Interaction of a neurotropic strain of Borrelia turicatae with the cerebral microcirculation system

Abstract

Relapsing fever (RF) is a spirochetal infection characterized by relapses of a febrile illness and spirochetemia due to the sequential appearance and disappearance of isogenic serotypes in the blood. The only difference between isogenic serotypes is the variable major outer membrane lipoprotein. In the absence of specific antibody, established serotypes cause persistent infection. Studies in our laboratory indicate that another consequence of serotype switching in RF is a change in neuroinvasiveness. As the next step to elucidate this phenomenon, we studied the interaction of the neurotropic Oz1 strain of the RF agent Borrelia turicatae with the cerebral microcirculation. During persistent infection of antibody-deficient mice, we found that serotype 1 entered the brain in larger numbers and caused more severe cerebral microgliosis than isogenic serotype 2. Microscopic examination revealed binding of B. turicatae to brain microvascular endothelial cells in vivo. In vitro we found that B. turicatae associated with brain microvascular endothelial cells (BMEC) significantly more than with fibroblasts or arachnoidal cells. The binding was completely eliminated by pretreatment of BMEC with proteinase K. Using transwell chambers with BMEC barriers, we found that serotype 1 crossed into the lower compartment significantly better than serotype 2. Heat killing significantly reduced BMEC crossing but not binding. We concluded that the interaction of B. turicatae with the cerebral microcirculation involves both binding and crossing brain microvascular endothelial cells, with significant differences among isogenic serotypes.

FIG. 1.

Cerebral microgliosis in scid mice persistently infected with serotype 1 of B. turicatae. Immunostaining with rat anti-mouse F4/80 monoclonal antibody shows extensive microgliosis in the brain of a scid mouse persistently infected with serotype 1 of B. turicatae (A). In comparison, little staining is seen in an uninfected control (B) or in a scid mouse persistently infected with serotype 2 (E) (magnification, ×20). Selected areas of the cerebral cortex (shown in squares in panels A, B, and E) are shown at higher magnifications in panels C, D, and F, respectively (×400). The arrow in panel D points to a perivascular F4/80-positive cell.

FIG.2.

Interaction of B. turicatae with cerebral microcirculation in vivo. Immunostaining with αVsp1 monoclonal antibody 1H12 of scid mouse brains 1 month after intraperitoneal inoculation with serotype 1 of B. turicatae is shown. Arrows point to spirochetes on the abluminal side of the leptomeningeal microcirculation within the subarachnoid space (A), bound to the luminal side of leptomeningeal cells (B), bound to brain parenchymal endothelial cells (C), and in the process of crossing leptomeningeal endothelial cells (D). 3,3′-Diaminobenzidine chromogen staining; magnification, ×1,000.

FIG. 3.

Interaction of serotype 1 of B. turicatae with brain microvascular endothelial cells (BMEC) in vitro. BMEC grown on cell culture slides were incubated with serotype 1 spirochetes, washed, incubated with DiI to label BMEC membranes orange, and immunostained with αVsp1 monoclonal antibody 1H12, followed by an FITC-labeled secondary antibody to label Vsp1 green. Microscopic examination with a dual FITC and rhodamine filter revealed green spirochetes next to BMEC. The spirochetes on the surface of BMEC show areas of yellow color, representing colocalization of Vsp1 and BMEC cytoplasmic membrane. Green signal, representing Vsp1, is seen not only extracellularly in spirochetes but also inside BMEC as amorphous material (open white arrow) (magnification, ×1,000).

FIG. 4.

Association of serotype 1 of B. turicatae with human brain microvascular endothelial, fibroblast, and arachnoidal cells. The association of serotype 1 of B. turicatae with human brain microvascular endothelial cells (BMEC), fibroblasts (IMR90), and arachnoidal cells (F5) was studied by measuring the radioactivity in the upper and lower chambers and the monolayer of smaller (6-mm) transwell chambers 24 h after inoculation of ³⁵S-labeled serotype 1 spirochetes in the upper chamber. The results are expressed as means (SD) of three to four separate transwells. Significant association of serotype 1 with the monolayers was observed only with BMEC (P < 0.001).

FIG. 5.

Protease pretreatment eliminates the association of serotype 1 with brain microvascular endothelial cells. (A) Distribution of radioactivity in the three compartments (upper, monolayer, and lower) of large (12-mm) transwell chambers with BMEC barriers inoculated with serotype 1, shown as mean of n = 3 replicates. The mean (SD) percentage of radioactivity associated with the BMEC monolayer was 16 (8.7), 14 (5.3), and 30 (1.8) after 3, 7, and 24 h of incubation, respectively. (B) The effects of pretreating the BMEC monolayer with proteinase K are shown, illustrating that pretreatment completely eliminated the association with serotype 1 even after 24 h of incubation.

FIG. 6.

Association of isogenic serotypes of B. turicatae with BMEC. Percentage of radioactivity associated with BMEC monolayers on transwell chambers 20 h after inoculation of ³⁵S-labeled serotypes 1 (Bt1), 2 (Bt2), and 3 (Bt3) of B. turicatae in the upper compartment. Results are expressed as means (SD) of three to four transwells each. Similar association with BMEC monolayers was observed with all serotypes tested (P, not significant).

FIG. 7.

Crossing of BMEC barriers by isogenic serotypes 1 and 2 of B. turicatae. Percentage (mean) of radioactivity in the three compartments (upper, monolayer, lower) of large (12-mm) transwell chambers with BMEC barriers inoculated with 1 × 10⁷ radiolabeled serotype 1 (Bt1) or serotype 2 (Bt2) spirochetes alive (A and C) or heat-killed (B and D) are shown. All percentages are estimates based on measuring the radioactivity of 20-μl samples removed from the upper and lower chambers at each time point. Serotype 1 alive crossed into the lower chamber significantly better than serotype 2 alive (P < 0.01 by one-way analysis of variance for all time points). Heat killing significantly reduced the crossing into the lower chamber (P < 0.01 by t test). However, heat killing had no effect on binding to the BMEC monolayers.