Neutrophils in Recovery after ICH

在脑出血(ICH)发作后不久，随后的几天里， 中性粒细胞(PMN)进入脑出血患者的大脑。在释放了各种细胞毒性部分后， 中性粒细胞经历快速的凋亡，然后被脑小胶质细胞/巨噬细胞(MΦ)清除。没有FAST 通过MΦ清除，凋亡性PMN(ANS)继续进行继发性坏死和财富溢出 有毒部分，对大脑造成伤害，加重血肿引起的脑损伤。 然而，考虑到PMN释放的个别部分的生物学特性，PMN很可能 还可以增加大脑修复。中性粒细胞的化学成分设定在中性粒细胞的前体阶段(PPMN)。 骨髓(BM)(BM-pPMN)成熟的PMN保留了预先建立的表型并充当航天飞机， 将内容物(包装成颗粒状)输送到PMN的渗透部位(这里是ICH损伤的脑)。 在这里，我们建议研究一种新的信号途径，特别是IL-27，修饰BM-pPMN 表型，使成熟的中性粒细胞进入脑出血后造成的损害较小，甚至 提供益处，包括促进吞噬介导的血肿清除和ANS。 乳铁蛋白(LTF)是一种多效性铁结合/免疫调节蛋白，由 PMN。我们的前期研究表明，脑出血诱导的IL-27是BM-pPMN表达更多LTF的信号。长期合作伙伴关系- 浓缩的中性粒细胞进入血肿后可释放更多的LTF。这种长效转运蛋白能够：(1)刺激 M-Φ吞噬ANS，(2)上调吞噬包被Φ的ANS清道夫受体M-CD91 和其他溶血产物，(3)隔离游离铁，(4)使M-Φ极化为有益的(类M2) 表型。除LTF外，IL-27还促进血红蛋白中和蛋白结合珠蛋白的表达， 帮助我信号分子Lipocalin-2，并通过抑制基质金属蛋白酶-9和NADPH-氧化酶的表达 PMN。最终，用重组IL-27(rIL-27)或重组LTF(RLTF)治疗动物，强有力地， 24小时治疗窗(对于rLTF)，减少脑出血引起的脑损伤和功能损失。 我们的假设是，在脑出血后，IL-27使中性粒细胞极化成有益的类型，当到达脑出血时- 损伤的大脑细胞毒性较小，甚至增强(部分通过长期转铁蛋白)M-Φ介导的清除(部分通过 上调CD91/CD36清道夫受体)，减少继发性脑损伤。具体目标是：(1) 鉴定IL-27诱导的骨髓和血液中的PMN表型；(2)通过原代细胞培养，确定 PMN表型和LTF在PMN/LTF清除/解毒ANS/HO/Fe中的有益作用 改良M-Φ；(3)评价重组人肝转移因子的治疗作用，明确CD91在肝转移因子辅助治疗中的作用 清理，在小鼠脑出血模型中。我们的目标是开发新的脑出血治疗靶点，基于 IL-27修饰的PMN释放更多的LTF以促进M-Φ介导的血肿清除。这种方法 不仅应该限制PMN介导的中枢神经系统损伤，而且还应该允许PMN以有益的方式行动。

Shortly after the onset of intracerebral hemorrhage (ICH) and then for several days, masses of polymorphonuclear neutrophils (PMNs) enter the ICH-affected brain. After releasing various cytotoxic moieties, the PMNs undergo rapid apoptosis and then are removed by brain microglia/macrophages (MΦ). Without fast clearance by MΦ, apoptotic PMNs (ANs) proceed to secondary necrosis and spillage of wealth of uniquely toxic moieties, causing harm to brain and augment brain injury caused by hematoma. However, given the biological properties of individual moieties released by PMNs, it is likely that PMNs can also add to brain repair. The PMN's chemical composition is set at the precursor stage of PMNs (pPMN) in bone marrow (BM)(BM-pPMN). The mature PMNs retain the pre-established phenotype and act as shuttles, delivering the content (packed in granules) to the sites of PMNs' infiltration (here ICH-injured brain). Here, we propose to study a novel pathway where the signal, specifically IL-27, modifies BM-pPMN phenotype, so that the mature PMNs upon entering the ICH-affected brain would cause less damage, or even provide benefit, including by promoting phagocytosis-mediated cleanup of hematoma and ANs. Lactoferrin (LTF) is a pleiotropic iron-binding/immunoregulatory protein that is synthetized/distributed by PMNs. Our pilot studies show that ICH-induced IL-27 is a signal for BM-pPMN to express more LTF. The LTF- enriched PMNs upon entering the hematoma could release more LTF. This LTF is capable of: (1) stimulating ANs engulfment by MΦ, (2) upregulating MΦ' CD91, a scavenger receptor for engulfment of LTF-coated ANs and other hemolysis products, (3) sequestrating the free iron, and (4) polarizing MΦ to beneficial (M2-like) phenotype. In addition to LTF, IL-27 promotes expression of hemoglobin neutralizing protein haptoglobin, “help-me-signal” molecule lipocalin-2, and suppresses the expression of MMP-9 and NADPH-oxidase by PMNs. Ultimately, treating animals with recombinant IL-27 (rIL-27) or with recombinant LTF (rLTF), robustly, with a 24h therapeutic window (for rLTF), reduces brain damage and functional loss caused by ICH. Our hypothesis is that after ICH, lL-27 polarizes PMNs to a beneficial type, which upon reaching ICH- injured brain is less cytotoxic and even enhances (in part through LTF) MΦ-mediated cleanup (in part through upregulating CD91/CD36 scavenger receptor) and lessens secondary brain damage. Specific aims are: (1) characterize the IL-27-induced PMN phenotype within BM and blood; (2) with primary cell culture, to define the beneficial role of the PMN phenotype and LTF in clearance/detoxification of ANs/heme/iron by the PMNs/LTF- modified MΦ; and (3) to assess the therapeutic role for rLTF and to define the role of CD91in LTF-assisted cleanup, in mouse ICH model. Our goal is to develop new therapeutic targets for ICH, based on the ability of IL-27-modified PMNs to release more LTF to promote MΦ-mediated cleanup of hematoma. This approach should not only limit PMNs-mediated CNS damage, but also allow PMNs to act in a beneficial fashion.