Inositol phosphorylceramide synthase null Leishmania are viable and virulent in animal infections where salvage of host sphingomyelin predominates.

Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt2-) and SLs survive and remain virulent, as infective amastigotes salvage host SLs and continue to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in Leishmania major (Δipcs-). Unexpectedly and unlike fungi where IPCS is essential, Δipcs- was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC were absent in Δipcs- promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian SM was highly abundant in purified amastigotes from both WT and Δipcs-, and salvaged SLs could be further metabolized into IPC. SM was about 7-fold more abundant than IPC in WT amastigotes, establishing that SM is the dominant amastigote SL, thereby rendering IPC partially redundant. These data suggest that SM salvage likely plays key roles in the survival and virulence of both WT and Δipcs- parasites in the infected host, confirmation of which will require the development of methods or mutants deficient in host SL/SM uptake in the future. Our findings call into question the suitability of IPCS as a target for chemotherapy, instead suggesting that approaches targeting SM/SL uptake or catabolism may warrant further emphasis.

Chronic Ly49H Receptor Engagement in vivo Decreases NK Cell Response to Stimulation Through ITAM-Dependent and Independent Pathways Both in vitro and in vivo.

Natural killer (NK) cells play an important role in the innate immune response. The summation of activation and inhibitory signals delivered through cell surface membrane receptors determines NK cell function. However, the continuous engagement of an activating receptor on NK cells appears to render the cells hyporesponsive to stimulation through other unrelated activating receptors. The mechanism by which this takes place remains unclear. Herein we demonstrate that continuous in vivo engagement of the Ly49H receptor with its ligand, m157, results in Ly49H+ NK cells that are hyporesponsive to further stimulation by other ITAM-dependent and independent receptors, while Ly49H- NK cells remain unaffected. The hyporesponsiveness of the NK cell correlates with the degree of Ly49H receptor downmodulation on its cell surface. We observe defects in calcium flux in the hyporesponsive NK cells following stimulation through the NK1.1 receptor. In addition, we observe differences in signaling molecules that play a role in calcium flux, including spleen tyrosine kinase (Syk) at baseline and phosphorylated phospholipase C gamma 2 (p-PLCγ2) at both baseline and following stimulation through NK1.1. We also demonstrate that various ITAM associated activation receptors, including Ly49H, remain associated with their respective adaptor molecules. With regard to in vivo NK cell function, we did not find differences in the formation of metastatic lung lesions following IV injection of B16 melanoma cells. However, we did observe defects in rejection of missing-self targets in vivo. The data suggest that continuous engagement of the Ly49H activating receptor on NK cells results in hyporesponsiveness of the NK cells to all of the ITAM-dependent and independent receptors we analyzed due to altered signaling pathways downstream of the receptor and adaptor molecule.

Expression of the inhibitory receptor NKG2A correlates with increased liver and splenic NK cell response to activating receptor engagement.

INTRODUCTION: Natural killer (NK) cells play a critical role in the innate immune response to viruses and tumors, and comprise a large proportion of the hepatic lymphocyte population. They must remain tolerant to non-pathogenic antigens while protecting the host from harmful agents. Herein, we investigate how the NK cell response to activation receptor engagement is altered in the liver. METHODS: In this study, we assess IFN-γ production and degranulation of splenic NK cells and selected subsets of liver NK cells. Flow cytometry (FCM) was used to asses IFN-γ production and degranulation following stimulation of the NK cells with plate bound antibodies to activating receptors. RESULTS: We show that smaller percentages of hepatic NK cells produce interferon (IFN)-γ and/or degranulate than do splenic NK cells upon stimulation through activating receptors. We also found that smaller percentages of the circulating NK (cNK) cells in the liver produce IFN-γ and/or degranulate, compared to the liver tissue resident NK (trNK) cells. In addition, IFN-γ production by liver cNK cells is not increased in IL-10 deficient mice, suggesting that their hyporesponsiveness is not mediated by the presence of this anti-inflammatory cytokine in the hepatic microenvironment. On the other hand, liver trNK cells express higher levels of the inhibitory receptor NKG2A than do cNK cells, correlating with their increased IFN-γ production and degranulation. CONCLUSIONS: Liver cNK cells' hyporesponsiveness to stimulation through activating receptors is independent of IL-10, but correlates with decreased NKG2A expression compared to trNK cells. In addition, we demonstrate that liver NK cells become further hyporesponsive upon continuous engagement of an activating receptor on their cell surface.