional rhesus macaques.

FIG. 5.

Internalization of B. burgdorferi correlates with the enhancement of TLR expression. Microglia were incubated for 12 h with B. burgdorferi cells or carboxylate-modified microspheres. Confocal microscopy images show the expression of TLR1 (A and E), TLR2 (B and F), TLR5 (C and G), and TLR4 (D and H) after stimulation with B. burgdorferi (E, F, G, and H) or carboxylate beads (A, B, C, and D). Antibody to B. burgdorferi was labeled with FITC (green) and 488-nm FluoSpheres (yellow green), TLR1, -2, -5, and -4 were labeled with Alexa 568 (red), IBA1 was labeled with Alexa 633 (blue), and cell nuclei were labeled with BoPro1 (gray). Bars represent 20 μm. (I) TNF-α, IL-6, IL-8, CCL3, and CCL4 production by microglia in response to B. burgdorferi or carboxylate beads. Values are means (n = 3) ± standard deviations. Asterisks (**, P < 0.005) indicate a significant difference between unstimulated and stimulated cells. ELISA results were similar with specimens from three additional animals.