Research Projects

Although an association between cell death and inflammation has been recognized, the underlying mechanism(s) remains unclear. We have earlier reported that cell-free chromatin particles (cfChPs) that circulate in the blood, or those that are released locally from dying cells, can readily enter into healthy cells to induce DNA damage and activate inflammatory responses. We hypothesised that these cfChPs are the elusive agents that trigger inflammation. In pursuit of this hypothesis, we have performed the following experiments. cfChPs activate immune response via STING pathway: Treatment of PBMCs with cfChPs isolated from healthy individuals led to their localization in nuclei of PBMCs within 2h. This was associated with; 1) immune activation in terms of CD69 expression, 2) cellular stress response and 3) production of cytokines. These events could be markedly inhibited by concurrent treatment with H151 [stimulator of interferon genes (STING) protein inhibitor]. This suggested that cfChPs induce inflammation via the STING pathway. cfChPs released from dying PBMCs activate immune response: When conditioned media of hypoxia-induced dying fluorescently dually labelled lymphocytes (BrdU in their DNA and histone-2B-GFP in their histones) was added to healthy PBMCs, dually fluorescently labelled cfChPs accumulated in their nuclei of recipient cells by 4h. This was associated with marked activation of CD69, which was inhibited by prior incubation of the conditioned media with three different de-activators of cfChPs, namely anti-histone antibody complexed nanoparticles, DNase I and a combination of the nutraceuticals Resveratrol and Copper. Conclusion: Data from the above two experiments indicated that cfChPs may activate immune response via the STING pathway. Future experiments planned: 1. To investigate the immune modulatory effects of cfChPs released from dying cells on immune cells by analysing the cytotoxic activity, degranulation pattern and intracellular cytokine expression of T-and NK-cells in cfChPs (10ng) treated cells. 2. To investigate the mechanism of activation of the STING pathway by cfChPs by analysing the expression and cellular localization of different proteins involved in the downstream signalling cascade of the STING pathway. 3. To investigate the immune-modulatory effects of cfChPs on other cell types, viz. mesenchymal cells, epithelial cells and monocytes to evaluate if the effects of cfChPs vary with cell type. 4. To investigate the inhibitory effects of cfChPs deactivating agents (mentioned above) on cell death-induced inflammation by treating conditioned media of hypoxia-induced dying cells with cfChPs deactivating agents and applying it to healthy PBMCs. Anticipated outcome: The above experiments will help in a deeper understanding of how cell death is involved in activating immune responses. If successful, the results may usher in new therapies for the multiple conditions that are associated with hyper-inflammation.