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Keratinocytes Gene Expression Of Innate And Adaptative Proinflammatory Cytokines And Members Of The Toll-Like Receptor Pathways In Severe Burned
ALFREDO GRAGNANI, Jr., MD, PhD, Silvana Aparecida Alves Correa de Noronha, PhD, Sarita Mac Cornick, master, Larissa Elias Lanziani, student, Marcus Vinicius Boaretto Cezillo, student, Samuel Ribeiro de Noronha, PhD, Lydia Masako Ferreira, MD, PhD, Full Professor.
UNIVERSIDADE FEDERAL DE SÃO PAULO, São Paulo, Brazil.
Purpose: Burn injuries caused by various types of agents occur in average around one percent of world population annually. There are more than one million burns in the United States every year and about 5,000 of these injuries are fatal, making of burns the fourth leading cause of death from unintentional injuries in that country. No data about gene expression of skin cells of burned patients. The objective was to assess the expression profile of genes related to Innate and Adaptative Immune System (IAIS) and Toll-Like Receptors (TLR) in cultured primary human epidermal keratinocytes from patients with severe burns.
Methods: Isolation and culture of human epidermal keratinocytes were performed with normal marginal-devitalized-skin fragments removed by the surgeon from burn injuries at day 4 after injury (n=6). Control group was patient undergoing to aesthetic surgery (n=3). The collected tissue was immediately immersed in sterile DMEM medium supplemented with penicillin and streptomycin. The culture was initiated by enzymatic method using dispase and then collagenase. Cells in the third passage were homogenized in Trizol reagent (Invitrogen). Total RNA extracted was dried and dissolved in RNase-free water, purified with Qiagen RNeasy Mini kit and subjected to DNAse treatment. The quantity and quality of extracted RNA were assessed by spectrophotometry using Nanodrop. Samples from each group were processed to perform PCR Array plates (SA Biosciences) containing 84 genes related to IAIS or to TLR pathways. The experiments were made in technical triplicates.
Results: After the expression analysis of the 84 studied genes for each pathway, we observed, for IAAS pathway, that 63% of these genes were differentially expressed, among these 77% were down-regulated and 23% were up-regulated. Moreover, we could also observe for TLR pathways, that 21% of these genes were differentially expressed, and all of these genes (100%) were down-regulated.
Among significant differentially expressed genes, we highlight the following ones (fold change): IAAS pathway= IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (-73), CD86 (-41) and HLA-A (-35); TLR pathway= HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), and FOS (-16).
Conclusions: These results provide a new insight into the potential role played by keratinocytes to drive inflammatory responses in severe burned patients. These epithelial cells play a key role in triggering the formation of several innate and adaptative proinflammatory cytokines and in activating members of the toll-like receptor pathway that might be disrupted by extensive lesions of the skin.
Therefore, this study aims to contribute to understanding the molecular mechanisms underlying wound infection in severe burned patients and to provide new strategies that would restore the normal expression of these genes to enhance the inflammatory process and drive these patients to a better outcome.
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