Co-culture of human fibroblasts and Borrelia burgdorferi enhances collagen and growth factor mRNA (very scientific/for nerds)
From this link: https://link.springer.com/article/10.1007/s00403-017-1797-1
Skin fibrosis has been reported in Borrelia burgdorferi infection in Europe, but has been questioned by several authors. The objective of the present study was to examine the interaction of skin fibroblasts with B. burgdorferi sensu stricto B31 (BB) and B. afzelii (BA) in vitro by electron microscopy. We also determined the expression of collagen type I, TGF-β, FGF-1, calreticulin (CALR), decorin (DCN), and PDGF-α at the mRNA level in Borrelia/fibroblast co-cultures. Intact Borrelia attach to and transmigrate fibroblasts, and undergo cystic transformation outside the fibroblasts. Fibroblasts preserve their vitality and express a prominent granular endoplasmic reticulum, suggesting activated protein synthesis. On two different semi-quantitative real-time PCR assays, BB- and BA/fibroblast co-cultures showed a significant induction of type I collagen mRNA after 2 days compared to fibroblasts (fourfold for BA and 1.8-fold for BB; p < 0.02). In addition, there was a significant upregulation of mRNA expression of TGF-β, CALR, PDGF-α, and DCN in BA and BB co-cultures compared to control fibroblasts in monolayer cultures after 2 days (p < 0.01). The BA/fibroblast co-culture induced a considerably greater upregulation of collagen and growth factor mRNA compared to BB/fibroblast co-culture. In contrast, a significant down-regulation of FGF-1 (20-fold for BA and 4.5-fold for BB) mRNA expression was detected in co-cultures compared to controls (p < 0.01). The results of the study support the hypothesis that BB sensu lato, and BA in particular, enhances collagen mRNA expression and can stimulate growth factors responsible for increased collagen production.
B. burgdorferi B. afzelii Fibroblast Co-culture mRNA type I collagen TGF-β FGF-1 PDGF Decorin Calreticulin Fibrosis
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Borrelia burgdorferi sensu lato, a bacterium transmitted by ticks into the skin of humans and animals, has a high affinity for connective tissue and binds to decorin, fibronectin, integrin, plasminogen, glycosaminoglycans, and other components of connective tissue [9, 28, 33]. Borrelia may invade type I collagen fibres  and also fibroblasts, where they are protected from the action of antibiotics such as ceftriaxone .
Lyme borreliosis in Europe is caused by three main genospecies of B. burgdorferi sensu lato, namely, B. burgdorferi sensu stricto (BB), B. afzelii (BA), and B. garinii (BG) . Only BB exists in the United States. A certain organotropism of the different genospecies has been observed. BA, the most common detected genospecies in Europe, has a high affinity for the skin. Its persistence causes the chronic infection known as acrodermatitis chronica atrophicans [10, 35]. Skin infection of long duration is characterized by atrophy on one hand and skin sclerosis on the other, as seen in ACA with pseudoscleroderma . In the brain, reactive gliosis or astrogliosis evidenced by condensation of astrocytic fibres was observed in the rhesus macaque infected with a neurotropic B. burgdorferi strain . Degenerative lesions and fibrosis were seen in peripheral nerves . Experimental studies on monkeys have shown that BB infection of heart tissue also induces fibrosis, as shown by a significantly increased density score .
Fibrosis of the skin has been repeatedly observed in association with a Borrelia infection in Europe only, namely, circumscribed scleroderma (cSc), lichen sclerosus et atrophicus, exacerbation of systemic scleroderma, trigger finger, or the carpal tunnel syndrome [5, 6, 15, 40, 48]. Since sclerotic skin lesions have not been reported in the United States, the question arises as to whether the predominant genospecies in Europe, BA or BG, can influence and exaggerate collagen turnover. In cSc, the BA genospecies was isolated from skin biopsies and DNA of BA- and BG-, but not BB-DNA has been detected in cSc patients’ skin biopsies from patients in Germany, Austria, and Japan but not in the United States [6, 11].
CSc and SSc are caused by excessive deposits of type I collagen , manifested by more than a threefold elevation of collagen I mRNA levels in SSc skin compared to controls . This phenomenon is promoted by several cytokines and growth factors. Isolated scleroderma fibroblasts produce elevated mRNA levels of type I collagen and transforming growth factor (TGF-ß) in culture . Furthermore, platelet-derived growth factor (PDGF-α) , calreticulin (CALR) [27, 54], decorin (DCN) , and other molecules are thought to be involved in the increased collagen syntheses . Basic fibroblast growth factor (FGF-1) induces collagen production by stimulating skin fibroblasts and connective tissue growth factor (CTGF) .
Based on these reports, we studied the behaviour of fibroblasts in co-culture with different Borrelia genospecies. We looked for a difference between collagen syntheses in co-culture compared to fibroblasts only, induced either by BB or by BA. Our aim was to simultaneously show the interaction of fibroblasts with Borrelia by electron microscopy and investigate the synthesis of mRNA collagen type I as well as the synthesis of different growth factors and molecules known to stimulate collagen production compared to fibroblasts only.
Materials and methods
Culture of Borrelia
Three strains of B. burgdorferi, BB strain B31 (gifted by Bettina Wilske of the Max von Pettenkofer Institute in Munich, Germany), and two strains of BA, isolated from skin biopsies of patients with erythema migrans at the department of dermatology, Medical University of Graz (ethical approval No. 181 99/00), were used. The two BA isolates were typed by RFLP analysis at the Institute of Hygiene and Microbiology, Medical University of Graz, by Doris Stünzner .
Borrelia were stored at − 70 °C, thawed at room temperature, and cultured in 8-ml Falcon tubes at 34 °C in BSK-H medium . Cultures were monitored once a week in regard of growth, vitality, movement of Borrelia, and the purity of the medium by dark-field microscopy. Borrelia were counted in a Petroff-Hausser counting chamber. For subcultures, 100–1000 μl of Borrelia suspension was inoculated in fresh BSK-H medium. Borrelia were subcultured to a density of 108 cells/microscopic field.
Culture of fibroblasts
The FF2462 fibroblast cell line was isolated from human foreskin [provided by Dr. Meenhard Herlyn (The Wistar Institute, Philadelphia, PA, USA)] . The cells were stored at − 70 °C, thawed in warm water, and dissolved in 8 ml DMEM (500 ml DMEM supplemented with 50 ml FCS und 10 ml glutamine). Fibroblasts were centrifuged at 1200 rpm for 5 min, and the pellet was dissolved in 4 ml DMEM and applied to culture tubes of 75 cm2. Ten millilitres of DMEM was then added and the cells were cultured at 37 °C with 5% CO2. After 4 days, the supernatant was replaced by 10 ml fresh DMEM and cultured for another 3 days. Subcultures were performed once a week to a cell count of 105 cells.
Co-culture of fibroblasts with B. burgdorferi for studying cell morphology by electron microscopy
Four parallel analyses were started for the co-culture experiments using the BB B31 and the BA 1 strain; the latter showed better growth than BA2. Cultures could be harvested after 24, 48, 72 h, and 7 days. For the experiments, a polypropylene lattice was placed in each well of a 6-well plate, and then, 105 fibroblasts in 2 ml DMEM were pipetted in the well. After 24 h, DMEM was withdrawn and replaced by 1 ml RPMI. After centrifugation of Borrelia cultures at 1500 rpm, the pellet was resuspended in 1 ml of RPMI containing 108Borrelia/ml, added to the wells, and incubated for 24, 48 and 96 h, and 7 days, respectively.
For morphological analysis, the fluid of the plate was withdrawn and the cells were removed from the lattices, fixed for 30 min in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 2 h, rinsed in cacodylate buffer for 2 h at 4 °C, and centrifuged at 800 rpm for 5 min. The pellet was placed in a 2% agarose solution, cooled to 4 °C, and then cut into small blocks which were again fixed in 2.5% glutaraldehyde in cacodylate buffer for 2 h. The specimens were dehydrated and embedded in TAAB embedding resin (TAAB, Aldermaston). They were sectioned at 60 nm using a Leica UCT ultramicrotome (Leica Microsystems, Vienna, Austria), stained with lead citrate and uranyl acetate, and analysed with a Zeiss EM 902 transmission electron microscope (Carl Zeiss Oberkochen).
RNA isolation and semi-quantitive RT-qPCR
Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using the RevertAid™ H Minus first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany). To investigate the mRNA synthesis of collagen type I, two different primer pairs were used (col 1–1 fw: 5′-AAA CAA TGG TGC TCA GGG AC-3′ and col 1–1 rev: 5′-AGG ACC AGG GAG ACC AAA CT-3′; Col 1–2 fw: 5′-CAG CAC CTT CTC TCA GAC CC-3′ and Col 1–2 rev: 5′-GCA TCC TTG GTT AGG GTC AA-3′). The mRNA of FGF1, PDGF-α and TGF-β, CALR, and DCN were measured with a commercially available primer assay (Qiagen, Hilden, Germany).
Semi-quantitative real-time PCR (RT-qPCR) was performed in triplicate using an ABI Prism 7000 Detection System (Applied Biosystems, Carlsbad, CA, USA). Reaction mix (25 μl): 1 × SYBR® Green PCR Master Mix (Invitrogen, Carlsbad, CA, USA), forward and reverse primer (1 mM each), 3 μl cDNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine–guanine phosphoribosyltransferase (HPRT1) served as housekeeping genes. The results are expressed as relative units based on calculation 2− ΔΔCT, which yields the relative amount of target gene normalised to the endogenous control (mean of two housekeeping genes) and relative to peripheral blood mononuclear cells. The cycling protocol was as follows: one cycle of 50 °C for 2 min and 95 °C for 10 min, followed by 50 cycles consisting of denaturation for 15 s at 95 °C, annealing of primers and elongation for 1 min at 60 °C.
All statistical analyses were performed using the Statistical Package for Social Sciences, version 17.0 (IBM, NY, USA). Differences in expression levels were analysed using the Mann–Whitney U test. Spearman’s correlation test was performed to examine any correlation of collagen type I expression to FGF-1, PDGF-α, TGF-β, CALR, and DCN. A p value lower than 0.05 was considered to indicate statistical significance. Expression levels are presented as means ± standard deviation. All statistical tests were two-sided.
Borrelia bind to fibroblasts, invade them, and undergo extracellular cystic transformation
Morphologic changes were identical when using BB B31 and BA in co-culture with fibroblasts in RPMI. Owing to better tissue preservation, the presented images are shown for the BA/fibroblast co-culture. The first changes were noted after 24 h of co-culture. BA bind to fibroblast membranes, forming tethers or being surrounded by a cytoplasmic extension (Fig. 1a). Even at this stage, some Borrelia seem to be enclosed in fibroblasts, thus preserving the membranes of both Borrelia and fibroblasts. Invagination and the passage of Borrelia through the fibroblast become evident after 48 h (Fig. 1b). Borrelia are surrounded by a dense microfilament network (diameter ~ 6 nm) showing condensations at the fibroblast`s plasma membrane (Fig. 1b).
Now look at this research and the 700 plus articles that show that Lyme/Morgellons/ and other co infections are killing people. Wake Up. This is your wake up call Mr. Smith and Mr. Anderson. Stop playing games!