Elizabeth Germino

THE P110 d AND g SUBUNITS OF PI3K INHIBIT MACROPHAGE ACTIVATION AND MAY ACCOUNT FOR THE INHIBITION OBSERVED IN DAP12-/- CELLS. Elizabeth Germino¹, Isaiah Turnbull², Marco Colonna.² Department of Biology, Washington University, St. Louis, MO.¹ Department of Pathology, Washington University School of Medicine, St. Louis, MO.²

Mononuclear phagocytes such as microglia, osteoclasts and macrophages function in the immune system to ingest and clear microbes, damaged host tissue and cell debris. These cells both react to and release cytokines, proteinaceous signals important for the control of inflammation and effective clearance of invading pathogens. A wide variety of membrane receptors are involved in regulating the function of mononuclear phagocytes. Toll-like receptors (TLRs) are the primary receptors for recognition of microbial products, and activation of TLRs elicits a proinflammatory response in macrophages, leading to production of proinflammatory cytokines such as TNF-α and IL-6 which function to initiate an antimicrobial response. However, prolonged inflammation can damage host tissue, and thus inhibitory mechanisms must be in place to prevent unnecessary or excessive macrophage activation.

DAP12 is a signaling adaptor for a wide range of cell surface receptors expressed on innate immune cells, and was initially defined by its ability to activate NK cells, monocytes and granulocytes. In contrast, recent studies have demonstrated that DAP12 can also attenuate proinflammatory cytokine production by macrophages in response to TLR ligation by microbial products such as lipopolysaccharide (LPS). The mechanism underlying these contrasting roles for DAP12 in macrophages is not yet clear; however, previous studies have demonstrated that the phophoinositide-3 kinase signaling pathway, which can be activated by DAP12, inhibits TLR activation. We hypothesized that the DAP12-mediated activation of PI3K may account for the inhibition of TLR activation by DAP12-associated receptors.

Class I PI3Ks have four types of catalytic subunits, p110α, β, δ and γ; deletion of the p110α and β subunits in mice is lethal. However, p110δ and γ knockouts are viable, providing a system in which to examine PI3K function in vivo. To test our hypothesis, bone marrow-derived macrophages were obtained from mice deficient in both the p110δ and γ catalytic subunits of PI3K (PI3K-/-). These cells were then stimulated in vitro with varying doses of LPS and the supernatants were assayed for levels of the proinflammatory cytokine TNF-α. At certain doses, the PI3K-/- cells displayed increased levels of cytokine compared to wild type cells, demonstrating that PI3K p110d and g subunits attenuate the response to LPS. In contrast to data on DAP12, which only inhibits cytokine production at low doses of LPS, PI3K appears to inhibit TLR activation across a broad range of doses of LPS.

To determine if there was a correlation between this phenotype and that of DAP12-/- mice, the experiment was then repeated with DAP12 and FCεR1γ (a related receptor signaling chain) knockouts, in addition to cells deficient in both DAP12 and FCεR1γ. Interestingly, preliminary data suggest that the DAP12/ FCεR1γ double knockout mice show a further augmentation in TNF-α levels compared to DAP12-/- mice, suggesting that in the absence of DAP12, FCεR1γ also has some inhibitory function (FCεR1γ-/- cells do not display increased cytokine response compared to wild type). Currently, experiments are underway comparing the DAP12-/- and DAP12/FceR1g-/- cells with the PI3K-/- cells to determine if PI3K accounts for the inhibitory function of DAP12 and FCeR1g.