Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Oxidized phospholipids

• Infection

• Effector-triggered immunity

Main Findings

The preprint from Di Gioia et al. investigated the potential role of a host-derived DAMP, oxidized phospholipids (oxPLs) during infection. Non-enzymatic oxidation of lipids generating oxPLs could occur due to general oxidative stress during inflammatory conditions. These oxPLs have established roles in driving atherosclerosis and non-alcoholic steatohepatitis, but their function in the context of infection is still disputed.

Using several mouse models ranging from polymicrobial sepsis to exposure to a viral RNA analog, the authors demonstrated that circulating levels of oxPLs are increased in acute inflammatory conditions. Accumulation of oxPLs was detected in blood and lymphoid organs such as the spleen, where they were particularly enriched in macrophages.

Using a transgenic mouse expressing an antibody that binds and neutralizes oxPLs, the impact of oxPL depletion was tested in vivo. In all infection models, oxPLs promoted worse morbidity and survival rates. Bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS) +/- oxPLs suggested that IL-10, a potent anti-inflammatory cytokine, was suppressed by oxPLs. Indeed, blockade of the IL-10 receptor was sufficient to abrogate the protective effect seen in E06-scFv mice. To understand the action of oxPLs in IL-10 repression, the authors conducted a series of ex-vivo experiments in phagocytes and concluded that oxPLs prevent the phosphorylation of AKT, which normally occurs within minutes of exposure to LPS. Additionally, oxPLs were found to directly bind to AKT.

To further understand how AKT inhibition results in IL-10 repression, a metabolomics approach was performed in BMDMs, which showed and enrichment of intermediates of the methionine cycle such as S-adenosyl-homocysteine (SAH) in LPS + oxPLs treated cells. Given that the conversion of S-adenosyl-methionine to SAH releases methyl groups that can be used for epigenetic modifications, the methyltransferase EZH2 was tested as an epigenetic regulator of the Il10 locus. Indeed, oxPL treatment suppressed the phosphorylation of EZH2 induced by LPS and increased H3K27 trimethylation. Furthermore, pharmacologically inhibiting EZH2 restored IL-10 secretion in vitro and increased survival in murine sepsis.

Limitations

Major comments:

• The study is very IL10-centric even though there are lots of differentially regulated genes in the RNA-seq dataset. It would be good to get a better overview of the differentially regulated genes in this dataset, as there are likely other pathways involved. Also, it would be good to have a WT + anti-IL10R control in the survival experiment (Figure 2C) to account for a possible deleterious effect of IL-10 blockade that is independent of oxPLS.

• Testing EZH2-specific effects in vivo for Figure 7 with GSK343 is an important part of the manuscript. To strengthen this, how would GSK343 effect the E06-scFv model tested in the earlier Figures?

Minor comments:

• The link between AKT and EZH could have been explored in more detail. It is unclear whether AKT is sufficient to induce the epigenetic regulation of IL10. For example, would a AKT phospho-mimetic cancel the deleterious effect of oxPLs in vivo? Along these lines, the phosphorylation of EZH2 is shown in Figure 6, but what are the total EZH2 levels?

• The metabolomics approach focusses only on polar metabolites but a wider analysis that captures lipid species would be useful, especially considering that oxPLs could potentially be metabolized into other bioactive lipids.

• A genetic model of EZH2 knockout in macrophages could be an interesting way to strengthen the EZH2 part of the manuscript.

• It is still unclear how oxPAPC is being “sensed” by macrophages, although this is not at a lack of effort from the authors. This could be expanded on more in the Discussion.

Significance/Novelty

This preprint is expected to have a high impact in the innate immunity field with clinical implications. OxPLs emerge for the first time as a DAMP of host origin that triggers a life-threatening inflammatory response. This is reminiscent of mechanisms of ETI initiated by microbial virulence factors, except that in this case the DAMP is host-derived and acts independently of the type of pathogen or pathogen burden. The fact that human data was used to complement mouse experiments strengthens the findings and warrants further investigation for the potential of oxPLs as a pharmacological target in severe infections.

Credit

Reviewed by Henrique Colaço and Kelsey Voss as part of a cross-institutional journal club between the Vanderbilt University Medical Center (VUMC), the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.