Abstract
The NLRP3 inflammasome activation is a key signaling event for activation and secretion of pro-inflammatory cytokines such as IL-1β from macrophages. p58IPK is a molecular chaperone that regulates protein homeostasis through inhibiting eIF-2α kinases including double-stranded RNA–dependent protein kinase (PKR), which has been recently implicated in inflammasome activation. Herein we investigate the role of p58IPK in TLR4 signaling and inflammasome activation in macrophages. Primary bone marrow-derived macrophages (BMDM) was isolated from p58IPK knockout (KO) and wildtype (WT) mice and treated with lipopolysaccharide (LPS) and ATP to activate TLR4 signaling and stimulate inflammasome activation. Compared to WT macrophages, p58IPK deficient cells demonstrated significantly stronger activation of PKR, NF-κB, and JNK and higher expression of pro-inflammatory genes TNF-α and IL-1β. Coincidently, p58IPK deletion intensified NLRP3-inflammasome activation indicated by enhanced caspase 1 cleavage and increased IL-1β maturation and secretion. Pretreatment with specific PKR inhibitor or overexpression of p58IPK largely abolished the changes in inflammasome activation and IL-1β secretion in p58IPK null macrophages. Furthermore, immunoprecipitation assay confirmed the binding of p58IPK with PKR, but not other TLR4 downstream signaling molecules. Collectively, these results suggest a novel and crucial role of p58IPK in regulation of inflammasome activation and IL-1β secretion in macrophages.
Highlights
(WT) mice and treated with lipopolysaccharide (LPS) and ATP to activate TLR4 signaling and stimulate inflammasome activation
Using bone marrow derived macrophages (BMDM) from wild-type and p58IPK knockout mice, we demonstrate that p58IPK inhibits lipopolysaccharide (LPS)-induced activation of NF-κ B and c-jun N-terminal kinase (JNK) and decreases the expression of pro-inflammatory cytokines TNFα, interleukin 1β (IL-1β), and interleukin 6 (IL-6)
We show that p58IPK suppresses NLRP3 inflammasome activation and reduces IL-1β production through inhibition of PKR
Summary
P58IPK is a key regulator of protein translation[8,9,10] and protein folding[11] depending on its subcellular localization[26]. Using BMDM isolated from p58IPK knockout mice and pharmacological PKR inhibitor, we demonstrated that deletion of p58IPK augments PKR, NF-κ B p65 and JNK MAPK activation, but reduces p38 activation, while inhibition of PKR by specific pharmacological inhibitor largely abolished NF-κ B p65 and JNK, but not p38 MAPK activation These results support that manipulating PKR by either endogenous or exogenous inhibitors differentially regulates p38 and JNK MAPKs and NF-κ B p65 activation in macrophages. In addition to regulation of canonical inflammatory pathways, recent studies provide compelling evidence that supports a new and potential role of PKR in inflammasome activation[31]. Our study demonstrates an important role of p58IPK in regulation of PKR-mediated TLR4 downstream NF-κ B and JNK MAPK pathways, inflammasome activation, and IL-1β secretion from macrophages. Enhancing the function of p58IPK could lead to a new approach to prevent inflammasome and IL-1β -related tissue injury and pathological consequences such as neurodegeneration
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