Hi all,
Recently I tried to determine NO production in differentiated and undifferentiated THP-1 cells under several exposure conditions. I was not able to do so however, also under exposure to LPS (1 and 10 ug/mL) there was no increase compared to the solvent control.
I expected this already, because there are several articles that indeed state that human macrophages express little iNOS and thereefore produce very low levels of NO ( Epigenetic Silencing of the Human NOS2 Gene: Rethinking the Role of Nitric Oxide in Human Macrophage Inflammatory Responses | The Journal of Immunology (jimmunol.org) , Protein kinase C-eta (PKC-η) is required for the development of inducible nitric oxide synthase (iNOS) positive phenotype in human monocytic cells - ScienceDirect).
However, on the other side there are multiple articles that have been able to show a clear increase in NO production in THP-1 cells ( Berberine Decreased Inducible Nitric Oxide Synthase mRNA Stability through Negative Regulation of Human Antigen R in Lipopolysaccharide-Induced Macrophages | Journal of Pharmacology and Experimental Therapeutics (aspetjournals.org) , Anti-Inflammatory Effects of a Mytilus coruscus α-d-Glucan (MP-A) in Activated Macrophage Cells via TLR4/NF-κB/MAPK Pathway Inhibition (nih.gov)). Does someone have an explanation for this? Is this like a different subtype of THP-1 cells then or something? I'm very confused.
Kind regards
It's possible that you need to differentiate your THP-1 cells further into the macrophage lineage. Because you mentioned that an isoform of the PKC is needed, you might try pushing the differentiation pathway with a phorbol ester like TPA or TMA. BE EXTRA-CAREFUL with these compounds, they're tumor promoters. Add some 1,25-dihydoxyvitamin D (calcitriol). Once you've differentiated a bunch of cells, compare with undifferentiated ones, test whether their NO production increase after proper stimulation (IgG opsonized zimosan Gross et al 1998). Get an iNOS primary antibody to confirm by a Western blot.
Hello Martine
I've worked with THP1 and the arrest of PMA stimulation after differentiation cell to macrophage, help to study inflammatory pathways. Stimulation by PMA promotes an activation of inflammatory genes and after your stimulation by LPS, these genes already had their maximum activation. You should test IL1 or some target gene with or without arrest of PMA after macrophage differentiation and you can see this effect.
Best regards
Dear Martine,
We could not detected NO production in both differentiated and undifferentiated THP-1 cells. We lived the same hesitation as you described. Please see our latest publication on the issue.
Article Dataset on the Differentiation of THP-1 Monocytes to LPS Ind...
Regards..
Here is my answer from another question, basically, THP-1 isnt producing NO. :
https://www.researchgate.net/post/Do-THP-1-have-less-robust-nitric-oxide-production-compared-to-other-macrophage
research papers which I have read found that M1-activated THP-1 macrophages have very little NO or at least not differing significantly to control
example:
Article Umbelliprenin Increases the M1/M2 Ratio of Macrophage Polari...
Meanwhile, NO measurement in inflammation situations in human has found it at higher concentrations, and macrophages derived from human monocytes are shown to produce NO when challenged.
example :
Article Proinflammatory Cytokine and Nitric Oxide Production by Huma...
If your goal is to see NO production, I would advise against the use of THP-1. And I think that is where the controversies come from, because people still want to use THP-1 as a human macrophage model while it isnt producing NO, so it renders many paper useless unless the NO effect is something that they dont want to see
- Badges
- Science topic