Nous utilisons le pour les macrophages et les souris le LPS de E Coli 0127B8 (Ref L3129 Sigma) ou E Coli 0111 B4 (Ref L2630 Sigma)

Nicolas,

I would advice against the use of LPS to mimic obesity excepted if your hypothesis is that fatty acids cause inflammation through TLR4 or that bacterial infection is a exacerbating factor in obesity. Even with this asumption various ligands for a same receptor can have bias toward subsets of signaling pathways. LPS could be also use as a positive control as an proinflammatory stimulus, but again its all depend of your hypothesis as there are many pathways that can led to the acquisition by monocyte of a inflammatory phenotype. In my opinion, your hypothesis will dictate the use of LPS or any other stimulus. Hope it sort of help.

Jacques

I agree with Jacques. You should use a fatty acid such as palmitate, not LPS.

It has been demonstrated that plasma endotoxins are increased during obesity, so I was wondering what type of LPS/endotoxins would mimic the best this effect in vitro or in animals. For instance, I guess LPS from E. Coli would be more relevant than LPS from Salmonella.

Palmitate indeed induces inflammation, but has many other effects which are due to metabolism. I would like to study low-grade inflammation, independently of metabolism.

Dear Nicolas,

Your question is very relevant. I do agree that different types of LPS may have different inflammatory effects. Choosing just one type of LPS to mimic the effect of all possibilities of mixtures of LPS types which probably occur in vivo is always a limitation. So either you give the best possible justification for using the LPS you choose (eg based on the prevalence of bacterial strains in feces from obese humans), or you try to get hold of an LPS sample (mixture) obtained from humans serum. I would choose for the first option since getting reproducible in vitro data with pure compounds is difficult enough....

I disagree with Jacques. I think it has been clearly demonstrated that LPS is present in significant amounts in the blood, even in healthy individuals, and that LPS levels increase in various diseases, including diet-induced obesity. I also think that you cannot say that palmitate is a more relevant endogenous ligand for TLR4 than LPS. I would not hesitate to use a bread and butter E. coli LPS, since it is the lipidA moiety that interacts with TLR4, not the immunogenic sugar moiety. And the variation in lipidA is not very big.

I did not want to create a polemic. My advice was simply that Nicolas' hypothesis should dictate the choice, or not, of a particular LPS. It is beyong raisonable doubt that LPS levels are significant in vivo. That these levels of LPS can affect TLR4-initiated behavior. It is also accepted that plasma concentration of LPS increase after a meal and are increased in many disease states including obesity. Therefore the study of the biology of LPS in the context of obesity is of outmost importance. I still maintain that LPS cannot be use to mimmick inflammation in obesity as we could argue for various other cause of inflammation. However, in my opinnion, it is very appropriate to study LPS-induced proinflammatory response in the contect of obesity.

Thank you all for the comments. I will use LPS from E Coli as suggested by several of you. Do any of you have references about the fact that it is the lipidA and not the sugars that interact with TLR4?

Jacques, no polemic here at all! This is a complex problem, and LPS and saturated fatty acids both play a role in it. What is interesting is that many obese people have normal insulin sensitivity, so obesity per se is perhaps not the cause. Maybe some obese people have inflamed adipose tissue, and others not, and then the question is, where does the inflammation come from. And since we all sysnthesize large amounts of palmitic acid ourselves, I just find it hard to understand how in some people PA would cause inflammation, and not in others. We, ahum, recently found that some obese people have more IgG against E. coli than other equally obese people. And those with IgG against E. coli where the ones that had diabetes... Doesn't prove that bacteria or LPS are the cause of the disease, but it is intriguing, I think...

Cheers,

E

Perhaps it would be good to use both LPS and PA and compare responses? In my opinion, one should maintain physiological relevance, I am not an expert in the domain but do obese individuals exhibit increased intestinal permeability, correlating with BMI? Is this linked with mode of birth, vaginal versus cesaerian section? If that is the case then LPS from a common type of intestinal bacteria would be useful.. In that case if there is circulating LPS which would come into contact with the relevant cell types. Appropriate concentrations should be used to stimulate cells.

However, it is true that the different types of bacteria possess different types of LPS and can modify their LPS in order to modulate the immune response.

It seems to me that we are more accustomed to perceiving inflammation as due to an infectious agent or pathogen...but one must keep in mind that sterlle inflammation is certainly of relevance here. Another layer of complexity is that receptors such as Tolls may bind more diverse ligands that was previously appreciated. There may be an "inflamamtory tone" that is altered in obese patients etc. I agree that the metabolic aspect is crucial and the manifestion of chronic low-grade inflammation likely depends on many factors.

This is a truly intriguing question. I would add that to use of LPS in culture should always consider the possibility to get sensitivity or tolerance responses. Particularly I suggest using low LPS (to induce sensitivity) concentration and restimulation in the case of obesity related inflammation.