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Restoration of IFN?R subunit assembly, IFN? signaling and parasite clearance in Leishmania donovani infected macrophages: role of membrane cholesterol.

ABSTRACT: Despite the presence of significant levels of systemic Interferon gamma (IFN?), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFN? 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 IFN? signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFN? receptor (IFN?R) 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. IFN?R subunit 1 (IFN?R1) and subunit 2 (IFN?R2) colocalize in raft upon IFN? stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFN?R1 and IFN?R2 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 IFN? 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)-X(1-5)-Y-X(1-5)-(R/K)] in the transmembrane domain of IFN?R1 was also noted. The interaction of peptides representing this motif of IFN?R1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (K(D): 4.27×10(-9) M versus 2.69×10(-7) M). These observations reinforce the importance of cholesterol in the regulation of function of IFN?R1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFN?R1 and IFN?R2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol.

SUBMITTER: Sen S

PROVIDER: S-EPMC3169561 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Restoration of IFNγR subunit assembly, IFNγ signaling and parasite clearance in Leishmania donovani infected macrophages: role of membrane cholesterol.

Sen Subha S Roy Koushik K Mukherjee Sandip S Mukhopadhyay Rupkatha R Roy Syamal S

PLoS pathogens 20110908 9

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ 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 IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their l ...[more]

PMID: 21931549

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Project description:BackgroundPreviously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activation.Methodology/principal findingsMΦ of CBA/j mice were infected with Leishmania donovani (I-MΦ). Two different anti-Aκ mAbs were used to monitor the status of MHC-II protein under parasitized condition. One of them (11.5-2) was conformation specific, whereas the other one (10.2.16) was not. Under parasitized condition, the binding of 11.5-2 decreased significantly with respect to the normal counterpart, whereas that of 10.2.16 remained unaltered. The binding of 11.5-2 was restored to normal upon liposomal delivery of cholesterol in I-MΦ. By molecular dynamics (MD) simulation studies we found that there was considerable conformational fluctuation in the transmembrane domain of the MHC-II protein in the presence of membrane cholesterol than in its absence, which possibly influenced the distal peptide binding groove. This was evident from the faster dissociation of the cognate peptide from peptide-MHC complex under parasitized condition, which could be corrected by liposomal delivery of cholesterol in I-MΦ.ConclusionThe decrease in membrane cholesterol in I-MΦ may lead to altered conformation of MHC II, and this may contribute to a faster dissociation of the peptide. Furthermore, liposomal delivery of cholesterol in I-MΦ restored its normal antigen presenting function. This observation brings strength to our previous observation on host directed therapeutic application of liposomal cholesterol in experimental visceral leishmaniasis.

Dataset Information

Restoration of IFN?R subunit assembly, IFN? signaling and parasite clearance in Leishmania donovani infected macrophages: role of membrane cholesterol.

Publications

Restoration of IFNγR subunit assembly, IFNγ signaling and parasite clearance in Leishmania donovani infected macrophages: role of membrane cholesterol.

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