Colony-stimulating factor 2 (CSF2) as a gut microbiome dependent immune factor that alters molecular and behavioral responses to cocaine

Cocaine use disorder is a condition that leads to tremendous morbidity and mortality for which there are currently no FDA-approved pharmacotherapies. Previous research has demonstrated an important role for the resident population of bacteria of the large intestine, collectively dubbed the gut microbiome, in modulating brain and behavior in models of cocaine and other substance use disorders. Importantly, previous work has repeatedly shown that depletion of the gut microbiome leads to increased cocaine taking and seeking behaviors in multiple models. While the precise mechanism of these gut-brain signaling pathways in models of cocaine use is not fully clear, and intriguing possibility is through gut microbiome influences on innate immune system function. In this manuscript we identify the cytokine colony stimulating factor 2 (CSF2) as an immune factor that is increased by cocaine in a gut microbiome dependent manner. Peripherally injected CSF2 crosses the blood-brain barrier into the nucleus accumbens, a brain region central to behavioral responses to cocaine. Treatment with peripheral CSF2 reduces acute and sensitized locomotor responses to cocaine as well as reducing cocaine place preference at high doses. On a molecular level, we find that peripheral injections of CSF2 alter the transcriptional response to both acute and repeated cocaine in the nucleus accumbens. Finally, treatment of microbiome depleted mice with CSF2 reverses the behavioral effects of microbiome depletion on the conditioned place preference assay. Taken together, this work identifies an innate immune factor that represents a novel gut-brain signaling cascade in models of cocaine use and lays the foundations for further translational work targeting this pathway. Overall design: To define the contribution of CSF2 on the transcriptomic landscape of acute and repeated-cocaine treated mice, we administered exogenous CSF2 (or PBS control) in mice treated with cocaine (or saline control). With a 2x2 design, we transcriptionally profiled whole nucleus accumbens (NAc) tissue of n=5-6 samples (mice) per group. DIfferential gene expression and gene ontology was performed on the aforementioned RNAseq data.

可卡因使用障碍（Cocaine use disorder）是一种可引发极高发病率与死亡率的疾病，目前尚无经美国食品药品监督管理局（FDA）批准的药物疗法。既往研究证实，大肠常驻细菌群落（统称为肠道微生物组（gut microbiome））在可卡因及其他物质使用障碍模型中，对大脑功能与行为发挥着关键调控作用。

尤为重要的是，既往多项研究反复表明，肠道微生物组缺失会在多种模型中增强可卡因的摄取与寻求行为。尽管可卡因使用模型中此类肠-脑信号通路的具体机制尚未完全阐明，但一种颇具吸引力的可能性是，其通过肠道微生物组对先天免疫系统功能的调控得以实现。

本研究鉴定出细胞因子集落刺激因子2（colony stimulating factor 2, CSF2）是一种免疫因子，其表达会以肠道微生物组依赖的方式被可卡因上调。外周注射的CSF2可透过血脑屏障（blood-brain barrier）进入伏隔核（nucleus accumbens）——该脑区是可卡因行为应答的核心区域。外周给予CSF2可降低小鼠对可卡因的急性运动应答与敏化运动应答，且在高剂量下可减弱可卡因条件性位置偏好。

在分子层面，我们发现外周注射CSF2可改变伏隔核内急性与反复可卡因处理后的转录应答模式。最后，用CSF2处理肠道微生物组缺失的小鼠，可逆转微生物组缺失对条件性位置偏好实验中行为学表型的影响。

综上，本研究鉴定出一种先天免疫因子，其代表了可卡因使用模型中全新的肠-脑信号级联反应，为靶向该通路的后续转化研究奠定了基础。

整体实验设计：为明确CSF2对急性与反复可卡因处理小鼠的转录组图谱的调控作用，我们对可卡因（或生理盐水）处理的小鼠施加外源性CSF2（或磷酸盐缓冲液（PBS）对照）。采用2×2实验设计，对每组n=5-6只小鼠的全伏隔核（NAc）组织进行转录组分析。基于上述RNA测序（RNA-seq）数据，我们开展了差异基因表达分析与基因本体论（gene ontology）富集分析。