12/15-Lipoxygenase orchestrates murine wound healing via PPARg-activating oxylipins acting holistically to dampen inflammation.

Key results

1. ShownAlox15 deletion gives a pro-fibrotic, pro-inflammatory wound: more collagen and myofibroblast actin, more SSEA3+ stem cells, raised pSTAT3, pSMAD3 and IFNγ, lower CD206 and lower matrix metalloproteinase (MMP) collagenase activity. Keratinocyte re-epithelialization is unchanged.

   Masson trichrome collagen higher in Alox15-/- wounds on days 7 and 14 (n = 10-14/group); by immunohistochemistry pSTAT3 (n = 5-6/group), pSMAD3 (n = 10-11/group) and IFNγ (n = 9/group) up and CD206 (n = 5/group) down; day 7 gelatin zymography showed significantly lower active MMP2, pro-MMP2 and MMP9 activity (n = 6/group). IL6 protein and day-4 keratinocyte migration did not differ significantly. · Alox15 deletion alters the phenotype of the…; Elevated TGFβ/IFNγ/IL6 activity is seen in the…; Alox15-/- wounds show reduced matrix…; Figure legends

2. ShownWounding induced 12/15-LOX by 24 hrs, mainly in F4/80+ macrophages and hair-follicle stem cells. Its free monohydroxy products peaked at day 1 and were lost or halved in Alox15-/- wounds. Canonical specialized pro-resolving mediators (SPMs) were essentially absent.

   Of ~100 oxidized fatty acids targeted by LC/MS/MS, 68 were detected. 15-HEPE, 14-HDOHE, 17-HDOHE and 13-HOTrE were absent in Alox15-/-, and 12-HETE/12-HEPE and 15-HETE/15-HETrE fell by 50%; 5-LOX and COX products were unaffected. Esterified 12-HETE-PE was reduced by >50%. Only trace RvD5 was found (max 1.5 pg/wound, around 4 fmol/wound). · Tissue 12/15-LOX is acutely induced by wounding…; The temporal profile of oxylipins is altered by…; Enzymatically-oxidized phospholipids (eoxPL) are…; Discussion

3. ShownA topical high-oxylipin mixture of abundant 12/15-LOX-dependent lipids, dosed to match peak wound levels, raised MMP activity and reduced collagen deposition relative to vehicle in Alox15-/- wounds. An oxidized phospholipid (PE 18:0a/12-HETE) had no effect.

   Lipids were applied on days 0, 2, 4 and 6; wounds analyzed at day 7. MMP9, active MMP2 and pro-MMP2 activities rose to levels between wild type and Alox15-/- (zymography, n = 4 pools of 4-5 wounds). The non-significant collagen increase seen with vehicle was completely suppressed by the high-oxylipin mixture (Masson trichrome, n = 5-6 wounds/group). Total oxylipin dose was 6.5 ng per wound per day. · MMP activities and collagen deposition are…; Discussion; Animal model; Figure legends

4. ShownUnwounded Alox15-/- skin has lower Pparg and Adipoq expression. The inflammatory response at day 4 starts normally, but a co-regulated pro-inflammatory gene module fails to return to baseline by day 7.

   DESeq2 (adjusted p < 0.05): 143 genes differed at day 0, around 42 at day 4 and 79 at day 7 (60 higher in Alox15-/-). A 45-gene module (Pearson |r| > 0.8; e.g. Il1b, Il6, Nlrp3, Ptgs2, Cxcl2, Trem1) stayed around 50% of day 4 levels in Alox15-/- at day 7 but returned to basal in wild type. Ingenuity Pathway Analysis (IPA) predicted higher IL1, IFNβ and inflammasome activity. · Transcriptional analysis reveals reduced…; Late wound healing in Alox15-/- wounds shows a…; Animal model; Statistical Analysis

5. SuggestedThe high-oxylipin mixture activated PPARγ in a cell reporter assay, but only at concentrations around 100-fold above those found in vivo. Doses matched to a single wound did not reliably activate it.

   HEK293 cells expressing mouse PPARγ and a 3x PPRE-luciferase reporter: 0.4-1.2 μM total oxylipins (content of single day-1 wounds) gave no reliable activation; 40-120 μM total (12-HETE 12-36 μM) activated PPARγ, significantly at 80 μM (3 independent experiments). The PPARγ coactivator Helz2 rose significantly at days 4 and 7 in both strains; it is proposed as a sensitizer but not tested. · Late wound healing in Alox15-/- wounds shows a…; Wounding induces significant increases in gene…; Discussion; Cell transfection and reporter assays