Signaling pathway of autophagy associated with innate immunity

Autophagy has recently been shown to be an important component of the innate immune response. The signaling pathways leading to activation of autophagy in innate immunity are not well studied. Our recent study shows that Toll-like receptor 4 (TLR 4) serves as an environmental sensor for autophagy. We define a new molecular pathway in which lipopolysaccharide (LPS) induces autophagy in human and murine macrophages by a pathway regulated through Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-dependent, myeloid differentiation factor 88 (MyD88)-independent TLR4 signaling. Receptor-interacting protein (RIP1) and p38 mitogen-activated protein-kinase (MAPK) are downstream components of this pathway. This signaling pathway does not affect cell viability, indicating that it is distinct from an autophagic death signaling pathway. We further show that LPS-induced autophagy can enhance mycobacterial co-localization with the autophagosomes. The above study raises important questions. (1) What is the complete signaling pathway for LPS-induced autophagy? (2) Does TLR3 mediate autophagy? (3) What are the mechanisms that determine whether autophagy acts as a pro-death or pro-survival pathway? (4) What are the physiological functions of LPS-induced autophagosomes? Future studies examining the above questions should provide us with important clues as to how autophagy is regulated in innate immunity, and how autophagy can be utilized in pathogen clearance.     

Lipoxin A4 receptor agonist BML-111 induces autophagy in alveolar macrophages and protects from acute lung injury by activating MAPK signaling

Background

Acute lung injury (ALI) is a life-threatening lung disease where alveolar macrophages (AMs) play a central role both in the early phase to initiate inflammatory responses and in the late phase to promote tissue repair. In this study, we examined whether BML-111, a lipoxin A4 receptor agonist, could alter the phenotypes of AM and thus present prophylactic benefits for ALI.

Methods

In vitro, isolated AMs were treated with lipopolysaccharide (LPS) to induce ALI. In response to BML-111 pre-treatment, apoptosis and autophagy of AMs were examined by flow cytometry, and by measuring biomarkers for each process. The potential involvement of MAPK1 and mTOR signaling pathway was analyzed. In vivo, an LPS-induced septic ALI model was established in rats and the preventative significance of BML-111 was assessed. On the cellular and molecular levels, the pro-inflammatory cytokines TNF-α and IL-6 from bronchoalveolar lavage were measured by ELISA, and the autophagy in AMs examined using Western blot.

Results

BML-111 inhibited apoptosis and induced autophagy of AMs in response to ALI inducer, LPS. The enhancement of autophagy was mediated through the suppression of MAPK1 and MAPK8 signaling, but independent of mTOR signaling. In vivo, BML-111 pre-treatment significantly alleviated LPS-induced ALI, which was associated with the reduction of apoptosis, the dampened production of pro-inflammatory cytokines in the lung tissue, as well as the increase of autophagy of AMs.

Conclusions

This study reveals the prophylactic significance of BML-111 in ALI and the underlying mechanism: by targeting the MAPK signaling but not mTOR pathway, BML-111 stimulates autophagy in AMs, attenuates the LPS-induced cell apoptosis, and promotes the resolution of ALI.

     

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Endosome-mediated autophagy

Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4B^C74A mutant and atg4b^−/− bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival.     

Impaired autophagy and delayed autophagic clearance of transforming growth factor β-induced protein (TGFBI) in granular corneal dystrophy type 2

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease characterized by a progressive age-dependent extracellular accumulation of transforming growth factor β-induced protein (TGFBI). Corneal fibroblasts from GCD2 patients also have progressive degenerative features, but the mechanism underlying this degeneration remains unknown. Here we observed that TGFBI was degraded by autophagy, but not by the ubiquitin/proteasome-dependent pathway. We also found that GCD2 homozygous corneal fibroblasts displayed a greater number of fragmented mitochondria. Most notably, mutant TGFBI (mut-TGFBI) extensively colocalized with microtubule-associated protein 1 light chain 3β (MAP1LC3B, hereafter referred to as LC3)-enriched cytosolic vesicles and CTSD in primary cultured GCD2 corneal fibroblasts. Levels of LC3-II, a marker of autophagy activation, were significantly increased in GCD2 corneal fibroblasts. Nevertheless, levels of SQSTM1/p62 and of polyubiquitinated protein were also significantly increased in GCD2 corneal fibroblasts compared with wild-type (WT) cells. However, LC3-II levels did not differ significantly between WT and GCD2 cells, as assessed by the presence of bafilomycin A₁, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A₁ caused a similar change in levels of SQSTM1. Thus, the increase in autophagosomes containing mut-TGFBI may be due to inefficient fusion between autophagosomes and lysosomes. Rapamycin, an autophagy activator, decreased mut-TGFBI, whereas inhibition of autophagy increased active caspase-3, poly (ADP-ribose) polymerase 1 (PARP1) and reduced the viability of GCD2 corneal fibroblasts compared with WT controls. These data suggest that defective autophagy may play a critical role in the pathogenesis of GCD2.     

MCPIP1 negatively regulates toll-like receptor 4 signaling and protects mice from LPS-induced septic shock

Septic shock is one of leading causes of morbidity and mortality in hospital patients. However, genetic factors predisposing to septic shock are not fully understood. Our previous work showed that MCP-induced protein 1 (MCPIP1) was induced by lipopolysaccharides (LPSs), which then negatively regulates LPS-induced inflammatory signaling in vitro. Here we report that although MCPIP1 was induced by various toll-like receptor (TLR) ligands in macrophages, MCPIP1-deficient mice are extremely susceptible to TLR4 ligand (LPS)-induced septic shock and death, but not to the TLR2, 3, 5 and 9 ligands-induced septic shock. Consistently, LPS induced tumor necrosis factor α (TNFα) production in MCPIP1-deficient mice was 20-fold greater than that in their wild-type littermates. Further analysis revealed that MCPIP1-deficient mice developed severe acute lung injury after LPS injection and JNK signaling was highly activated in MCPIP1-deficient lungs after LPS stimulation. Finally, macrophage-specific MCPIP1 transgenic mice were partially protected from LPS-induced septic shock, suggesting that inflammatory cytokines from sources other than macrophages may significantly contribute to the pathogenesis of LPS-induced septic shock. Taken together, these results suggest that MCPIP1 selectively suppresses TLR4 signaling pathway and protects mice from LPS-induced septic shock.     

Impaired autophagy and delayed autophagic clearance of transforming growth factor β-induced protein (TGFBI) in granular corneal dystrophy type 2

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease characterized by a progressive age-dependent extracellular accumulation of transforming growth factor β-induced protein (TGFBI). Corneal fibroblasts from GCD2 patients also have progressive degenerative features, but the mechanism underlying this degeneration remains unknown. Here we observed that TGFBI was degraded by autophagy, but not by the ubiquitin/proteasome-dependent pathway. We also found that GCD2 homozygous corneal fibroblasts displayed a greater number of fragmented mitochondria. Most notably, mutant TGFBI (mut-TGFBI) extensively colocalized with microtubule-associated protein 1 light chain 3β (MAP1LC3B, hereafter referred to as LC3)-enriched cytosolic vesicles and CTSD in primary cultured GCD2 corneal fibroblasts. Levels of LC3-II, a marker of autophagy activation, were significantly increased in GCD2 corneal fibroblasts. Nevertheless, levels of SQSTM1/p62 and of polyubiquitinated protein were also significantly increased in GCD2 corneal fibroblasts compared with wild-type (WT) cells. However, LC3-II levels did not differ significantly between WT and GCD2 cells, as assessed by the presence of bafilomycin A₁, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A₁ caused a similar change in levels of SQSTM1. Thus, the increase in autophagosomes containing mut-TGFBI may be due to inefficient fusion between autophagosomes and lysosomes. Rapamycin, an autophagy activator, decreased mut-TGFBI, whereas inhibition of autophagy increased active caspase-3, poly (ADP-ribose) polymerase 1 (PARP1) and reduced the viability of GCD2 corneal fibroblasts compared with WT controls. These data suggest that defective autophagy may play a critical role in the pathogenesis of GCD2.     

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy

ABSTRACT

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.     

Autophagy dysfunction and regulatory cystatin C in macrophage death of atherosclerosis

Autophagy dysfunction in mouse atherosclerosis models has been associated with increased lipid accumulation, apoptosis and inflammation. Expression of cystatin C (CysC) is decreased in human atheroma, and CysC deficiency enhances atherosclerosis in mice. Here, we first investigated the association of autophagy and CysC expression levels with atheroma plaque severity in human atherosclerotic lesions. We found that autophagy proteins Atg5 and LC3β in advanced human carotid atherosclerotic lesions are decreased, while markers of dysfunctional autophagy p62/SQSTM1 and ubiquitin are increased together with elevated levels of lipid accumulation and apoptosis. The expressions of LC3β and Atg5 were positively associated with CysC expression. Second, we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE‐deficient mice, demonstrating that CysC deficiency (CysC^?/?) in these mice results in reduction of Atg5 and LC3β levels and induction of apoptosis. Third, macrophages isolated from CysC^?/? mice displayed increased levels of p62/SQSTM1 and higher sensitivity to 7‐oxysterol‐mediated lysosomal membrane destabilization and apoptosis. Finally, CysC treatment minimized oxysterol‐mediated cellular lipid accumulation. We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC. The deficiency of CysC causes autophagy dysfunction and apoptosis in macrophages and apoE‐deficient mice. The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis.     

Retracted: TNF-α–TNFR signal pathway inhibits autophagy and promotes apoptosis of alveolar macrophages in coal worker's pneumoconiosis

Objective : Exposure to coal dust causes the development of coal worker's pneumoconiosis (CWP), which is associated with accumulating macrophages in the lower respiratory tract. This study was performed to investigate the effect of tumor necrosis factor-α (TNF-α)–tumor necrosis factor receptor (TNFR) signal pathway on autophagy and apoptosis of alveolar macrophages (AMs) in CWP. Methods: AMs from controls exposed to coal dust and CWP patients were collected, in which expressions of TNF-α and TNFR1 were determined. Autophagy was observed by transmission electron microscopy, and apoptosis by light microscope and using terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. AMs in CWP patients were treated with TNF-α or anti-TNF-α antibody. Besides, expressions of autophagy marker proteins, apoptosis-related factors, FAS, caspase-8, and receptor-interacting serine–threonine-protein kinase 3 (RIPK3) were determined by western Blot. Activities of caspase-3 and caspase-8 were determined by a fluorescence kit. Flow cytometry was applied to measure the expression of TNFR1 on the surface of the AM. Results: TNF-α expression and TNFR1 expression on the surface of AM, as well as autophagy and apoptotic index were significantly increased in AMs of CWP patients. In response to the treatment of TNF-α, TNF-α expression and TNFR1 expression on the surface of AM as well as LC3I expression were increased, autophagy was decreased, and LC3, LC3II, Beclin1 and B-cell lymphoma 2 expressions decreased, whereas FAS expression and activity and expression of caspase-3 and caspase-8 increased, and apoptotic index increased. Moreover, the situations were reversed with the treatment of anti-TNF-α antibody. Conclusion: TNF-α–TNFR signal pathway was involved in the occurrence and development of CWP by activating FAS–caspase-8 and thus inhibiting autophagy while promoting apoptosis of AM.     

Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/GRP78/BiP

The N-end rule pathway is a proteolytic system, in which single N-terminal residues act as a determinant of a class of degrons, called N-degrons. In the ubiquitin (Ub)-proteasome system, specific recognition components, called N-recognins, recognize N-degrons and accelerate polyubiquitination and proteasomal degradation of the substrates. In this study, we show that the pathway regulates the activity of the macroautophagic receptor SQSTM1/p62 (sequestosome 1) through N-terminal arginylation (Nt-arginylation) of endoplasmic reticulum (ER)-residing molecular chaperones, including HSPA5/GRP78/BiP, CALR (calreticulin), and PDI (protein disulfide isomerase). The arginylation is co-induced with macroautophagy (hereafter autophagy) as part of innate immunity to cytosolic DNA and when misfolded proteins accumulate under proteasomal inhibition. Following cytosolic relocalization and arginylation, Nt-arginylated HSPA5 (R-HSPA5) is targeted to autophagosomes and degraded by lysosomal hydrolases through the interaction of its N-terminal Arg (Nt-Arg) with ZZ domain of SQSTM1. Upon binding to Nt-Arg, SQSTM1 undergoes a conformational change, which promotes SQSTM1 self-polymerization and interaction with LC3, leading to SQSTM1 targeting to autophagosomes. Cargoes of R-HSPA5 include cytosolic misfolded proteins destined to be degraded through autophagy. Here, we discuss the mechanisms by which the N-end rule pathway regulates SQSTM1-dependent selective autophagy.     

Fas/FasL pathway-mediated alveolar macrophage apoptosis involved in human silicosis

In vitro and in vivo studies have demonstrated that lung cell apoptosis is associated with lung fibrosis; however the relationship between apoptosis of alveolar macrophages (AMs) and human silicosis has not been addressed. In the present study, AM apoptosis was determined in whole-lung lavage fluid from 48 male silicosis patients, 13 male observers, and 13 male healthy volunteers. The relationships between apoptosis index (AI) and silica exposure history, soluble Fas (sFas)/membrane-bound Fas (mFas), and caspase-3/caspase-8 were analyzed. AI, mFas, and caspase-3 were significantly higher in lung lavage fluids from silicosis patients than those of observers or healthy volunteers, but the level of sFas demonstrated a decreasing trend. AI was related to silica exposure, upregulation of mFas, and activation of caspase-3 and -8, as well as influenced by smoking status after adjusting for confounding factors. These results indicate that AM apoptosis could be used as a potential biomarker for human silicosis, and the Fas/FasL pathway may regulate this process. The present data from human lung lavage samples may help to understand the mechanism of silicosis and in turn lead to strategies for preventing or treating this disease.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62

Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Autophagy-dependent PELI3 degradation inhibits proinflammatory IL1B expression

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.