Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8

Abstract

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBPb, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTFmarked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/ POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor kB (NF-kB) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation.

SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion

“…After incubation, neutrophils were pelleted by centrifugation and then total RNA was extracted by RNeasy mini kit (QIAGEN, Venlo, The Netherlands) [ 19 ]. To completely remove any possible contaminating DNA, an on-column DNase digestion with the RNase-free DNase set (QIAGEN) was performed during total RNA isolation [ 19 ].…”

“…After incubation, neutrophils were pelleted by centrifugation and then total RNA was extracted by RNeasy mini kit (QIAGEN, Venlo, The Netherlands) [ 19 ]. To completely remove any possible contaminating DNA, an on-column DNase digestion with the RNase-free DNase set (QIAGEN) was performed during total RNA isolation [ 19 ]. Purified RNA was reverse-transcribed into cDNA using PrimeScript™ RT Reagent kit (Takara Bio, Kusatsu, Japan), while qPCR was carried out using TB green ® premix Ex Taq ™ (Takara Bio).…”

“…Libraries for transcriptome analysis were prepared using the Smart-seq2 protocol [ 24 ], as already described [ 19 ]. Briefly, 2 ng of total RNA were copied into first strand cDNA by reverse transcription and template-switching oligo (dT) primers and an LNA-containing template-switching oligo (TSO).…”

“…Computational analysis of transcriptome datasets generated by Smart-seq2 has been performed using the bioinformatic pipeline utilized in a previous study [ 19 ], with minor modifications. Briefly, binary base call (BCL) files generated by the Illumina sequencer were converted into FASTQ files using bcl2fastq v2.20 software.…”

“…However, recent studies indicate that human neutrophils may also react to viral infections [ 11 ], as they not only express numerous PRRs known to recognize PAMPs of viral origin, including TLR8, MDA5, RIGI, and IFI16 [ 12 , 13 , 14 ], but also because they promptly respond to type I interferons [ 15 , 16 , 17 ]. Moreover, we uncovered that small molecules mimicking the action of ssRNA, acting via TLR8 (i.e., R848, CL075 or VTX-2337), dramatically reprogram human neutrophils at the transcriptomic level through mechanisms involving chromatin remodeling and activation of transcription factors such as NF-kB, AP-1, and OCT2 [ 18 , 19 ]. As mentioned, natural ligands of TLR8 typically consist of GU-rich ssRNA [ 20 ], which is noticeable since we recently demonstrated that SARS-CoV-2 genome contains several GU-rich sequences [ 21 ].…”