Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function.

IL-10 is a potent anti-inflammatory cytokine that is crucial for down-regulating pro-inflammatory genes, which are induced by Toll-like receptor (TLR) signaling. In this study, we have examined whether modulation of microRNAs plays a role in the inhibitory effect of IL-10 on TLR4 signaling. Analyzing microRNAs known to be induced by TLR4, we found that IL-10 could inhibit the expression of miR-155 in response to lipopolysaccharide but had no effect on miR-21 or miR-146a. IL-10 inhibited miR-155 transcription from the BIC gene in a STAT3-dependent manner. This inhibitory effect of IL-10 on miR-155 led to an increase in the expression of the miR-155 target, SHIP1. This is the first example of IL-10 playing a role in microRNA function and suggests that through its inhibitory effect on miR-155, IL-10 has the ability to promote anti-inflammatory gene expression.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1β in vitro. Accordingly, HIF-1α and IL-1β are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2^−/− mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

Fine-tuning of inflammatory responses by microRNAs (miRNAs) is complex, as they can both enhance and repress expression of pro-inflammatory mediators. In this study, we investigate inflammatory responses following global miRNA depletion, to better define the overall contribution of miRNAs to inflammation. We demonstrate that miRNAs positively regulate Toll-like receptor signaling using inducible Dicer1 deletion and global miRNA depletion. We establish an important contribution of miR-19b in this effect, which potentiates nuclear factor-κB (NF-κB) activity in human and mouse cells. Positive regulation of NF-κB signaling by miR-19b involves the coordinated suppression of a regulon of negative regulators of NF-κB signaling (including A20/Tnfaip3, Rnf11, Fbxl11/Kdm2a and Zbtb16). Transfection of miR-19b mimics exacerbated the inflammatory activation of rheumatoid arthritis primary fibroblast-like synoviocytes, demonstrating its physiological importance in the pathology of this disease. This study constitutes, to our knowledge, the first description of a miR-19 regulon that controls NF-κB signaling, and suggests that targeting this miRNA and linked family members could regulate the activity of NF-κB signaling in inflammation.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4^−/− and Myd88^−/−, but not TRIF^−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system. Clostridium difficile is the leading cause of antibiotic-associated diarrhoea among hospital patients and in severe cases can cause pseudomembranous colitis and even death. There is currently limited information regarding how this pathogen is recognised by the immune system and the key mechanisms necessary for clearance of the pathogen. C. difficile expresses a paracrystalline surface protein array, termed an S-layer, composed of surface layer proteins (SLPs). Their location on the outer surface of the bacteria suggests that they may be involved in immune recognition of the pathogen. In this study we demonstrate that these SLPs are recognised by toll-like receptor 4 (TLR4). Activation of TLR4 by SLPs resulted in maturation of dendritic cells and subsequent activation of T helper cell responses which are known to be important in clearance of pathogens. Furthermore, using a murine model of C. difficile infection we show that mice display increased severity of infection in the absence of TLR4. This is the first study to demonstrate a role for TLR4 in infection associated with C. difficile and suggests an important role for SLPs in the generation of the immune response necessary for clearance of this bacterium.