0 Abstract: Interaction of Borrelia burgdorferi in coculture with human fibroblasts

I haven't seen this paper mentioned elsewhere - this is from a conference held last year. Need to find full text - not available on ASM or Scholar...

Interaction of Borrelia burgdorferi in coculture with human fibroblasts 
Daniel Wilfinger, Gerd Leitinger, Anna Maria Pabst, Helmut Schaider, Elisabeth Aberer

Kurzfassung/Background: B. burgdorferi (B.b.) can be recovered long after initial infection from antibiotic-treated patients; a protective effect of fibroblasts was assumed. Outcome of previous studies differ whether spirochetes are able to invade fibroblasts. Skin fibrosis has repeatedly been observed in European chronic Lyme borreliosis. So the aim of our study was to examine the interaction of B.b. with fibroblasts in coculture ultrastructually and to investigate key factors for fibrosis such as transforming growth factor ß (TGF-ß) and the production of type I collagen.

Materials and Methods: Human skin fibroblasts were propagated in Dulbeccos’s modified medium at 37°C and 5% CO2 up to 10 5 cells per culture. Bb sensu stricto and B. afzelii were cultured at 34°C in BSK-H medium up to 10 8 cells per culture. Borreliae and fibroblasts were then coincubated in RPMI medium at 37°C for 14 days. Cocultures and fibroblasts only were harvested between 2 and 14 days 3-4 times, fixed in glutaraldehyde and prepared for electon microscopy analysis. Supernatants were investigated for TGF-ß by ELISA. mRNA levels for type I collagen were measured by real time PCR.

Results: Electron micrographs showed borreliae which were able to attach to the fibroblast membrane through protein bridges. Single spirochetes seemed to pervade fibroblast cytoplasm by invagination surrounded by an intact fibroblast membrane. Structural changes of extracellular borreliae due to the adverse culture condition were observed. Shedding of outer membrane blebs forming granules and tubules were seen as well as cystic degeneration of borreliae.

After 2 days of coculture with B.b.s.s. the total amount of TGF-ß in the supernatants was approximately the same as in fibroblast-cultures, after 14 days of coculture production of TGF-ß was decreasing in cocultures with B.b.s.s.. In supernatants of cocultures with B. afzelii TGF-ß was not detectable at any time.

The mRNA-levels for type I collagen were elevated after 2 days of coculture with B.b.s.s. as well as in cocultures with B. afzelii in comparison to fibroblast-cultures. After 7 days the mRNA-levels for type I collagen were decreasing in cocultures in comparison to fibroblast-cultures, a stronger decrease was observed in cocultures with B. afzelii.

Conclusions: The interaction of B.b.s.s. and B. afzelii with human fibroblasts was verified by electron microscopy. Fibroblast integrity was not disturbed by borreliae. Intracellular accumulation of spirochetes was not detectable.

We demonstrated differences in the two species B.b.s.s. and B. afzelii concerning production of type I collagen and TGF-ß. In contrast to cocultures with B.b.s.s. TGF-ß was not detectable in cocultures with B. afzelii at any time. In line with these findings in cocultures with B. afzelii a stronger decrease of collagen production was observed in comparison to cocultures with B.b.s.s. after 7 days of coculture. The elevation of mRNA-levels for type I collagen in cocultures after 2 days is confirmed by literature: Scleroderma-fibroblasts also show an increase in collagen production after 2 days of culture.

Schlagwörter/Keywords : Borrelia, B. burgdorferi sensu stricto, B. afzelii, electronmicroscopy, morphology, TGF-ß, mRNA collagen, morphea, fibrosis, ACA