Fig 2: The therapeutic effect of rIL-27 in a clinically relevant mouse model of ICH. ICH in mice was induced using autologous blood injection. The rIL-27 treatment was initiated at 30 min after ICH by i.v. injection and then daily by s.c. injection for 6 days, each treatment at 50 ng/kg. The outcome include: a the individual behavioral/NDS of footfault, circling, wire, postural flexing, and forward placing test, and grand NDS, a composite NDS score on day 1–14 after ICH (n = 8) The data were expressed as mean ± SEM and *p < 0.05 was established using one-way ANOVA followed by Newman–Keuls post-test. b Brain edema (water content) on day 3 (n = 5); e hemoglobin (Hb) and f iron content in ICH-affected brain (index of hematoma removal) on day 7 (n = 5). c The grand NDS on day 3 after ICH in mice treated with rIL-27 and with Ly-6G or rat isotype IgG, control. Twenty-four of C57/BJ6 mice were treated with rIL-27 (Biolegend, 577404, a heterodimer of mouse recombinant IL-27 EBI3 and p28 linked by a GGGSGGGSGGGTGGGS linker), 50 ng/kg by i.v. at 30 min and then daily s.c., on day 1–3 after ICH. To deplete neutrophils, 12 mice were injected with Ly-6G, a neutrophil neutralizing antibody (BioXCell, 1A8, BE0075) at 500 µg/mouse at 2, 24, and 72 h after ICH (i.p.). And 12 mice were injected with rat isotype IgG (IgG2a, BioXCell, 2A3, BE0089). The NDS were quantified with three behavioral tests (postural flexing, circling, and footfault) 3 days after ICH. d The percent change of body weight on day 3 (compared with the body weight on day 0, right prior to ICH). In b–f, all data were expressed as mean ± SEM. *p = 0.05, compared with the corresponding vehicle control, established using paired t-test

Fig 3: IL-27 induces LTF in PMNs. a Photomicrograph of representative LTF, immunostaining (green) in normal mouse BM-PMNs. The nuclei are stained with DAPI (red). b, c MFI of LTF, staining per cell was determined in BM-PMNs by flow cytometry, with T cells and monocytes serving as negative controls. Analyzed cells were from mice treated with saline or rIL-27 (50 ng/kg, i.v., at 30 min, 24 h, 42 h after ICH) and harvested at 48 h after the ICH. d Photomicrograph of representative LTF staining (green) in peripheral blood mature PMNs. The nuclei were stained with DAPI. Scale bar = 10 µM. e Bar graph of LTF, protein content (established with ELISA) in peripheral blood PMNs in naïve rats, and rats 48 h after ICH with and without treatment with rIL-27 (50 ng/kg, i.v., at 30 min, 24 h, 42 h after ICH). Data are expressed as mean ± SEM (n = 5). *p = 0.05, vs. naïve rat, **p = 0.05, vs. naïve rats and rats after ICH without IL-27 treatment. The p-value was established using one-way ANOVA followed by Newman–Keuls post-test. f Immunofluorescence of LTF, (red) and neutrophil elastase, ELANE (green) in a mouse brain at hematoma-affected area at 24 h after ICH. The nuclei were labeled with DAPI (blue). Scale bar = 50 µm. The insertion window highlighted the co-localization of LTF, with ELANE+ neutrophil. g LTF, protein in the ICH-affected rat brains (harvested from animals perfused with saline to flash intravascular cells), in the same animals as in e, and established using Western blot (n = 5)

Fig 4: Microglia-derived IL-27 causes neutrophil polarization. Cell-specificity of IL-27 and IL-27R expression in the SD rat brain cells (a–d), and the gene expression profile (e, f) in IL-27-modified mouse BM-PMN precursors, and g STAT3/pSTAT3 protein in rat BM-PMN exposed to rIL-27, in vitro. a Photograph of representative gels demonstrating IL-27 p28 and EBI3 mRNA and IL-27Ra mRNA and gp130 mRNA expression in primary rat brain microglia (micro, identified with CD68), neurons (neuro, identified with neurofilament L, NF-L), astrocytes (astro, identified with glial fibrillary acidic protein, GFAP), and oligodendrocytes (oligo, identified with myelin basic protein, MBP) in culture. IL-27 p28 and EBI3 are exclusively expressed by microglia. The gp130 is expressed by all cell types, while IL-27Ra is mainly by astrocytes and neurons. GAPDH was used as an internal control. b, c Representative RT-PCR gels and bar graph of IL-27 p28 and EBI3 mRNA expression in rat brain cells in response to RBC. IL-27 p28 and EBI3 are upregulated in rat microglia (Micro), but not in astrocytes (Astro), oligodendrocytes (Oligo) or neurons (Neuro) at 6 h after exposure to RBC. The data were calculated as mean ± SEM, n = 4–6. *p = 0.05 (using paired t-test), compared with the media control group (without RBC). GAPDH was used as an internal control. d IL-27 p28 protein determined with ELISA in rat microglial culture medium at 6 h after exposure to RBC, LPS, and interferon gamma (IFN?). *p = 0.05, compared with the control group (n = 3), established using one-way ANOVA followed by Newman–Keuls post-test. e Photograph of RT-PCR gels and f bar graph demonstrating the gene expression profile in the PMNs harvested from mouse BM-PMNs at 24 h after ICH and then cultured in 10% mouse serum in RPMI1640 and treated with recombinant mouse IL-27 (150 pg/ml) or saline for 24 h, in vitro. The analyzed genes included: iNOS, MMP-9, NOX2, Hp, LTF, myeloperoxidase (MPO), neutrophil elastase (ELANE), and GAPDH. Data were expressed as mean ± SEM (n = 3–5). *p = 0.05, compared with the vehicle-treated group, established using paired t-test. g Photos of immune Western blotting of phosphorylated form (pSTAT3) and total STAT3 protein in purified rat BM-PMN in culture at 6 h after incubating in recombinant mouse IL-27 (150 pg/ml) with or without 10 µM STATTIC. GAPDH was used as an internal control

Fig 5: Increased IL-27 production after ICH. a Photograph of representative gels demonstrating temporal changes of IL-27 p28 and EBI3 mRNA in the ICH-affected corpus striatum, at 0 h (sham control) and 1 h to 14 days after ICH in SD rats, which was assessed using RT-PCR, and (a, right panel) a bar graph of densitometrical quantitation. GAPDH was used as an internal control. The data were calculated as mean ± SEM (n = 4). b Bar graph of IL-27 p28 protein levels in CSF in naïve rats and in rats at 3, 24, and 48 h after ICH. The data were calculated as mean ± SEM (n = 3/time point). c Bar graph showing timecourse of IL-27 p28 protein levels in blood serum at 1 h to 21 days after ICH in C57BJ6 mice. The data were expressed as mean ± SEM (n = 5). d Photograph of a representative gel demonstrating temporal changes in IL-27 p28 and EBI3 mRNA in the spleen of rats subjected to ICH, assessed using RT-PCR, and (d, right panel) a bar graph of densitometrical quantitation. GAPDH was used as an internal control. The data were calculated as mean ± SEM (n = 4). *p = 0.05, compared with the Sham group (0 h) for all the panels. The p-value in this figure was generated using one-way ANOVA followed by Newman–Keuls post-test