Interaction of a Neurotropic Strain ofBorrelia turicataewith 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.Infection of the central nervous system (CNS) is a characteristic feature of several pathogenic spirochetes, including the agent of syphilis, Treponema pallidum, and many of the Borrelia spp. that cause Lyme disease (LD) or relapsing fever (RF). RF is best known for antigenic variation due to spontaneous serotype switches by variable expression of immunodominant outer membrane lipoproteins, referred to as variable major proteins (VMP). The VMP switch allows RF borrelias to escape killing by the host's serotype-specific antibody response (1,9,19). In mice infected with the RF agents Borrelia hermsii strain HS1 (9) and B. turicatae strain Oz1 (12, 15), we have observed significant differences in the ability of isogenic serotypes to enter the brain. Since immunocompetent mice in most cases eliminate the predominant serotypes from blood and brain via production of VMP-specific antibodies, to be able to study CNS infection by specific serotypes over time we had to use B-cell-deficient mice. In mice with severe combined immunodeficiency (scid), which are B and T cell deficient, we isolated and characterized two isogenic serotypes of the Oz1 strain of B. turicatae of different virulence and CNS tropism: serotype 2 (formerly known as serotype B) is characterized by a VMP of 20 kDa, called Vsp2 for variable small protein 2, and serotype 1 (formerly known as serotype A) is characterized by a VMP of 23 kDa, called V...

IL-10 Prevents Apoptosis of Brain Endothelium during Bacteremia

“…Several previous studies of relapsing-remitting or persistent high-level infection in mice failed to find any evidence of cerebrovascular injury (9)(10)(11)(12)(15)(16)(17). This was the case even after 2-3 mo of persistent infection with 10 7 -10 8 bacteria per ml blood (15,17,18).…”

“…HBMEC passages 3-5 were cultured in RPMI 1640 medium (Sigma-Aldrich), supplemented with 10% NuSerum (BD Biosciences, Mountain View, CA), 10% FBS (Sigma-Aldrich), MEM vitamins (BioWhittaker), MEM nonessential amino acids (Sigma-Aldrich), 1 mM sodium pyruvate (BioWhittaker), 2 mM L-glutamine (Sigma-Aldrich), 30 mg/ml endothelial growth supplement (Sigma-Aldrich), and 5 U/ml heparin (Sigma-Aldrich) at 37˚C in 5% CO 2 as described previously (12). HBMEC were detached from culture flasks by trypsinization, counted by phase-contrast microscopy with a hemocytometer, and resuspended in fresh medium without FBS.…”

“…Spirochetes were cultured in Barbour-Stoenner-Kelly H medium (Sigma-Aldrich) with 12% rabbit serum and grown at 37˚C; all bacterial strains were used at passages ,6 for all experiments. Prior studies showed that HBMEC medium supports the survival of B. turicatae (12). Bacterial numbers were counted using hemocytometer in a dark-field microscopy, and final inoculum numbers were confirmed by TaqMan PCR.…”

“…For studies of brain endothelial cells we used SV-40-transformed HBMEC; HBMEC were chosen because they have been found to express features of blood-brain barrier endothelial cells and their interaction with bacterial and viral pathogens have been studied extensively (12,(21)(22)(23)(24)(25). HBMEC passages 3-5 were cultured in RPMI 1640 medium (Sigma-Aldrich), supplemented with 10% NuSerum (BD Biosciences, Mountain View, CA), 10% FBS (Sigma-Aldrich), MEM vitamins (BioWhittaker), MEM nonessential amino acids (Sigma-Aldrich), 1 mM sodium pyruvate (BioWhittaker), 2 mM L-glutamine (Sigma-Aldrich), 30 mg/ml endothelial growth supplement (Sigma-Aldrich), and 5 U/ml heparin (Sigma-Aldrich) at 37˚C in 5% CO 2 as described previously (12).…”

“…Our laboratory has been studying the neurologic complications of bacterial infection by using a neurotropic strain of the RF spirochete Borrelia turicatae (9)(10)(11)(12)(13)(14). Several previous studies of relapsing-remitting or persistent high-level infection in mice failed to find any evidence of cerebrovascular injury (9)(10)(11)(12)(15)(16)(17).…”