Abstract
Lipopolysaccharide (LPS)-induced activation of TLR4 (toll-like receptor 4) is followed by a subsequent overwhelming inflammatory response, a hallmark of the first phase of sepsis. Therefore, counteracting excessive innate immunity by autophagy is important to contribute to the termination of inflammation. However, the exact molecular details of this interplay are only poorly understood. Here, we show that PELI3/Pellino3 (pellino E3 ubiquitin protein ligase family member 3), which is an E3 ubiquitin ligase and scaffold protein in TLR4-signaling, is impacted by autophagy in macrophages (MΦ) after LPS stimulation. We noticed an attenuated mRNA expression of proinflammatory Il1b (interleukin 1, β) in Peli3 knockdown murine MΦ in response to LPS treatment. The autophagy adaptor protein SQSTM1/p62 (sequestosome 1) emerged as a potential PELI3 binding partner in TLR4-signaling. siRNA targeting Sqstm1 and Atg7 (autophagy related 7), pharmacological inhibition of autophagy by wortmannin as well as blocking the lysosomal vacuolar-type H(+)-ATPase by bafilomycin A1 augmented PELI3 protein levels, while inhibition of the proteasome had no effect. Consistently, treatment to induce autophagy by MTOR (mechanistic target of rapamycin (serine/threonine kinase)) inhibition or starvation enhanced PELI3 degradation and reduced proinflammatory Il1b expression. PELI3 was found to be ubiquitinated upon LPS stimulation and point mutation of PELI3-lysine residue 316 (Lys316Arg) attenuated Torin2-dependent degradation of PELI3. Immunofluorescence analysis revealed that PELI3 colocalized with the typical autophagy markers MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) and LAMP2 (lysosomal-associated membrane protein 2). Our observations suggest that autophagy causes PELI3 degradation during TLR4-signaling, thereby impairing the hyperinflammatory phase during sepsis.
Keywords:
ACTB, actin, beta; ATG7, autophagy-related 7; BECN1, Beclin 1, autophagy related; Baf A1, bafilomycin A1; CHX, cycloheximide; Epoxo, epoxomycin; HBSS, Hank's balanced salt solution; IL1B, interleukin 1, beta; IRAKs, interleukin-1 receptor-associated kinases; LAMP2, lysosomal-associated membrane protein 2; LPS; LPS, lipopolysaccharide; MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; MAP3K14, mitogen-activated protein kinase kinase kinase 14; MAP3K7, mitogen-activated protein kinase kinase kinase 7; MAPK1/3, mitogen-activated protein kinase 1/3; MAPK14, mitogen-activated protein kinase 14; MAPK8/9, mitogen-activated protein kinase 8/9; MTOR, mechanistic target of rapamycin (serine/threonine kinase); MYD88, myeloid differentiation primary response 88; MΦ, macrophages; NFE2L2, nuclear factor, erythroid 2-like 2; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B-cells; NLRP3, NLR family, pyrin domain containing 3; PELI3; PELI3, pellino E3 ubiquitin protein ligase family member 3; PRRs, pattern recognition receptors; RIPK1, receptor (TNFRSF)-interacting serine-threonine kinase 1; Rapa, rapamycin; SQSTM1; SQSTM1, sequestosome 1; TABs, TGF-beta activated kinase 1/MAP3K7 binding protein 1/2/3; TBK1, TANK-binding kinase 1; TICAM1, toll-like receptor adaptor molecule 1; TLRs, toll-like receptors; TNF, tumor necrosis factor; TRAF6, TNF receptor-associated factor 6, E3 ubiquitin protein ligase; TUBB, tubulin, beta class I; Torin2; UBB, ubiquitin B; WT, wildtype; Wortm, wortmannin; autophagy.