The progression of the innate response is inherently built to drive ROS production upon sensing PAMPs etc. It should be noted that inflammation is always secondary to oxidative stress and the formation of ROS since the primary transcription factor responsible for the generation of the pro-inflammatory cytokines, NF-kB is redox dependent. Therefore it is difficult to discern how one would get inflammation in the absence of ROS. I hope this helps.

Peace

fMLP has a dose-dependent effect on what it does to neutrophils (i.e calcium signaling, chemotaxis, degranulation, ROS). Mitochondrial Damage Associated Molecular Patterns will also activate neutrophils (p38 marker of activation). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843437/

LPS is not as good as PMA, but it does induce ROS, as all surface TLR ligands.

Endossomal TLRs (3/7/8/9) do not induce mitochondrial ROS. I don't know about other sources of ROS in this case, I think it depends on the stimulated cell type.

The progression of the innate response is inherently built to drive ROS production upon sensing PAMPs etc. It should be noted that inflammation is always secondary to oxidative stress and the formation of ROS since the primary transcription factor responsible for the generation of the pro-inflammatory cytokines, NF-kB is redox dependent. Therefore it is difficult to discern how one would get inflammation in the absence of ROS. I hope this helps.

Peace

Jon,

How does your answer fit with Chronic Granulomatous Disease (CGD) where NADPH Oxidase is non-functional and patients/animals suffer from chronic inflammation? Nrf2, which opposes NF-kB, is redox sensitive as well. In a sterile inflammation model using zymosan, CGD mice develop chronic inflammation due to a lack of ROS to trigger Nrf2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838778/

Maybe TLR3 ligands, since this triggers antiviral responses which do not require ROS-mediated killing. Also inflammasome ligands, such as NALP3 binders (some metal oxides) might work.

The problem is that most PRRs use redox signalling in their transduction pathways. So if we are not talking about great amounts of ROS such as respiratory burst ROS, it is difficult to find inflammation without ROS.

Evan, NOX2 is not the only source of ROS, so it is not surprising that CGD mice can still suffer from chronic inflammation (if we are to assume that inflammation always comes with ROS). Perhaps in the model you are talking about there is a lack of one (source of) ROS to trigger Nrf2-antioxidant defenses against (another source of) ROS.

As endossomal TLRs do not stimulate mitochondrial ROS as all others (surface TLRs) do, you can check if they do stimulate ROS production by other pathways, it seems a good starting point. I recall that they are not directly involved in NF-KB translocation, but mostly with type I IFN production.

I guess Jon's assumption that inflammation is always secondary to oxidative stress is related to natural situations such as infection, not to the experimental test of an endossomal TLR ligand?

The history of Toll-like receptors — redefining innate immunity

Luke A. J. O'Neill, Douglas Golenbock & Andrew G. Bowie

Nature Reviews Immunology 13, 453–460 (2013) doi:10.1038/nri3446

A important secondary issue is even if your initial stimulus dose not induce ROS, then it is almost certain that autocrine signals due to your stimulus will. We see that Poly I:C induces iNOS in murine microglia cultures. So timing will be critical and perhaps a vesicle release blocker would be needed in vitro to extent the time in which you could investigate your pathway in isolation. What system do you want to apply the stimuli in ?

Some chemotherapies such as 5 fluorouracile or gemcitabine are able to trigger the NLRP3 inflammasome and IL1b production without any ros production. What is the system you want to look at?

Ok guys thanks for all the inputs. I was trying to induce inflammatory signaling in macrophages and hopefully not induce ROS at the same time. I do find that although to a lesser degree compared to the whole pathogen, TLR2 and 4 ligands did induce ROS. I would not be interested in nucleic acid ligands as the do not generally induce the typical TNF, IL6 etc responses but rather more interferons! And i want to measure those. So all in all its a complicated issue for me as far as I can see. I will let it rest for a bit. Thanks again for all your input!

Hey Jane,

it depends on the compartment and the ROS probe you are using. We measured ROS with peritoneal macrophages (not elicited), bone marrow-derived macrophages and ex vivo microglia, and the pattern was always the same.

Nearly all PAMPs we tested (LPS, Pam3, Poly(I:C), OpG, Flagellin, FSL-1, Imiquimod) did not induce extracellular ROS production by Nox2 (in macrophages) or Nox1 (in microglia) by themselves, but primed the Nox2, which resulted in increased ROS prodcution after a bacterial stimulus (which induces a strong ROS production already by itself).

In contrast, all PAMPs were able to induce cytosolic ROS production.

We found that (at least after bacterial infection), the pro-inflammatory pathways resulting in NF-kB activation and cytokine secretion were highly dependend on cytosolic ROS. So at least in macrophages, if you find a stimulus that induces pro-inflammatory signaling, but no cytosolic ROS production, it will eventually end in no pro-inflammatory signaling at all. So I think that is pretty hard to solve. If you find a solution to this problem, I would be happy to know.

For further information on ROS production in macrophages (or microglia) please have a look at:

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO, February 2019, Science Signaling 2(568):eaar5926

DOI: 10.1126/scisignal.aar5926

The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes, March 2018, Cell Host & Microbe 23(3)

DOI: 10.1016/j.chom.2018.01.018

The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye, June 2020, Nature Communications 11(1)

DOI: 10.1038/s41467-020-16400-8

All the best and stay healthy,

Marc

Hey Jane,

I tried to stimulate primary microglia isolated from adult mouse brain with LPS to induce ROS production, as it not ally drives inflammation in these cells.

I tested different stimuli and LPS did not induce ROS.

For more infos you can have a look in our paper that has been recently published

https://rdcu.be/b4GjH.

Good luck,

Anne

7 ketocholesterol induces IL-6, 8 VEGF viaNFKB but without ROS production in RPE, endothelial cells and smooth muscle cells.

Article 7-Ketocholesterol-Induced Inflammation: Involvement of Multi...