Role Of Histone Deacetylase 6 (HDAC6) In The Modulation Of STAT Pathways and Immune Function Of APCs

Histone deacetylases (HDACs), originally discovered as histone modifiers are now proposed as important regulators of non-chromatin related processes, including the regulation of cellular pathways involved in the production of anti- and pro-inflammatory cytokines and the subsequent function of antigen-presenting cells (APCs). We have recently identified HDAC6 as a positive regulatory factor in the production of IL-10. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we present evidence of the important role of HDAC6 in the modulation of the JAK/STAT pathway through the IL-6 regulation.

We generated knockdown cell lines of HDAC6 (HDAC6KD) and non-target (NT) cells as a control in RAW264.7 murine macrophages using lentiviral shRNA. Two HADC6KD and two NT cell lines were treated with LPS or were left untreated and then analyzed by microarray. In HDAC6KD cells we found 1542 genes were down-regulated and 775 up-regulated in HDAC6KD cells. Their ontology distribution revealed significant changes in immune-related and apoptosis/cell cycle control genes. Importantly, we observed that most STAT3 and SP1 target genes were down regulated in HDAC6KD cells, suggesting the participation of HDAC6 in the regulation of these two transcription factors. Further analysis evidenced that the phosphorylation of STAT3 and the acetylation of Sp1 were diminished in HDAC6KD cells when compared against control cells. Chromatin immuneprecipitacion (CHIP) assays indicate that this particular effect of abrogation of HDAC6 involved histone modifications at the IL-6 promoter level, and more importantly, the recruitment of STAT3 and Sp1 to the IL-6 promoter was abrogated. Then, we analyzed the relevance of these findings by studying the tolerogenic JAK/STAT signaling pathway, which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. Our observations included a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in HDAC6KD cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6.

Our final results demonstrate a critical role of HDAC6 in the modulation of IL-6 and the potential role of HDAC6 in the regulation of the JAK/STAT3 pathway. In addition HDAC6 is a regulator of SP1 and STAT3 target genes. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation, and positioning HDAC6 as a potential therapeutic target.