Hi. I am having trouble interpreting the results of my experiment.
Chondrocytes were treated with LPS for 24 hours and then incubated with drugs that are expected to have anti-inflammatory effects and mRNA was extracted after 24 hours of incubation.
IL-6, TNF-a, and IL-1b mRNA expression levels were determined by qPCR. IL-6 was highest in the LPS(+)drug(-) group and decreased with increasing drug concentration.
However, TNF-a and IL-1b were expressed at levels similar to or lower than normal controls in the LPS(+)drug(-) group.
Comparatively, TNF-a and IL-1b were higher in the drug-treated group.
I'm curious that when I look at IL-6, it seems like the LPS signal is working, but I don't understand why TNF-a and IL-1b are lower in the LPS group. I'm wondering if this is possible.
If the mRNA peaked and then declined because of the longer time, would I need to reduce the LPS treatment time to get the desired results for TNF-a and IL-1b?
Hello Juhee Seo
Cells activated by LPS can produce high levels of IL-10 which is relatively unique in its ability to downregulate the production of multiple pro-inflammatory cytokines.
I would like you to know that IL10 has been shown in vitro to potently downregulate LPS induced production of TNFα, IL1, IL6, IL8, G-CSF, and GM-CSF. In fact, it functions primarily as an inhibitory cytokine to suppress Th1 mediated cellular responses and deactivate macrophages. Importantly, IL10 acts to diminish the production of pro-inflammatory cytokines such as TNFα and IL1, which are important mediators of joint inflammation.
Please refer to the paper attached below.
https://ard.bmj.com/content/58/suppl_1/I99
In one of the studies, human monocytes activated by LPS were able to produce high levels of IL-10, which was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. The kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, and G-CSF, which were all secreted at high levels 4-8 h after activation.
Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF. For more information you may also want to refer to the article attached below.
https://pubmed.ncbi.nlm.nih.gov/1940799/
TNF-a and IL-1b being lower in the LPS group is possible because they may be inhibited by IL-10 which is also produced when cells are treated with LPS.
To summarize, IL-10 has important regulatory effects on the inflammatory responses because of its capacity to inhibit the production of proinflammatory cytokines.
You may try reducing the LPS treatment time, or, if you need to observe the anti-inflammatory effect of the drug, you may treat the chondrocytes with LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies because you have observed TNF-a and IL-1b being expressed at levels similar to or lower than normal controls in the LPS(+)drug(-) group.
Best.
24h is too late for cytokine mRNAs.
Try to treat cells with LPS in a manner of time course (0, 2, 4, 6, 8, 12, 24h) and find the peak time (usually 4-8h), and then treat cells with LPS for the peak time before you add the drug. The time and concentration of the drug may need to be optimized as well. Usually LPS 100ng/mL is enough.
1.LPS is a potent stimulator of IL-6 synthesis /excretion from macrophages. How clean is your chondrocyte culture?
2. IL-6 increase would downregulate TNFa at least in short term (up to
60 min) experiments.see attachment; Figs.5&6. This effect is not known for 1L-1b (for me)
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