Regulation of cytokines by small RNAs during skin inflammation

Abstract

Intercellular signaling by cytokines is a vital feature of the innate immune system. In skin, an inflammatory response is mediated by cytokines and an entwined network of cellular communication between T-cells and epidermal keratinocytes. Dysregulated cytokine production, orchestrated by activated T-cells homing to the skin, is believed to be the main cause of psoriasis, a common inflammatory skin disorder. Cytokines are heavily regulated at the transcriptional level, but emerging evidence suggests that regulatory mechanisms that operate after transcription play a key role in balancing the production of cytokines. Herein, we review the nature of cytokine signaling in psoriasis with particular emphasis on regulation by mRNA destabilizing elements and the potential targeting of cytokine-encoding mRNAs by miRNAs. The proposed linkage between mRNA decay mediated by AU-rich elements and miRNA association is described and discussed as a possible general feature of cytokine regulation in skin. Moreover, we describe the latest attempts to therapeutically target cytokines at the RNA level in psoriasis by exploiting the cellular RNA interference machinery. The applicability of cytokine-encoding mRNAs as future clinical drug targets is evaluated, and advances and obstacles related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described.

Figure 1

Schematic representation of the cytokine network driving development of psoriasis. Cytokines mediate intercellular communication between skin-infiltrating activated immune cells and epidermal keratinocytes. Resident T-cells and antigen-presenting cells are activated upon stimulation by stress or infection (indicated by lightning symbol), leading to production of cytokines, as indicated by horizontal arrow labeled with boxed cytokines. Naïve CD4⁺ T-cells undergo differentiation upon exposure to different cytokine milieus, as indicated by the listed cytokines driving the differentiation into one of five T-helper (T_h) cell lineages, T_h1, T_h2, T_h17, T_h22, or T_reg. A subset of cytokines (listing is not intended to be absolute), produced by each of the T_h lineages and macrophages, orchestrates a multi-faceted stimulation of epidermal keratinocytes. This leads to production of an additional set of cytokines triggering epidermal remodeling through altered keratinocyte growth and differentiation as well as angiogenesis. Numbers in blue ovals above cytokine names indicate the number of AUUUA pentamer sequences (potential ARE sequences) that are located in the 3'UTR of each cytokine mRNA (or for heterodimeric cytokines the number of AUUUA sequences in mRNA species encoding each of the two subunits). Also shown is the development of pre-psoriatic, non-lesional skin (left insert) to a psoriatic plaque (right insert) driven by activated immune cells and cytokine-activated keratinocytes. The lesional skin section shows thickened epidermis with elongated retes and an abnormal outer skin layer (stratum corneum) with formation of epidermal scales. Skin-infiltrating immune cells (including activated T_h cells, dendritic cells, and macrophages) are visible in the dermis.

Figure 2

Overview of pentameric ARE motifs and potential miRNA target sequences in psoriasis-relevant cytokines. The pentameric sequence motif, AUUUA, has been shown to be a discriminating motif for AREs which generally consist of clustered and/or overlapping pentamer repeats. miR-16 and miR-369-3p target sequence motifs in the ARE of TNFα, which are also present in multiple psoriasis-relevant cytokines as indicated by green and red bars, respectively. Target sites are shown by blue and purple bars, respectively, for miRNAs that are predicted by the PicTar (pictar.mdc-berlin.de) and TargetScan (targetscan.org) algorithms to target psoriasis-relevant cytokines.

Figure 3

Overview of the p38-MK2-TTP pathway. Upon normal cellular conditions the p38 MAPK pathway is not activated, which leaves TTP able to bind the ARE in the 3'UTR of ARE-containing mRNAs and recruit mRNA decay enzymes. Upon a stress stimulus, the p38 MAPK pathway is activated, initiating a phosphorylation cascade which result in the phosphorylation of TTP and a consequent 14-3-3-mediated sequestration of TTP from the ARE.

Figure 4

Overview of RNA interference pathways. The RNA interference pathway is active in endogenous regulation of gene expression, in which pri-miRNAs are transcribed from the genome and processed in several steps ultimately leading to a mature miRNA which is loaded into the effector protein complex called RISC from where the guide strand of the miRNA guides RISC to mRNAs by sequence-specific target recognition. Depending on the degree of sequence similarity, RISC facilitates either translational suppression or mRNA degradation. The pathway can be exploited for sequence-specific down-regulation of a gene, by transfection of synthetic siRNAs or intracellular expression of shRNAs which are both efficiently processed by the RNAi machinery and enter the RNAi pathway.

Figure 5

TNFα regulation by miR-16 and miR-369-3p. Sequence alignment of part of the TNFα 3'UTR and miR-16 or miR-369-3p. The core, non-seed, recognition sequence is shown in bold. (A) TTP is associated with RISC and in a mutually dependent mechanism, TTP and miR-16 promote TNFα mRNA decay through binding to the TNFα ARE. The core non-seed miR-16 recognition motif is shown in bold. (B) miR-369-3p promotes canonical translational suppression or mRNA degradation during normal cellular conditions, but upon cell cycle arrest, FXR1 is associated with RISC to increase translational activity. The core miR-369-3p seed match is shown in bold.