Regulatory role of miroRNAs upon blistering agent-mediated innate immunity in skin keratinocytes

Skin is the body's predominant defense barrier against pathogens and environmental stimuli. Skin keratinocytes respond to toxic chemical exposure by secreting immunity factors that mobilize macrophages and T-cells. Upon the re-epithelialization process of chemically-damaged skin, cutaneous cell proliferation and inflammation cascades are activated. Increasing evidence has indicated that small, non-coding microRNAs (miR) regulate innate immunity, tissue repair, and inflammation. Currently, the signaling pathways that regulate the secretion of innate immunity soluble factors is unknown.

We used quantitative real-time PCR methodology (RT-QPCR) and a microRNA PCR array to amplify selected genes for the purpose of understanding the skin's protective response against nitrogen mustard (HN2)-mediated toxicity. The PAM212 mouse keratinocyte cell line was used as an in vitro skin model to investigate the interaction of innate immunity and skin inflammation. Cells were treated with 3µM of HN2 for 48h followed by RT-QPCR analysis. Interestingly, we found an overproduction of miR-183 and miR-21 in response to HN2 treatment in PAM212. These results correlate with an increase in pro-inflammatory signaling, IL-6 and TNF, and a decrease in the innate immunity receptors, TLR-2 and TLR-3. Furthermore, there was a decrease in anti-oxidant enzyme isotopes, GST-M1 and GST-T1, and the selected chemokine, CCL-27, post-HN2 exposure.

We conclude that HN2 may dysregulate cellular defense systems comprised of innate immunity receptors, antioxidant enzymes, T-cells, and chemokines. Furthermore, miR-21 and miR-183 may regulate these defense mechanisms in keratinocytes. Finally, TLR-2 and TLR-3 may serve as biomarkers to assess the extent of cellular damage and the success of therapeutic countermeasures under a clinical and public health surveillance paradigm.