Fig 1: In response to injury caused by ICH, activated brain microglia and spleen rapidly produce IL-27 (1), a cytokine, which modifies the developing PMNs in the BM to express more of a potent iron-sequestrating LTF and less of pro-inflammatory factors (2). This process is referred to as PMNs polarization. In consequence, these polarized PMNs may carry more cytoprotective LTF. Upon entering the injured brain, the PMNs may release more LTF that through sequestrating the cell-free ferric iron (produced due to intra-hematoma hemolysis) alleviates oxidative stress, ICH-deposited iron, and ultimately neurological damage
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
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