Neutrophils can cast NETs to trap microbial pathogens. NETs are beneficial to trap pathogens during acute infections. We are identifying how NETs kill ensnared pathogens. Excess NETs released into the tissues can severely damage organs particularly in chronic inflammatory conditions. NETs also contribute to autoantibody generation. We aim to understand the molecular mechanisms governing NETosis, and to identify drugs that regulate NET formation. We expect that our work will help to control inflammatory and autoimmune diseases.

Frequently Used Techniques

Below is a diagram outlining techniques we use regularly in our experiments regarding NETs. This work can be done with either HL-60 cells (in vivo) that can be differentiated into neutrophil-like cells using DMSO, or primary neutrophils obtained from volunteers (ex vivo).

Palaniyar - In vitro/ex vivo + HL60 cells + polymorph + cell counting

Once the cells are properly isolated, they will be stimulated by a variety of agonists. These include: PMA, A23187, ionomycin, LPS, P. a, S. a, PAF, Pyocyanin, IC, and HxA3. After they are stimulated, the cells will follow two different paths. They can be pipetted into 96-well plates for to be imaged, to have their ROS levels measured, or have cell viability measured among other things. They could also be left in the microtube so that other aspects of the cells can be measured such as protein production, and gene transcription. Possible methods are shown in the image below.

Palaniyar - methods + sytox + ROS + Live cell imaging + drug screening + transcriptomics + CyTOF + Flow cytometry + WB + Cytospin

Experimental models

Below are diagrams that depict the various experimental models used in our experiments. The majority of our models are in mice, though we do use a single horse model. The diagrams show the type of model, briefly how they are created, data depicting their use, and publications in which they are utilized.

Mouse

Palaniyar + Douda - LPS mediated inflammation + Mouse Model

Mouse

Palaniyar + Douda - Mouse Model + Chronic bacterial infection + agar bead

Mouse

Palaniyar + Yildiz - Ventilator Induced Lung Injury + Mouse model + BALF + LPS

Mouse

Palaniyar + Cheng - Mouse model + CF like lung disease + Nedd4L ubiquitin ligase KO

Mouse

Palaniyar + Jin - Polymicrobial sepsis cecal ligation + Mouse model + CLP + PF

Horse

Palaniyar + Cote - Horse Model + Recurrent airway obstruction + western blot