Identification of Innate Receptors of Inducing Neutrophilic Production of Tumor Necrosis Factor-Alpha

Neutrophils and inflammatory monocytes (IMs) are white blood cells (leukocytes) essential for the efficient function of the innate immune system, the body’s first line of defense against infection. Recognition of microbes often occurs through molecular patterns (ligands) binding to a class of proteins called Toll-like receptors (TLRs). When microbial ligands bind to a single TLR, IMs are able to produce proinflammatory cytokines, including tumor necrosis factor-alpha (TNFa). In contrast, neutrophils produce TNFa upon activation by bacteria, which contain multiple TLR ligands. This is important because dead body cells present ligands capable of activating TLRs, so production of TNFa by neutrophils might promote unnecessary inflammation. This project attempts to identify the specific signals neutrophils require for TNFa production in vitro. Neutrophils could either be responding through a combination of TLR ligands or through a different class of receptors altogether, such as NOD-like receptors or C-type lectin receptors (CLRs), which both signal differently than TLRs. During experimentation, naïve bone marrow cells were tested to ensure that leukocytes used had not yet been activated by any ligand. Naïve cells were subsequently activated by various ligands: (1) single TLR ligands such as LPS and CpG, (2) combinations of TLR ligands, (3) zymosan, a ligand for both dectin-1 (CLR) and TLR2, and (4) both live and heat-killed bacteria. Intracellullar cytokine staining illustrated TNFa production. Zymosan has induced TNFa production in neutrophils. Further research aims at identifying the mechanism by which neutrophil-TNFa production is regulated in addition to viewing TNFa production in vivo during an infection.