Restoration of IFNcR Subunit Assembly, IFNc Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Sen, Subha and Roy, Koushik and Mukherjee, Sandip and Mukhopadhyay, Rupkatha and Roy, Syamal (2011) Restoration of IFNcR Subunit Assembly, IFNc Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol. PLoS Pathogens, 7 (9).

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    Abstract

    Despite the presence of significant levels of systemic Interferon gamma (IFNc), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNc signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNc signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNc receptor (IFNcR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNcR subunit 1 (IFNcR1) and subunit 2 (IFNcR2) colocalize in raft upon IFNc stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNcR1 and IFNcR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNc allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNcR1 was also noted. The interaction of peptides representing this motif of IFNcR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.2761029 M versus 2.6961027 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNcR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNcR1 and IFNcR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol.

    Item Type: Article
    URI: http://www.eprints.iicb.res.in/id/eprint/1339
    Subjects: Infectious Diseases and Immunology
    Divisions: Indian Institute of Chemical Biology
    Depositing User: Ms Sutapa Ganguly
    Date Deposited: 31 Jan 2012 15:15
    Last Modified: 12 Jul 2012 11:52
    Official URL: http://dx.doi.org/10.1371/journal.ppat.1002229
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