Fig. 6: ALOX15^−/− T_regs display dysregulated pro-resolving actions in vivo.

(A) Human monocyte-derived macrophages were obtained from peripheral blood monocytes (see methods for details). These were incubated with T_regs differentiated in the presence or absence of an ALOX15 inhibitor (5 µM) for 4 days at a ratio of 4:1 respectively. Macrophages were then harvested, incubated with fluorescently labeled apoptotic cells (1:3 ratio) and efferocytosis was assessed in real-time using High content Imaging. (Left panels) representative immunofluorescence staining of labeled apoptotic cells (red) and nuclei (blue) taken at 60 min, (middle panel) kinetics of apoptotic cell uptake, (right panel) quantitation of apoptotic cell uptake measuring the area under the curve (AUC). Results are mean ± s.e.m. and expressed as change in signal intensity recorded at baseline (0 min); n = 5 healthy volunteers; *p < 0.05, 2-way ANOVA with Tukey multiple comparisons test; ****p < 0.0001, using Mann–Whitney test. (B, C) WT and Alox15^−/− naive CD4⁺ T-cells were differentiated to T_regs (see methods for details). Peritonitis was initiated in RAG^−/− mice with 0.1 mg of zymosan administered via i.p injection. After 30 h, 3 × 10⁵ WT or Alox15^−/− T_regs were injected i.p. Four days later 6 × 10⁶ PKH67-labeled apoptotic cells were injected i.p., after 1-h peritoneal macrophages were collected and efferocytosis assessed using flow cytometry. (left panel) representative images (central panel) abundance of macrophage positive for apoptotic cells (right panel) amount of apoptotic cells taken up per cell for (B) large peritoneal macrophages (C) small peritoneal macrophages. Results are mean ± s.e.m.; n = 8 mice per group. *p < 0.05, Unpaired t test with Welch’s correction. (D–K) WT and Alox15^−/− mice were fed Western Diet for 8 weeks. Aortic arches were then harvested for real-time PCR quantification of inflammatory markers, and lesions were stained using Oil red O and quantified using ImageJ. Blood was also collected for cholesterol quantification in the plasma. (D) Representative images for aortic arches obtained from WT and Alox15^−/− mice (E) Aortic lesion quantitation. (F) Ratio of Total HDL versus LDL/VLDL. (G) Icam1, Vcam1 and Tnf gene expression. (H–K) WT and Alox15^−/− T_regs were differentiated as outlined above and then transferred to Alox15^−/− mice (3 × 10⁵ cells per mouse) via i.v. injection. Mice were then fed Western Diet for 8 weeks and aortic arches and blood were collected for further analysis as in E–G. (H) Representative images for aortic arches obtained from Alox15^−/− mice administered WT and Alox15^−/− T_regs (I) Aortic lesion quantitation. (J) Ratio of Total HDL versus LDL/VLDL. (K) Icam1, Vcam1 and Tnf gene expression. Results are means ± s.e.m.; n = 4 mice per group from two distinct experiments and expressed for E, I as mean gray intensity; F, J as the ratio between HDL and LDL/VLDL concentration; G, K as fold change from WT or Alox15^−/− mice that received Alox15^−/− T_regs (2^−ΔΔC_T); *p < 0.05, **p < 0.01, Unpaired t test with Welch’s correction for E, F and I, J; One sample t test for G and K.