I have found in the literature that H154 is a specific inhibitor of TLR9. For this reason, I performed an experiment using 10uM H154 in macrophages (PMA-differentiated THP1 cells) before stimulation with 5uM CpG ODN 2216 (specific agonist of TLR9). In Figure 1 you can see the four conditions that I tested and the timepoints when I collected supernatant (3, 12, 24h).
I measured IFN beta using ELISA because this cytokine is suposed to be released after activation of TLR9 as one of the Type 1 IFNs.
The problem is that I could not see a real inhibition with H154 and I wondered which could be the problem (Figure 2).
Do you have any suggestions?
Thank you in advance!
H154 sequence: C*C*T*C*A*A*G*C*T*T*G*A*G*G*G*G
*Phosphorothioate bond.
This is what I would do.
1. IFN-b is expressed at super low levels after stimulation and this is normal. You can see that from your ELISA values where even with CpG alone, you are only hitting 40 pg/mL. This is not much higher than the LOQ and it would be hard to see an inhibition when you are starting off that low. Try doing an IFN bioassay. These cells express ISRE and produce a luminescent signal that is proportional to ISRE activation, which in turn is proportional to IFN-b levels in the supernatants. The bioassay is super sensitive and you may see differences that you do not see in ELISAs.
2. Measure alternate cytokines that are induced at higher levels e.g. IL-6, TNF and so on. You may be able to see a difference here.
3. If you measure alternate cytokines that are expressed at higher levels (>200 pg- >5000 pg) and if you fail to see an inhibition here, you can try the following.
- Add the inhibitor earlier (1-2 hours prior to adding CpG).
- Add more of the inhibitor. I used to have better inhibition with 40-50 µM for several inhibitors compared to 10 µM (although 10 µM seems to be the most widely used dose in literature).
- Do a western blot to see if you are inhibiting TLR-9 signaling with the inhibitor. Collect supernatants a few hours after you notice significant inhibition.
-Try alternate inhibitors. Some work better than others. If you are following a published protocol, follow it to the T. Minor changes can make a huge difference in results.
Thank you so much Julie for your time and effort answering my question. I could say that I am currently in the 3rd option that you mention. So I will try to increase the concentration and the time with the inhibitor.
Thanks a lot again!
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