Fig 2: eCIRP is released in ischemic stroke and causes efferocytic dysfunction in vivo. a Concentration of eCIRP in CSF collected from mice 24 h after sham or tMCAO surgery. (n = 5; mean ± SEM; * p = 0.0006 vs. Sham; Unpaired t-test). b Mertk expression Z-score in microglia cluster from GSE227651. c Bederson score in WT tMCAO and CIRP−/− tMCAO mice 24 h after surgery. (n = 8; mean ± SEM; * p = 0.0031; Unpaired t-test). d Representative confocal images of peri-infarct brain tissues in Sham, WT tMCAO, and CIRP−/− tMCAO mice, stained for DAPI (blue), Iba-1 (magenta), and MerTK (green). Magnification 630x, scale bar, 20 μM. e Quantification of MerTK expression in microglia per animal. (n = 6; mean ± SEM; * p < 0.0001 vs. Sham; # p = 0.0032 vs. WT tMCAO; one-way ANOVA, Tukey’s multiple comparisons test). f Representative confocal images of peri-infarct brain tissues in WT tMCAO and CIRP−/− tMCAO mice, with staining for nuclei (DAPI, blue), neurons (NeuN, yellow), dead/dying cells (TUNEL, green), and microglia (Iba-1, magenta); arrows indicate efferocytosis events with complete internalization of TUNEL+/NeuN+ cargo, arrowheads indicate incomplete efferocytosis events. Magnification 630x, scale bar, 20 μM. g Ratio of TUNEL+ neurons efferocytosed by microglia to total TUNEL+ neurons expressed as fold-change with WT tMCAO set as 1. (n = 8; mean ± SEM; * p = 0.0017 vs. WT tMCAO; Unpaired t-test). h Zoomed in high power reconstruction of 3D segmented surfaces in Imaris showing efferocytosis event indicated in white box in f, demonstrating TUNEL+/NeuN+ cargo engulfed by Iba-1+ microglia.

Fig 3: eCIRP is released in ischemic stroke and causes efferocytic dysfunction in vivo. (a) Concentration of eCIRP in CSF collected from mice 24 h after sham or tMCAO surgery. (n = 5/group; mean ± SEM; * p = 0.0006 versus Sham; Unpaired t‐test). (b) Mertk expression Z‐score in microglia cluster from GSE227651. (c) Bederson score in WT tMCAO and CIRP−/− tMCAO mice 24 h after surgery. (n = 8/group; mean ± SEM; * p = 0.0031; Unpaired t‐test). (d) Representative confocal images of peri‐infarct brain tissues in Sham, WT tMCAO, and CIRP−/− tMCAO mice, stained for DAPI (blue), Iba‐1 (magenta), and MerTK (green). Magnification 630x, scale bar, 20 µM. (e) Quantification of MerTK expression in microglia per animal. (n = 6/group; mean ± SEM; * p < 0.0001 versus Sham; # p = 0.0032 versus WT tMCAO; one‐way ANOVA, Tukey's multiple comparisons test). (f) Representative confocal images of peri‐infarct brain tissues in WT tMCAO and CIRP−/− tMCAO mice, with staining for nuclei (DAPI, blue), neurons (NeuN, yellow), dead/dying cells (TUNEL, green), and microglia (Iba‐1, magenta); arrows indicate efferocytosis events with complete internalization of TUNEL+/NeuN+ cargo, arrowheads indicate incomplete efferocytosis events. Magnification 630x, scale bar, 20 µM. (g) Ratio of TUNEL+ neurons efferocytosed by microglia to total TUNEL+ neurons expressed as fold‐change with WT tMCAO set as 1. (n = 8/group; mean ± SEM; * p = 0.0017 versus WT tMCAO; Unpaired t‐test). (h) Zoomed in high power reconstruction of 3D segmented surfaces in Imaris showing efferocytosis event indicated in white box in (f), demonstrating TUNEL+/NeuN+ cargo engulfed by Iba‐1+ microglia.

Fig 4: CIRP‐deficiency improves outcomes in acute ischemic stroke. WT and CIRP−/− mice were subjected to Sham or tMCAO surgery, and outcomes assessed 3‐day post‐tMCAO. (a) Representative images of 2 mm thick brain sections stained with TTC for infarction detection and quantification 3‐day post‐tMCAO. Infarcted brain tissue is stained pale white, while healthy brain tissue is stained in red. (b) Infarct volume in WT and CIRP−/− mice 3 days after tMCAO quantified with NIH ImageJ. (n = 7/group; mean ± SEM; * p = 0.0005; Unpaired t‐test). (c) IL‐6 levels in 3‐day post‐tMCAO total brain lysates expressed as IL‐6 pg/mg total protein. (n = 6/group; * p < 0.0001 WT sham versus WT tMCAO; ns. p = 0.2885 CIRP−/− sham versus CIRP−/− tMCAO; Mixed‐effects model (REML) with Sidak's multiple comparisons test). (d) Bederson score 3‐day post tMCAO in WT and CIRP−/− mice (n = 7/group; mean ± SEM; * p = 0.0005; Unpaired t‐test). (e) WT and CIRP−/− mice subjected to tMCAO‐induced ischemic stroke were monitored for 3 days for humane endpoints, and differences in survival were determined using Kaplan‐Meier survival plots and a log‐rank test. n = 8‐16 mice/group. Hazard ratios (HR): WT, 5.557; CIRP−/−, 0.1799. *p = 0.0452 versus WT.

Fig 5: miR‐155 is induced and MafB is suppressed by eCIRP in ischemic stroke. (a,b) miR‐155 host gene (a) MiR155hg and (b) Mafb expression Z‐scores in microglia cluster from GSE227651. (c) Relative levels of miR‐155 in hippocampus 24 h after stereotactic injection of PBS or 1 µg eCIRP (Contra. & Ipsi. indicate hippocampus laterality relative to eCIRP injection). (n = 7–8/group; mean ± SEM; * p = 0.0009 versus Sham & p = 0.0002 versus Contra.; ns. p = 0.9632 versus Sham; one‐way ANOVA, Tukey's multiple comparisons test). (d) Representative Western blot images and relative MafB protein levels in hippocampal tissues 24 h after stereotactic injection of PBS or 1 µg eCIRP (n = 4–6/group; mean ± SEM; * p = 0.0288 versus Sham & p = 0.0153 versus Contra.; ns. p = 0.989 versus Sham; one‐way ANOVA, Tukey's multiple comparisons test). (e) Relative brain levels of miR‐155 in WT and CIRP−/− mice subjected to sham and tMCAO surgery. (n = 5/group; * p = 0.0217 WT sham versus WT tMCAO; ns. p = 0.903 CIRP−/− sham versus CIRP−/− tMCAO; two‐way ANOVA, Tukey's multiple comparisons test). (f) Representative Western blot images and relative MafB protein levels normalized to β‐actin in whole brain lysates of WT and CIRP−/− mice subjected to sham and tMCAO surgery. (n = 3–7/group; * p = 0.046 WT sham versus WT tMCAO; ns. p = 0.7657 CIRP−/− sham versus CIRP−/− tMCAO; Mixed‐effects model (REML) with Sidak's multiple comparisons test).