I am planning to study the epigenetics of TB infection in the host system. I have to quantitatively analyze the expression pattern of certain genes viz., IFN-g, TNF-a, TLR-4, FOXP3...etc. and their corresponding epigenetic changes. I therefore need to know which kind of cell line would be useful; either human monolayers or a macrophage cell line? Is this feasible?
Macrophages cell lines, will be easier (U937, THP1). However primary cells are more precise to evaluate "natural" behaviour.
Best
THP-1 cells. these are monocytes. you will have to stimulate it to differentiate into Macrophages with PMA and then infect with M.tb. All those you have specified, i believe there is considerable amount of research already been explored. Being working in lab that does the same here, i would like to share my experience. PMA itself a potent antigen more than just a stimulating factor and complications arise with its use. again there had been contrasting results published with each set of strains that people use to infect. Epigenetic analysis is a must study area but dont know how precise the mechanism compared to the active disease pattern. Any way All the best.
Thanks balaji...I really appreciate your help. and one more thing could you please explain the procedure in detail or else would you mind cite the papers of the above explanation?That would help me a lot....
Thank you.
If you can get your hands on it, use "real cells" - ie human alveolar macrophages. Far more relevant than THP1, or if you work in mice, take the whole lung, wash - and there are your cells. None of the lines will give you the real answers, simply because they are immortalised one way or the other
I think to give a solid answer, we need to have a better idea of what specifically you are looking for.
As an example, we recently published some work on host gene activation in early infection with virulent and avirulent strains of M. tuberculosis: for this we used cell lines in preference to harvested mactophages from tissue, because we wanted a homogenous population and as little background "noise" as possible. This is particularly an issue with microarray studies since the number of potential hits means that your sensitivity is necessarily low.
We considered cell lines an acceptable tradeoff since we were only looking at event ocurring in the first few hours of infection. On the other hand, cell lines are probably not the way to go if you are looking at longer term changes, since immortalised cells don't really represent a precursor population and can't be expected to develop normally. They also don't really represent a specific host population such as alveolar macrophages. I'm guessing that for epigenetic changes, you will probably want a population of cells that more closely represent a natural population, so harvested macrophages are probably more relevant. Cells from buffy coats are probablythe most practical source. Of course that raises the problem of keeping them alive long-term!
@Timothy: I partially agree with you, and to get an idea on Whats going on, cell lines are definitively the best starting point. However, we are dealing with a very heterogeneous population, whatever mycobacterium we are working with, and our hosts (human, cattle) are very outbreed. Thus, the cell line experiments needs to be verified with "real" cells.
I think that depends on the question being asked: if the question involves real-life infection, then I'd agree: you need to look at cells reflective of the real process. If the question is more about activation of specific genes and delineating gene pathways and the proteins involved, then not so much. Those interactions tend to be much less context-sensitive: the DISC gene cascade, for example, plays a major role in the pathogeneisis of TB, but the genes involved are the same in a cell line as in an activated macrophage - and for that matter, they are the same if the triggering factor is M. tuberculosis, or L. monocytogenes.
That's why I asked for a better sense of what the question being asked is: when working with human cells, I'm a big fan of keeping the methods as simple as possible ... but not simpler!
Ah, but not if you put BCG, M.tuberculosis or M.bovis on the same cells. We studied that :-)
Oh, no question - we also published the effects of different pathogens on that pathway (which is why I chose it as an example!) I think we basically agree that the response to infection of this pathway in reality is very much dependant on pathogen, cofactors such as cytokines, state of activation of the macrophage, etc.
The genes themselves are the same, though: if you want to study the roles of those genes in infection, you need a population that resembles those in your patients (ideally, cells from patients and from appropriate controls). But if all you want to do is delineate the actual genes involved in the pathway, then cell lines will give you the much the same answer as harvested cells but much more cheaply and easily.
@Timothy Mark Doherty: I really thank for your words. The problem in my study is I am going to working on TB infection in Children. In our institute we won't be allow to draw more than 5 mL of Blood from the patient. Since, the number of circulating monocytes will be lesser in this, So i would like to start initially with cell lines. If the cell line would give the best sort of answer what I am looking for then i would like to go with cell culture from the patients. Anyhow I have to confirm with patient derived cell population works at last. So for the sake of initial standardization i asked. I thought i would be better to draw outline of the transcriptome analysis at first with commercial cell line. Since I am new to this area i am need of more technical pitfalls in this...
@Timothy: yeap, I completely agree with that. As Kathirvel wants to work with human cells, THP-1 it is.
@Dirk Werling: Agreed!
The good news is that 5 ml of blood is plenty for transcriptome analysis, once you are ready to move to patient material.
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