Analysis of mechanosensor RNA expression during the activation of T cells and differentiation of macrophages in response to chemical and mechanical stimuli

T cells and macrophages are involved in many autoimmune pathologies. One suchautoimmune condition is alopecia areata, which results from immune responses targetinghair follicles and impairing the hair growth cycle. The immune infiltrates in alopecia areataare mainly lymphocytic, with CD8+ T cells being the main contributors to the disease inhumans and mouse models. This study looked at the mechanosensitivity of immune cells,which is a factor that can influence inflammatory responses.In this project, CD8+ T cell activation was studied at transcriptional level, looking into theregulation patterns of many genes encoding cytokines, effector proteins, mechanosensors,cell surface markers and transcription factors. This study demonstrated that the mRNAencoding the mechanosensor PIEZO1 was downregulated in activated Jurkat and CD8+ Tcells. Based on the regulation patterns of IL2RA, effector proteins such as GZMB, andreceptors such as IL7R and KLRG1, the PIEZO1 downregulation was occurring simultaneouslyto the CD8+ T cell activation and early effector differentiation.In addition, this study investigated T cell responses to 1000Hz nanovibrations. The resultsfrom these experiments were inconclusive and lacking reproducibility. In contrary, when thecells were activated chemically, the results were highly reproducible. Jurkat cell responseswere also assessed when cross-linking CD3 with PHA-L and applying nanovibrations at 24hours. Discrepancies in responses were observed, with IL-2 found upregulated anddownregulated in the supernatant of stimulated cells in two identical experiments, whereasthe IL2RA expression was not affected in any experiment. Furthermore, many of the assayscould not tested for reproducibility, as the device used for the nanovibrational stimulationfailed and was not suitable for further work.Other experiments assessed the macrophage differentiation upon stimulation of monocyteswith vitamin D3, 1000Hz nanovibrations, and both. The vitamin D3 induced macrophagedifferentiation, which was characterised by upregulated macrophage markers such as CD14,downregulation of HLA-DRA, and an inverse regulation of LEF-1 and TCF7L2. The vitamin D3also upregulated PKD2 (TRPP2), encoding for a non-selective cation channel withmechanosensory abilities. The nanovibrational stimulation caused upregulation of PIEZO1and TCF3 in monocytes, showing response towards this mechanical stimulus. However, the1000Hz vibrations did not induce macrophage differentiation because no regulation of CD14and TCF/LEF transcription factors took place. Furthermore, in the presence of vitamin D3,the effects of 1000Hz stimulation were cancelled and the cell responses were comparable tothose induced by the vitamin D3 when applied alone.Overall, this study identified interesting patters of gene expression upon activation anddifferentiation of T cell and macrophages. The experiments did not demonstrate that thenanovibrational stimulation, which was studied as alternative therapy for alopecia areata,could modulate T cell responses. In monocytes, the presence of vitamin D3 cancelled theobserved response towards the nanovibrational stimulation, which may indicate that in anenvironment rich in chemical signals such as the site of inflammation, the cells would not beaffected by its application.