MicroRNA-223 suppresses inflammation by down regulating NF-kB activation in epithelial cells

MicroRNA-223, a principal myeloid-specific anti-inflammatory microRNA, is dysregulated in numerous inflammatory conditions and cancers. We found that miR-223 deficient zebrafish displayed augmented neutrophilic inflammation, which was due primarily to elevated activation of the canonical NF-B pathway. To our surprise, the NF-B over-activation was restricted to the basal epithelial cells, a squamous layer permeable to oxygen and chemicals. MiR-223 was expressed in epithelial cells and directly down-regulated multiple components in the NF-B signaling pathway in zebrafish and human. Both phagocytic and epithelial miR-223 suppressed neutrophil wound response and NF-B activation in the basal epithelial cells. Together, our data provide the mechanism of the multifaceted role of miR-223 and highlight the previously overlooked relevance of epithelial cells in miR-223 related diseases. The zebrafish experiment was conducted in accordance to the internationally accepted standards. The Animal Care and Use Protocol was approved by The Purdue Animal Care and Use Committee (PACUC), adhering to the Guidelines for Use of Zebrafish in the NIH Intramural Research Program (Protocol number: 1401001018). To generate a miR-223 deficient fish line, two individual single guide RNAs (sgRNAs) targeting pre-miR-223 were designed: sgRNA1 binding site: 5’-TTAGAGTATTTGACAGACTG-3’, and sgRNA2 binding site: 5’-TTTGTCAAATACCCCAAGAG-3’. The T7 promoter and the scaffold were added using overlapping PCR with the following primers listed from 5’ to 3’: sgRNA1-P1: GCGGCCTCTAATACGACTCACTATAGGGTTAGAGTATTTGACAGACTGGTTTTAGAGCTA, sgRNA1-P2: TGACAGACTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC, sgRNA1-P3: GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACT; sgRNA2-P1: GCGGCCTCTAATACGACTCACTATAGGGTTTGTCAAATACCCCAAGAGGTTTTAGAGCTA, sgRNA2-P2: ACCCCAAGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC, sgRNA2-P3: GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACT. sgRNAs were synthesized by in vitro transcription using MEGAshortscript T7 kit (Invitrogen) using the purified PCR products as template. To make cas9 mRNA, the plasmid pT3TS-nCas9n (Addgene Plasmid #46757)1 was linearized by XbaI and purified. Cas9 mRNA was generated using mMESSAGE mMACHINE T3 kit (Invitrogen). The mix of sgRNA1 (100 ng/ul), sgRNA2 (100 ng/ul) and cas9 mRNA (150 ng/ul) was injected into one-cell stage embryos. The embryos were raised to sexual maturity and the F1s were screened by sequencing the miR-223 locus. The homozygous mutant were obtained after several generations of backcross and incross. To generate transgenic zebrafish lines, plasmids containing the tol2 backbone was coinjected with transposase mRNA into embryos at one-cell stage as described2. The construct tol2-lyzC-Gal4-Vp16-cyrs-CFP was generated with Gateway Cloning (Thermo Fisher) using the tol2 kit to make Tg(lyzc: Gal4-Vp16; crys: CFP)pu8. Founders were screened with green eyes. To generate a miR-223 over expression line, Tg(lyzC: RFP-miR-223) pu9, a 317 bp genomic DNA sequence containing miR-223 was PCR amplified using the following primers listed from 5’ to 3’: forward CTGGCAGTACGGGCTCAGGAGGAAGAGGGAGGAGTAAAATTGAAT, reverse TAACAGCAGTTGGCTAATGAATGTTGTCATCCTCCACATTTTGCA, and cloned into the BbsI site in the intron of the vector3. GFP was replaced by RFP and then inserted into a Tol2 backbone with lyzC promoter. A transgenic line expressing the empty backbone, Tg(lyzC: RFP) pu10 was generated as the control. To generate miR223 sponge line, Tg(krt4: RFP-miR-223 sponge)pu12, a sequence containing six copies of bulged miR-223 binding sites was synthesized as a gBlocks Gene Fragment (Integrated DNA Technologies) and inserted downstream of RFP in a pME construct. The final construct was generated by Gateway Cloning. Founders were selected with red apical epithelial cells. A line with RFP Tg(krt4: RFP) pu13 was made as the control. The line Tg(krt4: GFP) pu11 was also generated for cell sorting. More than one founders were obtained for each line and their offspring display similar phenotypes. Embryos were collected at 0 hpw, 1 hpw, and 6 hpw. Total RNAs were extracted with Trizol (Invitrogen) and further cleaned up with RNeasy MinElute kit (Qiagen). The quality and integrity of RNAs were confirmed using Agiloent Nano RNA QC chip (Sequencing Center of Purdue University). Transcriptomic microarray analysis was conducted using the one-color hybridization strategy to compare gene expression profiles among the different time points with a zebrafish V3 array (Agilent Technologies). Following hybridization, arrays were washed and scanned on an Agilent Technologies SureScan Microarray Scanner. Array image data was extracted using Agilent Feature Extraction Software and data was uploaded onto GeneSpring for statistical analysis. Microarray analysis was performed following MIAME guidelines.