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Comparative Study
. 2018 Feb 23;293(8):3003-3012.
doi: 10.1074/jbc.M117.809780. Epub 2017 Dec 26.

MicroRNA-146 and cell trauma down-regulate expression of the psoriasis-associated atypical chemokine receptor ACKR2

Kave Shams  1 , Mariola Kurowska-Stolarska  2 , Fabian Schütte  3 , A David Burden  4 , Clive S McKimmie  5 , Gerard J Graham  6
Affiliations
Comparative Study

MicroRNA-146 and cell trauma down-regulate expression of the psoriasis-associated atypical chemokine receptor ACKR2

Kave Shams et al. J Biol Chem. .

Abstract

Chemokines are the principal regulators of leukocyte migration and are essential for initiation and maintenance of inflammation. Atypical chemokine receptor 2 (ACKR2) binds and scavenges proinflammatory CC-chemokines, regulates cutaneous T-cell positioning, and limits the spread of inflammation in vivo Altered ACKR2 function has been implicated in several inflammatory disorders, including psoriasis, a common and debilitating T-cell-driven disorder characterized by thick erythematous skin plaques. ACKR2 expression is abnormal in psoriatic skin, with decreased expression correlating with recruitment of T-cells into the epidermis and increased inflammation. However, the molecular mechanisms that govern ACKR2 expression are not known. Here, we identified specific psoriasis-associated microRNAs (miRs) that bind ACKR2, inhibit its expression, and are active in primary cultures of human cutaneous cells. Using both in silico and in vitro approaches, we show that miR-146b and miR-10b directly bind the ACKR2 3'-UTR and reduce expression of ACKR2 transcripts and protein in keratinocytes and lymphatic endothelial cells, respectively. Moreover, we demonstrate that ACKR2 expression is further down-regulated upon cell trauma, an important trigger for the development of new plaques in many psoriasis patients (the Koebner phenomenon). We found that tensile cell stress leads to rapid ACKR2 down-regulation and concurrent miR-146b up-regulation. Together, we provide, for the first time, evidence for epigenetic regulation of an atypical chemokine receptor. We propose a mechanism by which cell trauma and miRs coordinately exacerbate inflammation via down-regulation of ACKR2 expression and provide a putative mechanistic explanation for the Koebner phenomenon in psoriasis.

Keywords: chemokine; immunology; inflammation; microRNA (miRNA); psoriasis.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
In silico analyses identified several psoriasis-associated putative ACKR2 3′-UTR–interacting microRNAs. a, summary of microRNAs predicted to bind the ACKR2 3′-UTR with associated microRNA-specific data from previous psoriasis publications. The 10 microRNAs most likely to target ACKR2 are included in the diagram with relevant findings in two papers given in the right-hand column. miR-10, miR-146, and miR-203 are all differentially regulated in psoriasis (27, 37). In the figure, Footnote 1 is Ref. , and Footnote 2 is Ref. . b, three microRNAs (miR-10, miR-146, and miR-203) are predicted to bind the ACKR2 3′-UTR and are differentially expressed in psoriasis. The diagram shows microRNAs that are predicted to bind the ACKR2 3′-UTR in silico and that have been shown to be differentially expressed in psoriasis by microarray studies.
Figure 2.
Figure 2.
miR-146 and miR-10 transfection reduced AKCR2 transcripts in KCs and LECs, respectively. a, absolute quantification of IRAK/TRAF6 mRNA (previously validated miR-146b targets) in primary healthy human keratinocytes that were stimulated with 100 ng/ml human recombinant IFNγ prior to transfection. b, absolute quantification of ACKR2 transcripts following transfection of KCs with miR-146b (i), miR-10 (ii), and miR-203 (iii). c, absolute quantification of ACKR2 transcripts following transfection of KCs with miR-146a and miR-146b. d, absolute quantification of ACKR2 transcripts following transfection of LECs with miR-10 (i), miR-146b (ii), and miR-203 (iii). In all cases, cells were transfected for 24 h and left for a further 24 h prior to lysis and RNA extraction. microRNAs were added at 10 nm; control is scrambled microRNA. Shown are representative experiments conducted in cells from various cell donors. Significance was assessed using Student's t test (*, p < 0.05; **, p < 0.01) except for d where significance was assessed using one-way ANOVA with Tukey's post-test (*, p < 0.05; **, p < 0.01). Error bars represent S.E. NS, not significant; TBP, TATA-binding protein.
Figure 3.
Figure 3.
miR-146b and miR-10b functionally interact with the ACKR2 3′-UTR. a, miR-146b can be successfully transfected into HEK293 cells. Absolute quantification of IRAK1 mRNA was assessed by Q-PCR in HEK293 cells following transfection with miR-146b as compared with scrambled control. b, ACKR2 3′-UTR was cloned into the downstream UTR of a firefly luciferase reporter, and interaction with transfected miRs was determined (bioluminescence inversely proportional to microRNA 3′-UTR binding) and normalized to Renilla luciferase. Results are representative from two different luciferase-expressing HEK293 clones following transfection with miR-10b and miR-146b (singly and in combination) and scrambled miR control. Significance was assessed using one-way ANOVA (***, p < 0.005). Error bars represent S.E. TBP, TATA-binding protein.
Figure 4.
Figure 4.
Transfection of KCs with miR-146b reduced cytoplasmic ACKR2 protein distribution. a, representative bright-field image of KCs grown as a confluent monolayer. b, representative immunofluorescence of KCs grown as confluent monolayers and stained with isotype control antibody. c and d, representative immunofluorescence microscopy images of confluent monolayers of KCs 48 h after transfection with scrambled miR control (c) or miR-146b (d).
Figure 5.
Figure 5.
Transfection of LECs with miR-10 reduced ACKR2 protein expression throughout the cytoplasm. a, representative bright-field image of LECs grown as a confluent monolayer. b, representative immunofluorescence of LECs grown as confluent monolayers and stained with isotype control antibody. c and d, representative immunofluorescence microscopy images of confluent monolayers of LECs 48 h after transfection with scrambled miR control (c) or miR-10 (d). White arrows indicate asymmetric distribution of ACKR2.
Figure 6.
Figure 6.
Effect of tensile stress on ACKR2 expression by primary human KCs. a–c, absolute quantification of ACKR2 mRNA normalized to TATA-binding protein (TBP) in keratinocytes. a, healthy human primary KCs that remained static or were subjected to tensile stress (flexed) at 0.8 Hz for 12 h and then allowed to rest for 12 h prior to cell lysis and RNA extraction. b, healthy human primary KCs that remained static or were subjected to tensile stress (flexed) at 0.8 Hz for 12 h and then allowed to rest for 12 h prior to cell lysis and RNA extraction. KCs were pretreated with either 1) tissue culture supernatant from activated human T-cells (1:8 dilution in fresh medium), 2) 100 ng/ml recombinant human IFNγ, or 3) tissue culture supernatant from activated human T-cells plus neutralizing anti-IFNγ antibodies overnight prior to flexing at 0.8 Hz for 12 h. Black bars, non-flexed static controls; gray bars, flexed cells. Significance was assessed using one-way ANOVA. c, treatment of healthy primary LECs overnight with tissue culture supernatant from activated human T-cells (diluted 1:8 with fresh medium) prior to flexing. d and e, effect of tensile stress on miR-146 expression in inflamed keratinocytes. -Fold change in miR-146a and miR-146b expression was assessed by Q-PCR and normalized to scrambled miR–treated static KCs. KCs were treated for 16 h with either tissue culture supernatant from activated human T-cells (1:8 dilution in fresh medium) (d) or recombinant IFN-γ at 100 ng/ml (e) prior to flexing at 0.8 Hz for 12 h. Error bars represent S.E. NS, not significant.
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