Plasmalogens (Pls) are glycerophospholipids with a hallmark vinyl ether bond that confers unique properties. Pls are present in numerous tissues, being abundant in the nervous, immune, and cardiovascular systems. Membrane organization, signaling, and antioxidant functions are attributed to these lipids. However, Pl functions remain enigmatic and are likely to be tissue and developmental stage specific. We recently discovered that Tmem189 is the long-sought enzyme responsible for incorporating the vinyl ether bond in a late step in Pl biosynthesis and demonstrated this activity for the two paralogs existing in zebrafish (1). This finding allows direct studies on Pls and their cellular functions in health and disease, which have thus far relied on targeting genes involved earlier in the biosynthetic pathway. Thus, we aim to evaluate the effects of tmem189 knock down in zebrafish embryos under different inflammatory conditions.

Genetic inhibition of tmen189 was performed using CRISPR/Cas9 technology. Pls levels were measured by mass spectrometry. Spint1a-/- zebrafish was used as a model of chronic cutaneous inflammation, which is characterized by neutrophil spreading and cutaneous aggregates. A tail wound was performed as a model of acute sterile inflammation in zebrafish larvae and a Genetic inhibition of tmen189 was performed using CRISPR/Cas9 technology. Pls levels were measured by mass spectrometry. Spint1a-/- zebrafish was used as a model of chronic cutaneous inflammation, which is characterized by neutrophil spreading and cutaneous aggregates. A tail wound was performed as a model of acute sterile inflammation in zebrafish larvae and a Salmonella sp infection as a model of non-sterile inflammation. Neutrophils and macrophages were assessed using the tg(lyz:dsred) and tg(mpeg:tomato) reporter, respectively. Inflammation markers were also analyzed using zebrafish reporter lines: tg(nfkb:gfp), tg(tnfa:gfp), tg(il1b:gfp).

Suppression of tmem189 by CRISPR/Cas9 technology resulted in a reduction of Pls levels as analyzed by mass spectrometry. Interestingly, in a sterile model of acute inflammation generated by tail injury, tmem189-deficient larvae showed impaired resolution of inflammation and tissue regeneration, accompanied by increased Il1b, Nfkb, and Tnfa activity at the wound site. Consequently, abnormal recruitment of immune cells, with a long-lasting persistence of neutrophils and a low number of macrophages with a predominantly pro-inflammatory phenotype, was found at 24- and 48-hours post-wounding. Similarly, under chronic skin inflammation, using the spint1a-/- zebrafish model, which is characterized by high Nfkb activity, neutrophil dispersion, and skin aggregates, tmem189 deletion worsened skin conditions by increasing all the above parameters in larvae at 3- and 5-days post-fertilization. Furthermore, exogenous Pls addition was able to rescue the exacerbated skin inflammation of Tmem189-deficient larvae. In addition, upon Salmonella sp infection, tmem189-deficient larvae showed higher susceptibility and mortality levels comparing with the controls. Moreover, the total number of neutrophils was significantly lower in tmem189-deficient larvae probably being the effect of increased apoptosis levels.

Overall, our results suggest an important role of Pls on immune cell biology and inflammation and highlight the potential anti-inflammatory properties of these unique lipids.