Interleukin-27-dependent transcriptome signatures regulation of the transcriptome during neonatal sepsis [ATAC-seq]. Interleukin-27-dependent transcriptome signatures regulation of the transcriptome during neonatal sepsis [ATAC-seq]

Human newborns exhibit increased vulnerability and risk of mortality from infection. Increased susceptibility to infection during the neonatal period reflects key differences in the innate and adaptive immune responses relative to those in adult cells. We have previously shown an increase in the pleiotropic immune suppressive cytokine, IL-27, in macrophages derived from human umbilical cord blood compared with those obtained from adult peripheral blood. Similar findings exist in the neonatal murine system, as well as elevated splenic transcripts and serum levels. Recently, we established a murine model of neonatal sepsis to explore the impact of early life IL-27 levels on the host response to infection. In this model, neonatal mice deficient in IL-27 signaling exhibit reduced mortality, increased weight gain, and better control of the bacterial burden with reduced systemic inflammation. These results suggest that there is a reprogramming of the host response in the presence of IL-27 signaling. To explore this hypothesis, we profiled the neonatal transcriptome of the spleen in the presence or absence of Escherichia coli-induced sepsis in wild-type (WT) and IL-27Rα-deficient mice. The spleen, a known site of infection, exhibits increased IL-27 expression and bacterial burdens in WT pups that were significantly lower in IL-27Rα KO pups during infection. We identified 549 genes that were differentially expressed in WT neonates in response to E. coli infection. The upregulated genes were associated with inflammation, cytokine signaling, and G protein coupled receptor ligand binding and signaling. The expression level of these genes generally failed to increase in IL-27Rα KO mice upon the E. coli infection. We observed similar changes in gene expression, aligned with changes in chromatin accessibility, for macrophages isolated from the spleens of control and infected neonates with or without IL-27Rα KO, supporting macrophages as an innate myeloid population contributing to the inflammatory profile in septic WT pups. Collectively, our findings highlight a less inflammatory environment in KO pups that is not expected given the anti-inflammatory/suppressive activity of IL-27. However, it is consistent with a lower bacterial burden. This suggests that improved bacterial clearance in the absence of IL-27 signaling does not require a heightened inflammatory state, and instead, there is a direct relationship between IL-27 signaling and bacterial killing. An improved response to infection that is not reliant upon heightened levels of inflammation offers new promise to the potential of antagonizing IL-27 as a host-directed therapy for neonates without concern for driving inflammatory responses that are pathological to host tissues. Overall design: Four-day old C57BL/6 pups were sorted into four groups based on IL27RA status (KO or wild type) and based on infection status with Escherichia coli strain O1:K1:H7 infection (with or without infection; 24 hours). For each of the four group, PolyA RNA-seq were performed for spleen tissue and for myeloid cells enriched from the spleen. Additional ATAC-seq assay to measure chromatin accessibility was performed for myeloid cells from each group.

人类新生儿对感染的易感性及死亡风险均显著升高。新生儿期对感染的易感性增加，反映出其固有免疫与适应性免疫应答相较于成人细胞存在关键差异。我们既往研究发现，相较于成人外周血来源的巨噬细胞，人脐带血来源的巨噬细胞中多效性免疫抑制细胞因子白细胞介素-27（IL-27）的表达水平显著升高。该现象在新生小鼠模型中亦得到验证，同时还观察到脾脏转录本与血清IL-27水平升高。
近期，我们构建了新生小鼠脓毒症模型，以探究生命早期IL-27水平对宿主抗感染应答的影响。在该模型中，IL-27信号通路缺陷的新生小鼠死亡率降低、体重增长更快，且可更好地控制细菌负荷，同时伴随全身性炎症反应减轻。上述结果提示，IL-27信号通路的存在会重编程宿主抗感染应答。
为验证这一假说，我们对野生型（wild-type, WT）及IL-27Rα缺陷型新生小鼠在大肠杆菌（Escherichia coli, E. coli）诱导脓毒症与否的状态下的脾脏转录组进行了测序分析。脾脏作为公认的感染靶器官，在受感染的野生型幼鼠中IL-27表达与细菌负荷均升高，而IL-27Rα敲除（knockout, KO）幼鼠的上述指标则显著降低。
我们共鉴定出549个在野生型新生小鼠中响应大肠杆菌感染的差异表达基因。上调基因主要富集于炎症反应、细胞因子信号通路以及G蛋白偶联受体配体结合与信号转导相关功能。在IL-27Rα缺陷型小鼠中，上述基因的表达水平在大肠杆菌感染后并未出现显著上调。
我们在分离自对照与受感染新生小鼠脾脏的巨噬细胞中观察到了类似的基因表达变化，且该变化与染色质开放程度的改变相一致；同时在IL-27Rα敲除小鼠中该变化未出现，这提示巨噬细胞作为固有髓系细胞群体，参与了脓毒症野生型幼鼠的炎症反应进程。
综上，我们的研究结果显示，IL-27Rα敲除幼鼠的炎症环境更弱——这与IL-27已知的抗炎/免疫抑制活性看似矛盾，但却与更低的细菌负荷相符。这表明在IL-27信号通路缺失的情况下，细菌清除能力的提升并不依赖于炎症反应的增强，反而提示IL-27信号通路与细菌杀伤能力存在直接负相关关系。这种不依赖于高强度炎症的抗感染应答优化策略，为以IL-27为靶点的宿主导向治疗带来了新希望：该疗法可用于新生儿感染治疗，且无需担心诱发损伤宿主组织的病理性炎症反应。
总体实验设计：将4日龄C57BL/6幼鼠按IL27RA基因型（敲除型与野生型）及大肠杆菌O1:K1:H7感染状态（感染与未感染，感染后24小时取样）分为4组。对4组小鼠的脾脏组织以及脾脏富集的髓系细胞进行PolyA RNA测序（PolyA RNA-seq）；同时对各组髓系细胞进行转座酶可及性测序（ATAC-seq）以检测染色质开放程度。