ABSTRACT
Cellular lipids determine membrane integrity and fluidity and are being increasingly recognized to influence immune responses. Cellular cholesterol requirements are fulfilled through biosynthesis and uptake programs. In an intricate pathway involving the lysosomal cholesterol transporter NPC1, the sterol gets unequally distributed across intracellular compartments. By using pharmacological and genetic approaches targeting NPC1, we reveal that blockade of cholesterol trafficking through the late endosome-lysosome pathway blunts NLRP3 inflammasome activation. Altered cholesterol localization at the plasma membrane (PM) in Npc1-/- cells abrogated AKT-mTOR signaling by TLR4. However, the inability to activate the NLRP3 inflammasome was traced to perturbed cholesterol trafficking to the ER but not the PM. Accordingly, acute cholesterol depletion in the ER membranes by statins abrogated casp-1 activation and IL-1ß secretion and ablated NLRP3 inflammasome assembly. By contrast, assembly and activation of the AIM2 inflammasome progressed unrestricted. Together, this study reveals ER sterol levels as a metabolic rheostat for the activation of the NLRP3 inflammasome.
Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Endoplasmic Reticulum/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Signal Transduction , Animals , Biological Transport, Active/physiology , Cell Membrane/genetics , Cholesterol/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/genetics , Inflammasomes/genetics , Intracellular Signaling Peptides and Proteins , Mice , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Niemann-Pick C1 Protein , Proteins/genetics , Proteins/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
Background: There is a recognized need for an improved diagnostic test to assess post-chemotherapeutic treatment outcome in visceral leishmaniasis (VL) and to diagnose post kala-azar dermal leishmaniasis (PKDL). We previously demonstrated by ELISA and a prototype novel rapid diagnostic test (RDT), that high anti-Leishmania IgG1 is associated with post-treatment relapse versus cure in VL. Methodology: Here, we further evaluate this novel, low-cost RDT, named VL Sero K-SeT, and ELISA for monitoring IgG1 levels in VL patients after treatment. IgG1 levels against L. donovani lysate were determined. We applied these assays to Indian sera from cured VL at 6 months post treatment as well as to relapse and PKDL patients. Sudanese sera from pre- and post-treatment and relapse were also tested. Results: Of 104 paired Indian sera taken before and after treatment for VL, when deemed clinically cured, 81 (77.9%) were positive by VL Sero K-SeT before treatment; by 6 months, 68 of these 81 (84.0%) had a negative or reduced RDT test line intensity. ELISAs differed in positivity rate between pre- and post-treatment (p = 0.0162). Twenty eight of 33 (84.8%) Indian samples taken at diagnosis of relapse were RDT positive. A comparison of Indian VL Sero K-SeT data from patients deemed cured and relapsed confirmed that there was a significant difference (p < 0.0001) in positivity rate for the two groups using this RDT. Ten of 17 (58.8%) Sudanese sera went from positive to negative or decreased VL Sero K-SeT at the end of 11-30 days of treatment. Forty nine of 63 (77.8%) PKDL samples from India were positive by VL Sero K-SeT. Conclusion: We have further shown the relevance of IgG1 in determining clinical status in VL patients. A positive VL Sero K-SeT may also be helpful in supporting diagnosis of PKDL. With further refinement, such as the use of specific antigens, the VL Sero K-SeT and/or IgG1 ELISA may be adjuncts to current VL control programmes.
