Hello reseachers,
I often stumble across discussions concering which macrophage type should be used for experiments, so I wrote down our experience over the last years for this topic.
THP-1-derived macrophages are easy to handle, they divide and they are much easier to transfect than primary human macrophages, but as the name implies it is a macrophage-like cell line derived from a cancer cell. The same goes for the macrophage-like cell line RAW.
A lot of people are argueing, if THP-1-derived and/or RAW macrophages are nearer to a tumor cell or a macrophage.
We use peritoneal macrophages as standard ex vivo model in nearly every experiment (besides BMDM and MEF sometimes) for a long time but I recommend to not use thioglycollate or any kind of elicited PMs.
I know everybody is using it to get more macrophages (because of limited mouse numbers) but my colleauge and I had to compare normal peritoneal macrophages and those attracted via thioglycollate injection during our PhD works. Thioglycollate-elicted macrophages respond in nearly every parameter we investigated (bacterial killing, ROS production, cytokine prodcution, phagocytosis capacity) much worse than normal peritoneal macrophages. They are more or less powered out. They used their whole pro-inflammatory potential on the thioglycllate and ate themselves (so to speak) to laziness.
They phagocytosed less bacteria, they prodcued nearly no ROS (Nox2-dervied phagosomal or cytosolic ROS from mitochondria) and they secreted nearly no cytokines after infection (all compared to PMs without thioglyllate). Moreover, the injection of thioglycollate is also a sterile inflammation, so other cells will be recruited from the blood stream (monocytes, neutrophils).
Again, I know everybody is using this technique, but this hardly represents an ex vivo peritoneal macrophage. Of course, in dependency of the research topic, not only PMs but different ex vivo macrophages should be picked for the experiments (e.g. Kupffer cells for liver, microglia for brain and so on).
For a comparison of ROS production and cytokine secretion between BMDM and naive (not elicited) peritoneal macrophages) please have a look at:
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO, February 2019 Science Signaling 12(568):eaar5926.
and
The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes, March 2018 Cell Host & Microbe 23(3).
I am looking foward to comments and experiences from other labs on this topic.
All the best and, in times of Corona, stay healthy :)
Marc
There are pro and anti-inflammatory macrophages.
Pro-inflammatory M1 macrophages can be induced in response to the cytokine Th1 interferon (IFN)-γ, as well as pathogen-associated molecular complexes (PAMP), lipopolysaccharides and lipoproteins. M1 macrophages produce pro-inflammatory factor proteins such as tumour necrosis factor (TNF)-α, interleukin-1β (IL-1β), IL-6, the chemokines CXCL9, CXCL10 and CXCL11, which attract IL-12, IL-23 and Th1 cells, as well as reactive oxygen species (ROS) and nitric oxide (NO). M1 macrophages stimulate and maintain the inflammatory response.
Other activated M2 macrophages are induced in response to Th2 infections the cytokines IL-4, IL-33 and IL-13. They secrete anti-inflammatory factors, such as IL-1RA and IL-10 receptor agonists and the chemokines CCL17, CCL22 and CCL24 . M2 macrophages are responsible for inflammation resolution, tissue repair and remodeling. M2 macrophages can be induced by IL-10, transforming growth factor (TGF-β) and glucocorticosteroids and have strong anti-inflammatory properties, producing pentraxin-3 (PTX3), TGF-β and IL-10.
I find your introduction and your research very interesting. I have a question here, what other alternative may you suggest to the thioglycollate? did you try some of these alternatives?
Hey Iskander,
thank you very much. Yes, we tried Biogel-injection, but it was the same as with thioglycollate. The PMs reacted very slowly or did not react at all after different stimuli (PAMPs or bacterial infection).
In my opinion, when an infection takes place in the body, macrophages (and neutrophils) are the first cells that have to deal with it and that quickly.
They start with ROS production in minutes (and it ceases after one and a half hour) and they secrete a a great amount of pro-inflammatory cytokines after 5 hours.
Elicited PMs react much slower (or did not react) after infection in our hands. We observed something similar with bone-marrow dervied macrophages (BMDM). They respond to infection but much slower than ex vivo macrophages (because they are artifically differentiated in a culture dish and have never seen something of the mouse (exept for M-CSF).
BMDMs produced ROS but much slower and not in the same amount than PMs and BMDMs take 24 hours to secrete the same amounts of cytokines, which PMs secrete after 5 hours (infected of course with the same MOI). I think, in the body you do not have 24 hours to wait for cytokines.
But BMDMs are still very useful for any basic research on macrophages for example on a new protein or pathogen to get an idea what is going on. But they also do not represent ex vivo macrophages. They are more like "Baby-macrophages".
Marc Herb , very interesting and detailed answer again! Thank you. Actually, I did not think about the use of alternatives to thioglycollate, guess I was following most of researchers, but thank you for introducing this question to my mind. I would search more about it and if I find anything useful about the matter I will share it here. Maybe even I will do my own research about it, looks promising!!
Hello Marc,
Thanks for sharing these experiences, I often find myself in similar circumstances and ponder on the same questions.
It is worth mentioning Zymosan-A as an alternative to TG. But of course, the essential limitations which you detailed - will persist.
If your experiment demands a large number of cells, you will always be in a controversy. On the one hand, a cell line or M-CSF culture which is large but unnatural. On the other hand, a small number of resident cells from each mouse. Or an expanded number which comes from ever-changing inflammation. So it will depend on the inflammation-inducing agent, its injected amount that will produce different reactions, the animal facility, and the point in time you decide to obtain the cells.
When u say they respond better or worse it really depends... So I guess that you keep all of them in a toolbox and choose according to what you try to do with them.
Hey Sergei,
you are absolutely right. Of course not everybody can affort the capacities to use ex vivo macrophages in every experiment and sometimes in vitro-generated macrophages (e.g BMDM) are even more suitable, for example when macrophages are altered already in the mouse because a molecule is missing due to a KO model. BMDM are highly useful when you want to analyze the role of a new protein or a new stimulus. And, in experimental dependency, you can prime them with TNF or IFNg to a more pro-inflammtory state or with IL-4 to an anti-inflammatory state.
I just wanted to make a statement that the most commonly used macrophages (RAW, BMDM and elicited PM) should not be generalized to the situation in the organism. And as I said, using BMDM or any kind of ex vivo macrophage is standard and good. I just would not recommend RAW and elicited PMs because they are highly and unnaturally altered before you start with your experiment.
Thank you very much for your contribution to this discussion. I would be happy, if you add further comments in the future :)
All the best and stay healthy,
Marc
In the context of this discussion, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages.
If you like, please have a look at:
Article Macrophages target Listeria monocytogenes by two discrete no...
All the best and stay healthy,
Marc