Fig 1: IL-27 neutralization in C57BJ6 mice alters gene expression in BM-PMNs and aggravates neurological outcome after ICH. a Photograph of representative gels (RT-PCR) and bar graphs demonstrating the expression profile of indicated genes in the BM-PMNs, purified from mice at 48 h after ICH that were treated with either mouse anti-IL-27 antibody (aIL-27Ab, 40 µg/mouse), recombinant mouse IL-27 (rIL-27; 50 ng/kg), or rat IgG isotype control antibody (40 µg/mouse), all by i.v. injection at 0.5, 24, and 42 h after ICH. Data are expressed as mean ± SEM (n = 5). *p = 0.05, established using one-way ANOVA followed by Newman–Keuls post-test. b The NDS in mice treated with anti-IL-27 antibody (aIL-27Ab) and rat IgG isotype control (IgG Cont) at days 1, 3, and 7 after ICH. The grand NDS is a composite score from four behavioral tests (Footfault, Postural Flexing, Wire, and Circling). *p < 0.05, established using one-way ANOVA followed by Newman–Keuls post-test. c Bar graph of the NDS on day 3 from the above study. In b, c, the data were expressed as mean ± SEM (n = 12). *p < 0.05, compared with the control, established using paired t-test. d Survival curve of day 1–day 7 after ICH in the above study in b. There is no difference between the groups. e, f Flow cytometry of peripheral blood taken from animals treated with anti-IL-27 antibody (aIL-27) or isotype control IgG (IgG Con) at three days post-ICH and stained for LTF. Data presented as MFI and given as mean +/- SEM (n = 3/group), established using paired t-test. An isotype control antibody (IgG Con) was used to determine the negative and positive gates for flow cytometry
Fig 2: 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 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: Neuroprotective effect of recombinant LTF in vitro (a, b) and in a clinically relevant rat (c–e) and mouse (f, g) model of ICH. a Representative immunofluorescence of MAP2+-neurons (green) and GFAP+-astrocytes (red) in rat brain cortical neuron-glial co-cultures at 24 h after exposure to RBC lysate (an ICH-like injury in vitro), demonstrating that lysed RBCs are toxic to neurons. Arrows show the swollen soma and broken dendrites. Astrocytes are less prone to the injury. b Representative images (left part of the panel) showing density (viability index) of NeuN+-neurons (green) in the rat neuron-glial co-cultures at 24 h after the exposure to lysed RBC in the presence or absence of rLTF, (5 µg/ml), which was added to the co-culture at 30 min prior to the insult (adding lysed RBCs). The nuclei were labeled with DAPI (blue). Scale bar = 50 µm. And a bar graph of NeuN+-neuron numbers at a 10× field. Data were mean ± SEM (n = 15). *p = 0.05 from all other groups; established using one-way ANOVA followed by Newman–Keuls post-test. c–e rLTF reduces ICH-mediated brain damage in rats after ICH. Rats were treated with rLTF (5 mg/kg, i.v. at 30 min after ICH plus 2.5 mg/kg p.o., at 24 and 48 h) and assessed for c NDS (a composite score for footfault, circling, wire, postural flexing, and forward placing test) at days 1 and 3 (n = 10), d oxidative burden (4-HNE; index of lipid peroxidation) at day 3 (n = 5), and e brain edema (% of water content) at day 3 (n = 5). f, g Recombinant mouse LTF, improves outcome after ICH in mice that were treated as late as 24 h after ICH (5 mg/kg, i.v. at 24 h after ICH plus 2.5 mg/kg p.o., on day 2–7) as determined with assessing f NDS on day 1, 3, 7, and 14 (n = 11) and g iron clearance from the hematoma-affected brain on day 7 after ICH (n = 7). All data were presented as mean ± SEM. *p = 0.05, compared with the vehicle control. The p-values in d, e, g were established using paired t-test; in c, f, the longitudinal data analysis was performed and corresponding p-values were provided using the mixed effects model
Fig 5: 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
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