Colonic cytokines and chemokines in DNBS colitis

Abdominal pain in irritable bowel syndrome and inflammatory bowel disease is thought to be driven by processes that sensitize sensory nerves innervating the gut. How sensory nerves become sensitized is not clear, but their terminals in the gut are surrounded by enteric glia. Here, we tested the hypothesis that intercellular enteric glia-to-nociceptor signaling contributes to visceral hypersensitivity during inflammation. In vivo and in vitro models of acute inflammation were used in combination with protein and RNA labeling, and cellular assays of activity and mediator release. Mechanisms of interaction between glia and nociceptors were studied using Trpv1Cre;GCaMP5gtdT;GFAP-hM3Dq mice, in which glial activity is controlled by chemogenetics while simultaneously recording nociceptor activity using calcium imaging. Mice lacking glial connexin-43 were used in combination with visceromotor reflex recordings to disrupt glial intercellular signaling and study its impact on visceral sensitivity. Acute colitis induces a transient increase in proinflammatory cytokines including IL-1β, which is produced in part by glia and facilitates glial connexin-43 function. Provoking glial activity under these conditions changes the normal benign influence of glia on nociceptors to one where glia have a sensitizing effect on gut-innervating nociceptors. The mechanisms responsible for glial-driven visceral hypersensitivity involve an upregulation of glial COX-2 and an increase in stimulated glial PGE2 release which acts on nociceptor EP4 receptors. In vivo recordings show that colonic IL-1β shifts normal innocuous stimuli toward a noxious range through mechanisms that require glial connexin-43. Enteric glia sensitize gut nociceptors during inflammation. Cell-specific therapies targeting the glial mechanisms identified here could benefit treatments for visceral pain.

Methods
Whole colon samples were collected from control and DNBS treated mice at 6 hours, 48 hours, and 3 weeks. Tissue was processed for multiplex immunoassay as previously described (50). Colonic samples were homogenized in a Tris-buffered saline Tween buffer solution to extract protein. After, homogenates were centrifuged at 10,000g for 10 minutes at 4°C. Cytokine/chemokine levels were evaluated using bead-based multiplex immunoassays (Eve Technologies, Calgary, AB, Canada).

肠易激综合征（irritable bowel syndrome）与炎症性肠病（inflammatory bowel disease）患者的腹痛，被认为由支配肠道的感觉神经致敏相关通路所驱动。目前尚不清楚感觉神经如何发生致敏，但肠道内的感觉神经末梢被肠胶质细胞（enteric glia）所包裹。本研究验证了这一假说：细胞间的肠胶质细胞-伤害感受器信号传导，可在炎症过程中介导内脏高敏感性。研究采用急性炎症的体内、体外模型，结合蛋白质与RNA标记技术，以及神经活动与介质释放的细胞检测方法。我们使用Trpv1Cre;GCaMP5gtdT;GFAP-hM3Dq小鼠开展胶质细胞与伤害感受器的相互作用机制研究：该小鼠模型可通过化学遗传学（chemogenetics）调控胶质细胞活性，同时利用钙成像（calcium imaging）记录伤害感受器的活动。此外，我们使用缺失连接蛋白43（connexin-43）的小鼠，结合内脏运动反射记录，以破坏胶质细胞间的信号传导，并探究其对内脏敏感性的影响。

急性结肠炎可诱导包括白细胞介素1β（IL-1β）在内的促炎细胞因子一过性升高，该细胞因子部分由胶质细胞产生，并可增强胶质细胞连接蛋白43的功能。在此条件下激活胶质细胞，会将胶质细胞对伤害感受器的正常良性调控作用，转变为对肠道支配型伤害感受器的致敏作用。胶质细胞介导的内脏高敏感性机制，涉及胶质细胞环氧合酶2（COX-2）的上调，以及受刺激的胶质细胞前列腺素E2（PGE2）释放增加——后者可作用于伤害感受器的EP4受体。体内记录结果显示，结肠内的IL-1β可通过依赖胶质细胞连接蛋白43的通路，将正常的无害刺激转化为伤害性刺激。本研究证实，炎症过程中肠胶质细胞会致敏肠道伤害感受器。针对本研究中明确的胶质细胞机制开发细胞特异性疗法，有望为内脏痛的治疗提供新方向。

实验方法
分别于造模后6小时、48小时及3周，从对照组与二硝基苯磺酸（DNBS）处理组小鼠体内采集全结肠样本。按照此前报道的方法（50）进行多重免疫检测样本处理：将结肠样本在Tris缓冲盐水吐温缓冲液中匀浆以提取蛋白质，随后将匀浆在4℃下以10000g离心10分钟。采用基于微球的多重免疫分析法（Eve Technologies，加拿大阿尔伯塔省卡尔加里市）检测细胞因子/趋化因子水平。