Date Published: April 23, 2019
Publisher: Public Library of Science
Author(s): David M. Ojcius, Ardavan Jafari, Laxmi Yeruva, Christian W. Schindler, Ali A. Abdul-Sater, Shibo Jiang.
Inflammation plays a critical role in initiation of adaptive immunity, pathogen clearance and tissue repair. Interleukin (IL)-1β is a potent pro-inflammatory cytokine and therefore its production is tightly regulated: its secretion requires the assembly of a macromolecular protein complex, termed the inflammasome. Aberrant activation of the inflammasome has been linked to debilitating human diseases including chronic inflammatory and autoimmune diseases. Thus, there is a great interest in understanding how inflammasomes are regulated. Here we show that Dicer, an enzyme necessary for the production of mature micro-RNAs (miRNAs), is required for optimal activation of NLRP3 inflammasomes in bone marrow macrophages. Our data indicate that miRNAs may play an important role in promoting inflammasome activation.
The innate immune system employs germ line encoded pattern recognition receptors (PRRs) that recognize “molecular patterns” from microbes, environmental stressors and damaged tissue. Once activated, these receptors initiate robust inflammatory responses that include the secretion of potent inflammatory cytokines such as Interleukin (IL)-1β, IL-6, TNF-α and Interferons (IFNs) [1–3]. IL-1β and IL-18 are among the most potent cytokines produced by human and murine innate immune systems, and they are released primarily as a part of the immune response to invading pathogens. However, excessive release of these cytokines, particularly IL-1β, is associated with autoinflammatory and autoimmune disorders [2–4]. Therefore, its secretion is tightly regulated, and typically requires two independent signals to be secreted. As with other cytokines, the first signal entails transcription of the IL-1β gene, but the gene product, pro-IL1β is not active. The second signal entails activation of the inflammasome, a multimeric protein complex typically composed of an NLR protein, the adapter protein ASC and the inactive zymogen caspase-1. Following inflammasome assembly, caspase-1 is activated and directs the cleavage of pro-IL1β into active and secreted IL-1β.
Given the significance of inflammasome regulation in controlling inflammation and associated human diseases, we explored whether microRNAs under the control of Dicer play a role in NLRP3 inflammasome activation. Bone marrow macrophages (BMMs) were prepared from Dicerfl/fl x ERT-Cre+ (germ line deletion of Dicer is embryonic lethal) and littermate control Dicerfl/fl x ERT-Cre- mice. Addition of tamoxifen led to the efficient deletion of Dicer in the Cre+ but not Cre- cells as evident by the deletion of the floxed allele (Fig 1A; [29, 32]). As expected, stimulation of WT control, LPS-pretreated (Signal 1) Dicerfl/fl x ERT-Cre- macrophages with two well-characterized NLRP3 activators, ATP and nigericin, led to a robust increase in the secretion of processed IL-1β p17 (Fig 1B and 1C) and active caspase-1 p10 (Fig 2) into culture supernatants. Stimulation with poly(dA:dT), an AIM2 inducer, yielded an analogous increase in secreted IL-1β p17 and caspase-1 p10 (Figs 1B, 1C and 2). In stark contrast, there was a marked reduction in the secretion of both IL-1β and caspase-1 in ATP- and nigericin-stimulated Dicer-deficient macrophages (i.e., from Tamoxifen treated Dicerfl/fl x ERT-Cre+ mice) (Figs 1B, 1C and 2). Remarkably, the response to poly(dA:dT) was not affected by the loss of Dicer. These results demonstrate that in the absence of Dicer, NLRP3 inflammasome but not AIM2 inflammasome activation is significantly impaired. Importantly, the ability of poly(dA:dT) to activate the AIM2 inflammasome in Dicer deficient macrophages provided strong evidence that ASC levels were not affected by Dicer deletion.
In this study, we explored whether miRNAs may regulate inflammasome activity. Using Dicer-deficient murine macrophages, we demonstrated, in the absence of miRNAs, a striking reduction in NLRP3 inflammasome activity, highlighting an overall positive role for miRNAs in this response.