miRNA-146a, miRNA-203a, and miRNA-223 Modulate Inflammatory Response in LPS- Acute Lung Injury in Sickle Cell Transgenic Mice

Introduction: Mouse models have allowed a wider investigation of the inflammatory process in Sickle cell disease (SCD). Previous studies have demonstrated that neutrophil recruitment inresponse to LPS is upregulated in Berkeley sickle mice and the development of acute LPS-induced airway inflammation is associated with enhanced expression of cytokines, chemokines and MMPs expression in lungs. MicroRNAs (miRNAs) belong to a regulatory class of RNAs with great importance in several biological processes, including the development and fate of immune cells, and in both innate and adaptive immune responses, whereby strong evidence links miRNAs expression to signalling pathways and receptors. Some miRNAs are positive regulators of inflammation and others are involved in the negative-feedback regulation of inflammatory processes. A growing amount of evidence indicates that these molecules are involved in various lung diseases. However, the expression profile of miRNAs in lung injury in SCD have not been investigated. Since pulmonary complications are an important cause of morbidity and mortality in SCD, the aim of this study was to determine whether miRNAs implicated in LPS-mediated inflammation are involved in neutrophil recruitment and inflammation in lung injury in SCD using the sickle mice model.

Methods: Acute lung inflammation and injury was induced in wild- type (WT, C57BL/6, n=5) and Berkeley SCD mice (n=5) by intranasal administration of LPS (50 ml of 250 mg/ml). The vehicle group received a similar volume of sterile PBS. BAL was performed 4h after LPS challenge. qPCR analysis was used to examine gene expression and ELISA was used to determine protein production. For miRNAs expression analysis, extraction of lung RNA was performed using mirVana^TM miRNA Isolation kit and TaqMan^TM MicroRNA Reverse Transcription.The expression level of miRNAs was determined using the Taqman miRNA expression assay by qPCR with U6 snRNA as an endogenous control.

Results: Levels of miR-15a-5p, miR-16-5p, miR-26a-5p and miR-98-5p in lung tissue increased almost twofold at 4h after LPS challenge in SS compared with SS PBS group (2.9, 3.7; 2.3; 2.9-fold change, respectively). The expressions of miR-106a-5p, miR-146a-5p, miR-146b-5p, and miR-181a-5p were not altered (2.0; 1.2; 1.55; 1.4-fold change, respectively). However, the expressions of miR-203a-3p and miR-223-3p were significantly up-regulated compared to animals from the PBS group (4.3 and 8.8 fold change, respectively, p<0.05). These increases correlated with the increased expressions of Tnf- α, Il-1 β, Il-6, Cxcl1 and Mmp-9 mRNA in the lungs (LPS: 0.27±0.05; 0.24±0.04; 0.18±0.007; 0.44±0.09, 0.46±0.09 vs PBS: 0.02±0.001; 0.03±0.02; 0.007±0.18; 0.052±0.03; 0.07±0.03, respectively) and increased BAL levels of Tnf-α, Cxcl1 and Mmp-9 (4250±636.4; 2491±454.3; 187.5±16.7 vs PBS: 20.8±8.9; 186.6±35.4, 5.4±2.9, pg/ml, respectively). LPS challenge also led to a higher expression of miRNAs in the lungs of WT animals; however only expression levels of miR-146a-5p and miR-223-3p were significantly up-regulated (2.6 and 3.2-fold change, respectively, p<0.05).

Conclusion: We have shown that theLPS-induced inflammatory response in SCD transgenic mice is associated with increases in miR-203a-3p and miR-223-3p expression in the mouse lung; suggesting a role of the miRNAs in the regulation of inflammatory mediator production, since these augmentations are associated with the increases in cytokines and chemokines. Furthermore, miR-146a-5p, a negative regulator of inflammatory gene expression, is not upregulated in SS mice and could contribute to the exaggerated inflammation observed in these animals. These data indicate that alterations in the miRNA expression may be associated with the inflammatory state in SCD. Financial Support: FAPESP