In-vitro screening of immune response with FTIR spectroscopy in a miRNA murine knock out model

MicroRNAs are small ~22 nucleotide RNA sequences that are guided to the 3' untranslated region (UTR) of protein-coding target mRNA sequences. One particular microRNA, miR-155, plays a remarkable role in the immune system, where it is essential for mounting appropriate immune responses. However, its dysregulation has been identified in multiple inflammatory disorders such as Multiple Sclerosis (MS), arthritis, psoriasis and colitis. More specifically, miR-155 has been found to be elevated in the serum and brain lesions ofMS patients. Importantly, therapeutic inhibition of miR-155 can inhibit progression of the MS disease model. One of us has identified that macrophages are major contributor to miR-155 elevation in the MS disease model, whilst its inhibition specifically in macrophages can limit the disease. Here macrophages were isolated from the femur and tibia of wild-type (WT) mice and mice with a knock-out (KO) of the gene regulating miR-155 production, and were cultured in-vitro and stimulated with lipopolysaccharide (LPS) to simulate an immune response. Cells were then prepared for spectral analysis by FTIR imaging with a Perkin-Elmer Spotlight 400 imaging microscope. After pre-processing the dimensionality of spectra were reduced using principal components analysis and classified using a support vector machine algorithm, delivering a classification performance approaching F1~0.89. Spectral features differentiating WT and KO classes were observed across the fingerprint region with no single spectral marker being the sole source of differentiation of the downstream molecular events. This study exemplifies the challenge in spectral discrimination of the complexity of molecular events in ex-vivo models of immune dysregulation.