Fibroblasts are important cells to study in relation to Lyme disease and other diseases.
|Mouse fibroblasts in cell culture|
Fibroblasts make collagens, glycosaminoglycans, reticular and elastic fibers, glycoproteins found in the extracellular matrix and the cytokine TSLP. On a more general note, they synthesize the extracellular matrix and collagen, the structural framework (stroma) for animal tissues, and play a critical role in wound healing.
To get a good idea of where fibroblasts are found in the human body, refer to this diagram:
In the above diagram:
epithelial cells = tissues which line the cavities and surfaces of structures throughout the body, and also form many glands. Functions of epithelial cells include secretion, selective absorption, protection, transcellular transport and detection of sensation.
basement membrane = a thin sheet of fibers that underlies the epithelium, which lines the cavities and surfaces of organs including skin, or the endothelium, which lines the interior surface of blood vessels.The primary function of the basement membrane is to anchor down the epithelium to its loose connective tissue underneath.
endothelium = the thin layer of cells that lines the interior surface of blood vessels.
interstitial matrix = a type of extracellular matrix found in interstitial connective tissue, characterized by the presence of fibronectins, proteoglycans, and different types of collagen.
A study from 2007, Fibroblasts as novel therapeutic targets in chronic inflammation, has this to say about fibroblasts in its abstract:
"A characteristic feature of many chronic inflammatory diseases is their persistence and predilection for certain sites. The molecular basis for such tissue tropism and failure of the inflammatory response to resolve has until relative recently remained obscure. Recent studies have strongly implicated fibroblasts as cells which contribute to disease persistence and which help define anatomical location. Therefore fibroblasts make an attractive therapeutic target as they help orchestrate the inflammatory infiltrate. Current anti-inflammatory therapies target immune cells in an attempt to inhibit the production of pro-inflammatory mediators. However an equally important target is the active induction of pro-resolution programmes responsible for the resolution of inflammation. Fibroblasts are likely to be an important source of these anti-inflammatory mediators. Therapeutic manipulation of fibroblasts and their biologically active products is an emerging concept in treating cancer and is likely to provide a novel method to achieve improved control of chronic inflammatory disease." Fibroblasts are likely to be a source of anti-inflammatory mediators, but they could also be a home for invading pathogens.
In yesterday's paper outline, Interaction of of Borrelia burgdorferi in coculture with human fibroblasts, the results stated:
"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."This recent research indicates that Borrelia burgdorferi (and afzelii) spirochetes attach to fibroblasts and individual spirochetes enter the cytoplasm by invagination while the fibroblast membrane remains intact.
Here, invagination means just what you imagine it would be: A single spirochete can enter the wall of the fibroblast without disrupting its integrity and happily live within its cytoplasm.
The conclusion reflects this statement:
"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." So according to this research, single spirochetes enter fibroblasts without disturbing the fibroblasts, and there was no evidence that more than one spirochete was found within the fibroblasts. I'm taking this to mean that spirochetes neither entered fibroblasts en masse nor did they find any reproducing spirochetes inside fibroblasts. It doesn't mean that isn't what might happen, but such behavior was not observed.
Another study was conducted in the same year, in Poland, in 2010, Interactions between Borrelia burgdorferi and Mouse Fibroblasts, and this study states more happens within those fibroblasts:
"Electron microscopic studies reveal consecutive stages of B. burgdorferi spirochetes penetration to mouse fibroblasts in vitro. It has been observed, as a first step attachment and engulfment of spirochetes followed by formation of vacuoles. After 48 hours of infection, vacuoles of fibroblastic cells have been seen full of B. burgdorferi bacteria and latter they have been released from infected cells to extracellular space. It can be the evidence that B. burgdorferi multiply intracellulary."Here the authors have stated that vacuoles within fibroblasts were full of Borrelia burgdorferi, and that was evidence that Borrelia burgdorferi multiplies within fibroblasts.
What portion of their lives Borrelia burgdorferi spends within fibroblasts remains to be seen, but if they spend any amount of time as intracellular obligate parasites and not just extracellular ones, this could explain why infection may be persistent.
Earlier studies on Borrelia burgdorferi in relation to fibroblasts have been completed, some of which indicate that perhaps Borrelia burgdorferi does not survive inside fibroblasts very long and instead destroys them. A 2001 study, Insights from a novel three-dimensional in vitro model of lyme arthritis - Standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts, states the following in its paper:
"Results: Both culture systems proved to be stable and reproducible. The host cells and spirochetes showed high levels of viability and maintained their physiologic shape for > 3 weeks, Bb invaded the synovial tissue and the artificial matrix in a time-dependent manner. Host cells were activated by Bb, as indicated by the induction of interleukin-1 beta and tumor necrosis factor alpha. Electron microscopic analysis revealed Bb intracellularly within macrophages as well as synovial fibroblasts, suggesting that not only professional phagocytes, but also resident synovial cells are capable of phagocytosing Bb. Most interestingly, the uptake of the spirochetes appeared to cause severe damage of the synovial fibroblasts, since the majority of these cells displayed ultrastructural features of disintegration.
Is this a reasonable conclusion to draw, based on this in vitro study? What other studies challenge the concept of intracellular persistence of Bb? What about findings from other studies including Klempner's earlier study on not only fibroblasts, but Borrelia burgdorferi's intracellular relationship to other cells?
Conclusion: A novel 3-D in vitro model has been established that allows the study of distinct aspects of Lyme arthritis under conditions that resemble the pathologic condition in humans. This reproducible, standardized model supplements animal studies and conventional 2-D cultures. The disintegration of synovial fibroblasts containing Bb or Bb fragments challenges the concept of an intracellular persistence of Bb and may instead reflect a mechanism that contributes to the inflammatory processes characteristic of Lyme arthritis."
In a 1992 study, Fibroblasts Protect the Lyme Disease Spirochete, Borrelia burgdorferi, from Ceftriaxone In Vitro, stated this in its abstract:
"... The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus several eurkaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival."Later on, within the study, the following statements are made:
"One of the regimens most commonly used clinically for treatment of Lyme disease is administration of ceftriaxone for 14 days. Our time course experiments showed that human skin fibroblasts can protect B. burgdorferi from ceftriaxone for 14 days. It will be of interest to examine the maximum duration of this protective effect."and
"It has been previously demonstrated that B. burgdorferi penetrates endothelial cell monolayers and can be observed inside and between these cells; however, the viability of those potentially intra- and intercellular spirochetes was not assessed."How many studies have been conducted since this study to see if spirochetes survive inside fibroblasts while exposed to more than 14 days of ceftriaxone? 28 days? 42 days?
Has anyone completed repeat studies which show Borreliae spirochetes surviving and replicating inside of other cell types?
How many intra- and intercellular spirochete studies have been completed, and what were the results?
A later, 1993 study by Klempner had this to say in its abstract:
"The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell surface by treatment with ceftriaxone (1 μg/mL) for 5 days. Despite the absence of visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy. Intracellular spirochetes specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent laser scanning confocal microscopy. These observations suggest that B. burgdorferi can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable."What do all the studies on Borrelia's interaction with fibroblasts to date suggest about its intracellular behavior? Is it possible that this is a major cause of some patients' persistent symptoms - even after antibiotic treatment?
This is only a sampling of studies on Borrelia burgdorferi and fibroblasts - what is needed is a meta analysis of the data on this phenomenon and further studies to confirm Borrelia burgdorferi's intracellular nature.
With confirmation of Borrelia burgdorferi's intracellular nature and its proclivity for fibroblasts, new treatments could be developed that help patients more effectively fight off infection.
 SJ Flavell, TZ Hou, S Lax, AD Filer, M Salmon, and CD Buckley. Fibroblasts as novel therapeutic targets in chronic inflammation. British Journal of Pharmacology. 153(S1): S241–S246.March 2008.
 Interaction of of Borrelia burgdorferi in coculture with human fibroblasts. International Conference of Lyme Borreliosis and Other Tick-borne Diseases. 2010.
 Chmielewski T, Tylewska-Wierzbanowska S. Interactions between Borrelia burgdorferi and Mouse Fibroblasts. Polish Journal Of Microbiology. Volume: 59 Issue: 3 Pages: 157-160. 2010.
 Franz JK, Fritze O, Rittig M, Keysser G, Priem S, Zacher J, Burmester GR, Krause A. Insights from a novel three-dimensional in vitro model of lyme arthritis - Standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts. Arthritis and Rheumatism. Volume:44 Issue:1 Pages: 151-162 Jan. 2001
 Kostis Georgilis, Monica Peacocke, and Mark S. Klempner. Fibroblasts Protect the Lyme Disease Spirochete, Borrelia burgdorferi, from Ceftriaxone In Vitro. Journal of Infectious Diseases. Vol. 166, pp. 440-444. 1992.
 Mark S. Klempner, Richard Noring and Rick A. Rogers. Invasion of Human Skin Fibroblasts by the Lyme Disease Spirochete, Borrelia burgdorferi. The Journal of Infectious Diseases. Vol. 167, No. 5 pp. 1074-1081. May 1993. http://www.jstor.org/pss/30112679
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