Hey Pavel,

do you have a theory why your MyD88 inhibiting peptide should inhibit ROS production ? So is it targeted to specific cells in the blood?

And more importantly, there are various extracellular and intracellular ROS sources in many different cells. Some but not all are triggered in a MyD88-dependent way.

When your control peptide lowers your ROS production it is likely that the peptide itself has ROS scavengening (not production inhibiting) properties. With a few more details I can perhaps make some further suggestions.

All the best and a happy new year,

Marc

Hi Marc,

thank you for your remarks and advice. I suppose that neutrophils are the major producers of ROS in whole blood, yet I agree that there are more ways of their production. To activate cells, I use zymosan which binds to TLR and activates intracellular pathway leading to ROS production. MyD88 is the component involved in this pathway and the use of MyD88 inhibitory peptide blocks it (it should prevent homodimerization of MyD88). To measure the level of ROS, I use their reaction with luminol and chemiluminescence detection.

The control peptide should be able to pass the cell membrane, but not to block homodimerisation of MyD88. The scavenging activity would mean a problem. I did some control measurements with bovine serum albumin to test the possible scavenging role of control protein in the system, but BSA did not influence level of ROS. However, it is difficult to compare BSA and peptide control, since both can act differently.

Any idea what to check further?

Pavel

Hey Pavel,

okay, a lot of information :) Luminol diffuses over membranes, so you measure overall ROS in the cell and there is no distinguishment between extra- and intracellular ROS prodcution. However, since neutrophils mainly produce extracellular/phagosomal ROS during the oxidative burst via Nox2 (in a MyD88-dependent manner), I suggest that this is your ROS source. So as basic ROS measurement setting this sounds good.

Your suggestion that neutrohpils are possibly the main source sounds also reasonable. However, at some point you will have to isolate them from blood and show that these cells are responsible for the ROS prodcution after zymosan treatement and your pepite (if it is working by then) targets the neutrophils.

Your control peptide control sounds very good to me (a non-functional, but similar peptide). I am note quite sure what BSA should do in this setting. BSA, as far as I know, has no scavening properties.

Moreover in our isoluminol measurements (so light emission as with Luminol) any kind of serum (BSA, mouse serum, FCS) scavenges the signal, but not due to ROS scavening but by quenching the light emission.

The only solution that I can come up with is to isolate your neutrophils from the blood. Measure ROS with Luminol in a plate reader with Zymosan and PMA (strong positive control for ROS meausrements) over one hour every minute. Leave one setting untreated, treat one setting with your control peptide and the third with your inhibitory peptide. I would also divide this whole setting again in one group that is incubated with the peptides (and then replaced with fresh medium, so that no quenching occurs) and one group that recives the peptides freshly right before the plate goes in the plate reader. If your peptides just quench or scavenge your ROS, this setting should reveal it.

It sounds a little complicated, but ROS measurements are mostly complicated :D

I hope that helps you. Please ask further questions and keep me informed.

If you like I can send you over a ROS plate how I would plan it via E-Mail.

All the best,

Marc

Hi Marc,

my intention was to measure overall ROS production, therefore it is good that you confirmed me that luminol was a good choice and also the set up is basically ok. You are right that neutrophil isolation will likely be another neccessary step instead of using the whole blood samples.

The suggested paralel measurement of non-treated, inhibited sample and blood with peptide control is understandable. I did actually do it in all measurements to have all data from one blood sample and eliminate individual differences among donors. But maybe I can play a bit more with incubation times (and possible injection of inhibitors when measurement is already running) and protein/peptide concentrations to see if there are any signs of quenching or scavenging.

Hopefully, I will find the correct interpretation of the results I get:) In case of some progress, I will send you an update. And thank you once again for the discussion and new ideas!

Greets,

Pavel

You are very welcome Pavel :) Anytime :)

In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".

In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).

If you like, please have a look at:

Functions of ROS in Macrophages and Antimicrobial Immunity

February 2021, Antioxidants 10(2):313

DOI: https://doi.org/10.3390/antiox10020313