Background & Aims

The innate immune system responds not only to bacterial signals, but also to non-infectious danger-associated molecular patterns that activate the NLRP3 inflammasome complex after tissue injury. Immune functions vary over the course of the day, but it is not clear whether these changes affect the activity of the NLRP3 inflammasome. We investigated whether the core clock component nuclear receptor subfamily 1 group D member 1 (NR1D1, also called Rev-erbα) regulates expression, activity of the NLRP3 inflammasome, and its signaling pathway.

Methods

We collected naïve peritoneal macrophages and plasma, at multiple times of day, from Nr1d1^–/– mice and their Nr1d1^+/+ littermates (controls) and analyzed expression NLRP3, interleukin 1β (IL1B, in plasma), and IL18 (in plasma). We also collected bone marrow–derived primary macrophages from these mice. Levels of NR1D1 were knocked down with small hairpin RNAs in human primary macrophages. Bone marrow–derived primary macrophages from mice and human primary macrophages were incubated with lipopolysaccharide (LPS) to induce expression of NLRP3, IL1B, and IL18; cells were incubated with LPS and adenosine triphosphate to activate the NLRP3 complex. We analyzed caspase 1 activity and cytokine secretion. NR1D1 was activated in primary mouse and human macrophages by incubation with SR9009; some of the cells were also incubated with an NLRP3 inhibitor or inhibitors of caspase 1. Nr1d1^–/– mice and control mice were given intraperitoneal injections of LPS to induce peritoneal inflammation; plasma samples were isolated and levels of cytokines were measured. Nr1d1^–/– mice, control mice, and control mice given injections of SR9009 were given LPS and D-galactosamine to induce fulminant hepatitis and MCC950 to specifically inhibit NLRP3; plasma was collected to measure cytokines and a marker of liver failure (alanine aminotransferase); liver tissues were collected and analyzed by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry.

Results

In peritoneal macrophages, expression of NLRP3 and activation of its complex varied with time of day (circadian rhythm)—this regulation required NR1D1. Primary macrophages from Nr1d1^–/– mice and human macrophages with knockdown of NR1D1 had altered expression patterns of NLRP3, compared to macrophages that expressed NR1D1, and altered patterns of IL1B and 1L18 production. Mice with disruption of Nr1d1 developed more-severe acute peritoneal inflammation and fulminant hepatitis than control mice. Incubation of macrophage with the NR1D1 activator SR9009 reduced expression of NLRP3 and secretion of cytokines. Mice given SR9009 developed less-severe liver failure and had longer survival times than mice given saline (control).

Conclusions

In studies of Nr1d1^–/– mice and human macrophages with pharmacologic activation of NR1D1, we found NR1D1 to regulate the timing of NLRP3 expression and production of inflammatory cytokines by macrophages. Activation of NR1D1 reduced the severity of peritoneal inflammation and fulminant hepatitis in mice.

中文翻译：

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核受体亚家族1 D组成员1调节NLRP3炎性小体的昼夜活动以降低小鼠恶性肝炎的严重程度

背景与目标

先天免疫系统不仅对细菌信号有反应，而且对与组织感染后激活NLRP3炎性体复合物的非传染性危险相关分子模式也有反应。免疫功能在一天当中会有所不同，但尚不清楚这些变化是否会影响NLRP3炎性小体的活性。我们调查了核心时钟组件核受体亚家族1 D组成员1（NR1D1，也称为Rev-erbα）是否调节NLRP3炎性小体的表达，活性及其信号传导途径。

方法

我们每天多次从Nr1d1 ^{– / –}小鼠及其Nr1d1 ^{+ / +中}收集幼稚的腹膜巨噬细胞和血浆小卵（对照）并分析了表达NLRP3，白介素1β（血浆中的IL1B）和IL18（血浆中的）。我们还从这些小鼠中收集了骨髓来源的原代巨噬细胞。NR1D1的水平被人类初级巨噬细胞中的小发夹RNA击倒。将来自小鼠和人类原代巨噬细胞的骨髓原代巨噬细胞与脂多糖（LPS）孵育，以诱导NLRP3，IL1B和IL18的表达。将细胞与LPS和三磷酸腺苷一起孵育以激活NLRP3复合物。我们分析了半胱天冬酶1的活性和细胞因子的分泌。通过与SR9009孵育，在原代小鼠和人类巨噬细胞中激活了NR1D1；一些细胞还与一种或多种NLRP3抑制剂或caspase 1抑制剂一起孵育。Nr1d1 ^{– / –}给小鼠和对照组小鼠腹膜内注射LPS以诱导腹膜炎症。分离血浆样品并测量细胞因子水平。注射SR9009的Nr1d1 ^{– / –}小鼠，对照小鼠和对照小鼠分别接受LPS和D-半乳糖胺诱导暴发性肝炎，MCC950特异性抑制NLRP3。收集血浆以测量细胞因子和肝衰竭的标志物（丙氨酸氨基转移酶）；收集肝脏组织，并通过定量聚合酶链反应，免疫组织化学和流式细胞术进行分析。

结果

在腹膜巨噬细胞中，NLRP3的表达及其复合物的激活随一天中的时间（昼夜节律）而变化-此调节需要NR1D1。与表达NR1D1的巨噬细胞相比，来自Nr1d1 ^-/-小鼠的初级巨噬细胞和具有NR1D1抑制作用的人类巨噬细胞已改变了NLRP3的表达模式，并改变了IL1B和1L18产生的模式。与对照小鼠相比，Nr1d1破坏的小鼠出现了更严重的急性腹膜炎症和暴发性肝炎。用NR1D1激活物SR9009孵育巨噬细胞可降低NLRP3的表达和细胞因子的分泌。与给予生理盐水的小鼠（对照组）相比，给予SR9009的小鼠出现较少的严重肝衰竭，并且存活时间更长。

结论

在对Nr1d1 ^{– / –}小鼠和具有NR1D1药理活性的人类巨噬细胞的研究中，我们发现NR1D1调节巨噬细胞NLRP3表达的时间和炎性细胞因子的产生。NR1D1的激活降低了小鼠腹膜炎症和暴发性肝炎的严重程度。