AUTOPHAGY 2016, VOL. 12, NO. 7, 1073–1082 http://dx.doi.org/10.1080/15548627.2016.1179410

REVIEW

Autophagy in sepsis: Degradation into exhaustion? Jeffery Hoa, Jun Yub, Sunny H. Wongb, Lin Zhangc, Xiaodong Liua, Wai T. Wonga, Czarina C. H. Leunga, Gordon Choia, Maggie H. T. Wangd, Tony Gina, Matthew T. V. Chana, and William K. K. Wua,b a Department of Anesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; bState Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences and Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; cSchool of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; dThe Jockey Club School of Public Health and Primary Care, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China

ABSTRACT

ARTICLE HISTORY

Autophagy is one of the innate immune defense mechanisms against microbial challenges. Previous in vitro and in vivo models of sepsis demonstrated that autophagy was activated initially in sepsis, followed by a subsequent phase of impairment. Autophagy modulation appears to be protective against multiple organ injuries in these murine sepsis models. This is achieved in part by preventing apoptosis, maintaining a balance between the productions of pro- and anti-inflammatory cytokines, and preserving mitochondrial functions. This article aims to discuss the role of autophagy in sepsis and the therapeutic potential of autophagy enhancers.

Received 5 February 2016 Revised 29 March 2016 Accepted 11 April 2016

Introduction Historically, macroautophagy/autophagy was recognized in yeast as a physiological response to starvation to sustain nutrient supply, during which cytoplasmic contents are nonselectively sequestered and directed to lysosomes for recycling into essential substrates for metabolism; there are also various types of selective autophagy during which misfolded/unfolded proteins and superfluous or malfunctioning organelles are degraded.1-3 In an animal model of neonatal starvation, autophagy-deficient mice die much sooner than their wild-type counterparts.4 This observation demonstrated the critical role of this evolutionarily conserved process (i.e., autophagy) in survival of mammals under stress. This pathway also counteracts microbial invasion by actively eliminating intracellular microbes, promoting antigen presentation, modulating inflammatory responses and removing damaged host cell organelles, such as mitochondria, in an attempt to maintain homeostasis, particularly during the course of sepsis.5 Sepsis, a condition characterized by systemic hyperinflammation and sometimes immunosuppression as a result of infection, causes significant mortality and morbidity worldwide. The sequential activation of these seemingly antagonistic pathways has been controversial. Recently, a genome-wide expression study of the critically ill has suggested that both pro- and anti-inflammatory cytokine genes are upregulated simultaneously.6 Although the physiological responses and molecular pathways between human and mice may not always be correlated,7 murine models of sepsis remain a valuable tool for understanding the disease course and testing potential therapeutics.

KEYWORDS

apoptosis; autophagy; immunity; mitochondrial function; sepsis

Investigation of risk factors has been focused on co-morbidities and therapeutic interventions administered, but the role of host factors is not well documented. Until recently, sporadic studies have suggested the involvement of autophagy in sepsis outcome. A multicenter prospective cohort study revealed that polymorphisms of the autophagy-related loci in the IRGM (immunity-related GTPase family, M) gene are associated with excess mortality in sepsis. In this respect, death rate in patients carrying the homozygous T allele was almost double compared with other genotypes, 48.1% vs 25.1%, p D 0.004.8 Further ex vivo experiments revealed that the mRNA expression of IRGM is significantly lower in the TT genotype, rendering it defective in autophagy.8 Accumulating evidence from in vitro and in vivo studies9-15 suggest that autophagy plays a protective role in sepsis, although contradictory findings have been reported.16,17 In support of this notion, newly tested therapeutic agents in animal models have targeted modulation of the same molecular pathway—autophagy.10,18-22 In this review, we discuss the role of autophagy in sepsis.

The process of autophagy Autophagy can be classified into nonspecific and targeted pathways. The former is characterized by nonselective sequestration of cytosolic cargo by double-membrane phagophores that mature into autophagosomes, which then fuse with lysosomes. In contrast, the targeted autophagy pathway requires receptor proteins to recognize specific cargoes for degradation. The

CONTACT Matthew T. V. Chan [email protected] 4/F, Main Clinical Building and Trauma Centre, Prince of Wales Hospital, Shatin, N.T., Hong Kong Special Administrative Region, China; William K. K. Wu [email protected] 4/F, Main Clinical Building and Trauma Centre, Prince of Wales Hospital, Shatin, N.T., Hong Kong Special Administrative Region, China. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kaup. © 2016 Taylor & Francis

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latter is particularly important in eliminating microbes and recycling damaged mitochondria through processes called xenophagy and mitophagy, respectively.23 Conventionally, autophagy is orchestrated by a collection of closely related macromolecules called autophagy-related (ATG) proteins and is therefore known as the canonical pathway. Recently, studies revealed that autophagic degradation could be activated without the involvement of some, if not all, of these ATG proteins. This alternative route is referred to as noncanonical autophagy.24 Nevertheless, both pathways are initiated by conversion of the cytosolic form of MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3; LC3-I) to LC3-II, which is bound to phagophores and autophagosomes. The process from sequestering molecular cargo to fusion with a lysosome leading to complete degradation is referred to as autophagic flux. The cellular and molecular mechanism of autophagy has been comprehensively reviewed by others.1,2,25 Autophagy is induced upon septic insult Sepsis-induced autophagy is initiated by binding of pathogenassociated molecular patterns within the microbial structure to pattern recognition receptors, such as toll-like receptors. This in turn activates different intracellular events and leads to increased autophagic activity by promoting the conversion of LC3-I to LC3-II.26,27 As such, binding of lipopolysaccharides of Gram-negative pathogens onto TLR4 (toll-like receptor 4) activates autophagy via the MAPK/p38 (mitogen-activated protein kinase) signaling axis whereas binding of lipoteichoic acid to TLR2 induces autophagy by the MAPK1/ERK2-MAPK3/ ERK1 pathway.26 Different TLR ligands exhibit variable autophagy-inducing abilities. Of these, single-stranded RNA binding to TLR7 is the most efficient inducer.27 Other features in sepsis include an increase of endoplasmic reticulum (ER) stress and the presence of damaged mitochondria.12,28 The initiation of autophagy upon microbial encounters is an important innate immune response. Infection triggers the release of pro-inflammatory cytokines, migration of polymorphs to the infection foci and production of reactive oxygen species that increase oxidative stress and mitochondrial damage. This is usually manifested by a loss of mitochondrial membrane potential and release of CYCS (cytochrome c, somatic) into the cytosol.29 Inflammatory factors including NFKB (nuclear factor of kappa light polypeptide gene enhancer in B cells), IRF1 (interferon regulatory factor 1) and inflammasomes AIM2 and NLRP3 modulate autophagy.30-32 These proteins are also involved in innate immunity. When the AIM2 inflammasome is activated in macrophages by transfection with double-stranded DNA poly(dA:dT), autophagosome formation increases significantly.32 Increased ER stress upregulates the expression of HSPA5/GRP78 proteins, which in turn phosphorylates ER-localized transmembrane proteins EIF2AK3/PERK and EIF2S1 (eukaryotic translation initiation factor 2, subunit 1 a).28 Damaged mitochondria recruit proteins such as PARK2, BNIP3L and BNIP3 targeting the organelle for mitophagy.12 At this point, mitophagy plays an important role in sequestering these malfunctioning organelles in order to prevent further aggravation of the pro-inflammatory response and minimize oxidative damage to the host.

The observation of autophagy induction in sepsis has largely relied on protein analysis of homogenized organs/tissues harvested from animal models of sepsis. This precluded the identification of cell types that predominantly elicited sepsis-induced autophagy. Nevertheless, animal studies have indicated differential induction of autophagy in vital organs. Of these, liver showed the highest level of autophagy induction, followed by heart and spleen.14 In this regard, it is anticipated that hepatocytes and cardiomyocytes may be the predominant cell types in response to sepsis-induced autophagy. Further cellular and differential expression studies may be useful in identifying the cell population responsive to sepsis-induced autophagy. Kinetics of autophagy in sepsis Sepsis is characterized by an initial overwhelming production of pro-inflammatory cytokines followed by immunosuppression.33,34 Accumulating evidence shows that autophagic activity is elevated during the hyperdynamic phase of sepsis followed by a decline.11,14,35,36 Cecal ligation and puncture (CLP)-induced peritonitis using C56BL/6 mice is the most widely used animal model of sepsis. During the initial 4 to 6 h after CLP, hepatocytes and cardiomyocytes show a considerably higher LC3-II:LC3-I ratio (a marker of autophagy induction), accompanied by an increased number of autophagosomes.11,14 However, the autolysosomal (the product of an autophagosome fusing with a lysosome) numbers decrease from 8 h onwards, leading to accumulation of SQSTM1/p62 receptor proteins. Survival of mice is increased by administration of carbamazepine, an autophagic flux modulator, as evidenced by an increase in LC3-II, RAB7 and a decreased level of the receptor protein SQSTM1. In heart and spleen, the same pattern of autophagic kinetics with an initial increase in the LC3-II:LC3-I ratio followed by a subsequent decline by 24 h is observed.14 This suggests that autophagic flux is inhibited after a short period of hyperactivity in sepsis. However, the occurrence of autophagic depression in relation to immunosuppression remains to be elucidated. Monitoring green fluorescent protein-LC3 transgenic mice, the number of LC3 puncta increases considerably in the liver, heart and spleen after CLP, peaking at 6 h.14 LC3 colocalizes with LAMP1 (lysosomal-associated membrane protein 1) in the liver up to 24 h after CLP, suggesting the autophagic process is complete, at least to the stage of autophagosomelysosome fusion. The authors attributed the sustainable flux to the 3-fold upregulation of SQSTM1 expression by 24 h.14 However, the autophagic process appears to be incomplete in the heart. A possible accumulation of autophagosomes could be the result of a downstream blockade of autophagosome-lysosome fusion. In support of this hypothesis, electron microscopy analysis of the left ventricle taken from mice after CLP-induced sepsis reveals numerous autophagosomes but only a few autolysosomes.37 In addition, colocalization of LC3 with LAMP1 is reduced by 60% at 24 h in septic mice. These data suggest that the effect of sepsis on autophagic flux might be tissue-dependent. Animal studies on autophagic kinetics in sepsis have been emphasized for the initial 24 h; no study has looked into 48 h and beyond. Late-stage suppression of autophagy shows a lethal

AUTOPHAGY

effect in animal models of sepsis, indicating that this late-stage autophagy may be more important than the acute phase in determining the clinical outcome of sepsis.36,37 The lagged suppression of autophagic activity may contribute to apoptosis of leukocytes and hence lead to unresponsiveness to microbial challenges and activation of otherwise latent viruses.33,38 Knocking out autophagy genes significantly reduces the number of circulating T- and B-lymphocytes in atg5¡/¡ mice, rendering CD4C and CD8C T cells unresponsive to stimuli.39 Autophagy and apoptosis also share common molecular regulators and appear to be antagonistic.40 To this end, inhibition of autophagy by interferon regulatory factor IRF1 has been shown to activates apoptosis in splenic macrophages.30 Physiologically, exhaustion of autophagy may result in extrusion of accumulated autophagosomes out of the cells, leading to a proinflammatory response.41 In addition, dysfunctional mitochondria cannot be eliminated properly by impaired autophagic flux. The interaction of pathogen and autophagy in relation to mitochondrial function and inflammatory response in sepsis is summarized in Figure 1A. Complete autophagic flux protects against sepsis The role of autophagy in sepsis has been examined in various animal models and cell culture experiments. Effects of autophagy on major organs in experimental sepsis are summarized in Table 1. Heart Recent studies suggest impairment of autophagy may contribute to contractile dysfunction and apoptotic cell death of cardiomyocytes. In the CLP model, despite elevation of LC3-II, the colocalization of LC3 and LAMP1 decreases over the course of sepsis.35,37 Electron microscopy confirms impaired autophagic flux at a late stage of sepsis with an increased number of autophagosomes, leaving a few autolysosomes.37 However, this can be readily rectified by administration of rapamycin. In addition, this restores the left ventricle ejection fraction and protects cardiomyocytes against apoptosis and necrosis.37 One of the key features of sepsis is the elevation of NOS2 (nitric oxide synthase 2, inducible) activity. Nitric oxide inhibits autophagy via suppression of MAPK8/JNK1 (mitogen-activated protein kinase 8) and BCL2 (B-cell CLL/lymphoma 2) phosphorylation, which subsequently strengthens the BCL2BECN1 interaction and prevents binding between PIK3C3/ Vps34 and BECN1.42,43 However, direct inhibition of NOS2 may result in cardiomyocyte atrophy and reduced contractility.44 Interestingly, co-inhibition of autophagy and reactive oxygen species formation protects CT (catalase) transgenic mice, namely FVB mice, against lipopolysaccharide (LPS)induced contractile dysfunction.45 Contractility can be influenced by the extent to which apoptosis takes place. In murine cardiomyocytes, apoptosis occurs as soon as LPS-induced autophagy returns to baseline level by 24 h. Both silencing of ATG5 with small interfering RNA (siRNA) and inhibiting autolysosome formation with 3-methyladenine hastens the occurrence of apoptosis at 4 h after endotoxin challenge.28

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Immune system The main culprit of sepsis was thought to be sudden overwhelming production of pro-inflammatory cytokines, leading to tissue edema and multiple organ injury. Autophagy interacts with these inflammatory pathways in response to sepsis. Deletion of ATG genes increases the production of inflammasome-associated IL1B (interleukin 1 b) and IL18 in Becn1C/¡ and lc3b¡/¡ mice, which had undergone CLP.46 In murine macrophages, knockdown of PELI3, a ubiquitin protein ligase involved in TLR4 signaling, downregulates the expression of IL1b.47 Likewise, BECN1-dependent autophagy reduces the production of NLRP3/NALP3 inflammasome-associated interleukins by eliminating damaged mitochondria.48 Conversely, blockade of mitophagy enhances the production of IL1B and IL18.48 The significance of the hyperinflammatory phase of sepsis has recently been challenged by the findings that dormant viruses, such as cytomegaloviruses, are reactivated at the late stage of sepsis.33 Concordantly, improved survival rate observed in Rubcn/rubicon-knockout mice share similar levels of pro-inflammatory cytokines to wild-type animals, suggesting that the beneficial effect of autophagic activation on sepsis may be independent of cytokines.35 It is highly possible that the immunosuppressive phase of sepsis is also important in determining mortality. Interestingly, autophagic suppression may result in a positive outcome. Inhibition of autophagy by chloroquine ablates the release of HMGB1 (high mobility group box 1) from macrophages and endothelial cells in Balb/C mice, which in turn prevents degradation of the repressor protein NFKBI/IkB. Consequently, NFKB (nuclear factor of kappa light polypeptide gene enhancer in B cells)-induced cytokine release is inhibited.49

Liver Examination of post-mortem liver specimens isolated from septic patients demonstrates considerable accumulation of autophagic vacuoles in hepatocytes.50 In septic animals, the mitochondrial fraction demonstrates insufficient oxygen utilization.50 To elucidate the protective effect of autophagy on sepsisinduced hepatic injury, liver tissues from C56BL/6 mice lacking SFTPA1 (surfactant associated protein A1) and SFTPD were analyzed for autophagic activity and histological morphology.51 The knockout mice show significant increases in LC3-II and autophagosomes while the expression of autophagy-related genes encoding ATG16L2, ATG12 and ATG7 are upregulated.51 Conversely, administration of chloroquine abolishes autophagic flux, which results in the elevation of serum transaminase levels.14 Compelling evidence confirms that autophagy in the liver is induced during the initial 4 h after CLP but declines after this point until 24 h.11,14,19,36,51 The blockade of autophagic flux is demonstrated by reduced LAMP1 expression, accumulation of SQSTM1 and lack of colocalization of autophagosomes with lysosomes.14,51 These observations suggest that fusion of autophagosomes and lysosomes may be impaired. Further studies

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Figure 1. Autophagy and its deregulation in sepsis. (A) The pathogen-autophagy interplay in relation to mitochondrial function and inflammatory response in sepsis. (B) Kinetics of autophagy and inflammatory response during sepsis. In addition to a hypoinflammatory response in protracted sepsis, the delayed autophagic depression may further contribute to mortality and morbidity by reduced microbial clearance, failure to sustain exotoxin tolerance and limited major histocompatibility complex IImediated antigen presentation. IL, interleukin; LPS, lipopolysaccharide; LTA, lipoteichoic acid; mtDNA, mitochondrial DNA; MYD88, myeloid differentiation primary response gene 88; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; NOD2, nucleotide-binding oligomerization domain containing 2; NLRP3, NLR family, pyrin domain containing 3; PGN, peptidoglycan; ROS, reactive oxygen species; SLR, sequestosome-like receptor; ssRNA, single-stranded ribonucleic acid; TNF, tumor necrosis factor; TLR, toll-like receptor; Cm, membrane potential.

should focus on the mechanisms that may prevent this process. The use of molecular tools to determine lysosomal function in addition to demonstrating their colocalization may be useful. Lung Respiratory failure usually occurs at the very early stage of sepsis. Consistent with this, the expression of autophagy-related proteins (e.g. LC3-II, ATG2, RAB7) declines very early (at 4 h) and this continues up to 24 h. This decline is associated with increased expression of pro-apoptotic proteins, such as FADD (Fas [TNFRSF6]-associated via death domain), BAX and cleaved CASP3 (caspase 3). Experimental induction of autophagy by rapamycin and activated PROC (protein C) mitigates

apoptosis and pro-inflammatory cytokines.52 In addition, mice overexpressing LC3 ablate apoptosis, inflammation, and neutrophil infiltration.53 Furthermore, the protective effect of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) against sepsis-related lung injury is mediated by ATG12-dependent autophagy enhancement.54,55 Deletion of Atg4b in LPS-challenged mice shows more prominent signs of pulmonary inflammation via modulation of ATF3 (activating transcription factor 3).56 Kidney Upon LPS challenge, young mice demonstrate a higher LC3-II level than old mice, accompanied with lower CST3 (cystatin C)

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Table 1. Effects of autophagy modulation on major organs in experimental sepsis. Organs

Interventions

Effects

Outcomes

Heart

Rubcn knockout

Induced autophagy

Immune

PARK2 knockout Silencing ATG5 with siRNA BECN1 hypomorphism and LC3B knockout Deletion of Atg7

Suppressed mitophagy Inhibited autophagy Reduced autophagy

Administration of chloroquine Knockout of genes encoding SFTPA1 and D Administration of carbamazepine

Inhibited autophagy Enhanced autophagy

Silencing ATG7 by siRNA

Suppressed autophagosome formation

Administration of activated PROC and rapamycin

Enhanced autophagic flux

LC3 over-expression Injection of GADPH

Increased clearance of autophagosomes Increased expression of ATG12

Silencing PIK3C3 by siRNA

Reduced autophagy

Suppression of GSK3B

CAMK4-MTOR dependent autophagic sustained Enhanced autophagy

Liver

Lung

Kidney

Brain

Administration of pyrrolidine dithiocarbamate

Reduced autophagosome formation

Enhanced autophagy

and blood urinary nitrogen (BUN).57 Silencing of PIK3C3/ Vps34 by siRNA results in the development of acute kidney injury subsequent to LPS exposure.57 Consistent with this finding, inhibition of autophagy by 3-methyladenine significantly enhances tubular injury.58 Phosphorylation of MTOR is downregulated by small hairpin RNA for genes encoding subunits of the 50 adenosine monophosphate-activated protein kinase (AMPK), which decreases autophagy and simultaneously increases apoptosis of renal tubular cells. The kidneys isolated from camk4¡/¡ mice show reduced expression of ATG5, ATG7 and LC3B upon LPS challenge.59 Skeletal muscle Mitochondrial damage in skeletal muscle occurs over the course of sepsis. Exposure of C57/BL6 mice to LPS for 24 h reveals dysmorphic mitochondria in tibialis anterior and soleus muscles. This goes along with an increased number of autophagosomes, LC3B and upregulation of ATG14, ATG12, BECN1 and LAMP2A. The elevation of autophagy activity is abolished by suppression of NFKBI/IkB, suggesting NFKB negatively regulates autophagy in skeletal muscles upon endotoxemia.31 Indeed, induction of autophagy may increase the rate of protein catabolism.60 Whether muscle wasting in septic patients is associated with elevation of autophagy requires further investigation. Brain Encephalopathy is associated with increased risk of mortality and morbidity in sepsis. This is characterized by a distorted blood brain barrier, an increased level of oxidative stress and

References

Improved diastolic filling and reduced apoptosis of cardiomyocytes Impaired cardiac contractility Hastened the occurrence of apoptosis Enhanced production of IL1B and IL18

35

Apoptosis of CD4C and CD8C lymphocytes Release of HMGB1 from macrophages Reduced serum transaminase

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Reduced level of peripheral cytokines and AST/SGOT Hepatocytes viability decreased upon challenged by TNF Reduced apoptosis and production of IL6 and TNF. Sustainable clearance of autophagosome Alleviated neutrophil infiltration and reduced pulmonary edema Serum level of pro-inflammatory cytokines and neutrophil infiltration were reduced Increased serum CST3 and BUN with signs of tubular injury Reduced renal tubular injury Reduced neuronal apoptosis in hippocampus

37 28 46

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43

53

54,55

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increased apoptosis.61 Wistar rats after CLP show increased autophagosome formation and lysosome activation in the hippocampus. These changes are accompanied by increased LC3-II and decreased BECN1, LAMP1 and RAB7. Inhibition of NFKB by pyrrolidine dithiocarbamate increases the level of LC3-II, BECN1, LAMP1 and RAB7.62 Mitophagy in sepsis Loss of mitochondrial membrane potential accompanied with the release of hydrogen peroxide and increased apoptosis have been documented in experimental sepsis.12,17,29 In addition, mitochondrial DNA is released into systemic circulation after organelle injury. In this regard, mitophagy plays an important role in eliminating defective mitochondria from the cytosol, thereby protecting the host against oxidative injury as well as inflammatory response. In a prospective cohort study, patients who developed post-traumatic sepsis had a significantly higher plasma level of mitochondrial DNA as compared to those who did not (1774 pg/mL vs 500 pg/mL, P< 0.001).63 In rats subjected to CLP-induced sepsis, mitochondrial grid intersects reduce significantly, accompanied by decreased activity of CS (citrate synthase) in both harvested heart homogenates and in the mitochondrial fraction.64-66 Sepsis-induced mitophagy requires MAPK9/JNK2 kinase through targeting the small mitochondrial form of the tumor suppressor CDKN2A/ARF for degradation.67 In the lungs, mitophagy in alveoli appears to be dependent on NFE2L2/ NRF2.68 Interestingly, reduced autophagic activity may prevent exocytosis of cytosolic mitochondrial DNA, perhaps conferring protection for the host. In Atg5C/C murine embryonic

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fibroblasts and rat hepatocytes, mitochondrial constituents are actively released from the cell, but not from atg5¡/¡ cells.17 In contrast, deletion of genes encoding Map1lc3b and Becn1 in macrophages enhances the release of mitochondria DNA mediated by the NRLP3 inflammasome.46 Late-stage suppression of autophagy is detrimental Collectively, studies reveal that autophagy increases transiently upon encounter of septic insult. This initial increase is followed by a prolonged decline of autophagic flux (Fig. 1B), contributing to organ dysfunction.36,37,69 The impaired autophagic function, as demonstrated by silencing of ATG7 using siRNA, aggravates TNF (tumor necrosis factor)-induced DNA fragmentation in proximal tubular cells and in hepatocytes.36,69 To date, the mechanisms contributing to the decline of autophagic activity in sepsis remain elusive. Possible mechanisms may include hyperactivity during the initial stage of microbial encounters, leading to the exhaustion of essential precursors for autophagy induction. Supporting this notion, overexpression of LC3 can successfully restore the autophagic insufficiency in septic lung.52 Given multiple roles of autophagy in microbial clearance,70 neutralizing microbial toxins,71 maintaining mitochondrial integrity46 and controlling cytokine release,32 suppression of the process would likely lead to poor outcome in sepsis. The progression of sepsis into immunosuppression may be explained by the role of autophagy in maintaining survival and proliferation of T-lymphocytes.39 Consequently, reduced lymphocyte counts increases the likelihood of acquiring opportunistic infections, including otherwise latent viruses.33 Microbe-host interactions and autophagy Autophagy involves direct clearance of intracellularly replicating bacteria by targeting the invaders for lysosomal degradation. Subsequent degraded microbial components will be presented on major histocompatibility complex (MHC) II molecules.5 Despite this robust degradation, certain microbes could express virulence factors, which either stall autophagosomelysosome fusion or inhibit the initiation of autophagosome formation.2,5,70 The role of autophagy in sepsis is also dependent on bacterial factors. Inoculation of wild-type Staphylococcus aureus strain USA300 confers higher mortality rate in lc3b¡/¡ and ATG16L1 hypomorphic mice as compared to wild-type groups. Conversely, administration of a-toxin-deleted S. aureus USA300 protects ATG16L1-deficient mice against sepsis.70 Interestingly, deletion of Atg16l1 in endothelial cells is more lethal than that in macrophages or dendritic cells.68 The interaction between bacteria and autophagy, or xenophagy, has been comprehensively reviewed by Huang and colleagues.72 Autophagy activator: An emerging therapeutic strategy for sepsis In critical care settings, the amount of caloric intake has long been a concern. Given that diabetes-independent blood sugar deregulation is rather common among the critically ill, overfeeding may result in poor clinical outcomes.73 As such, permissive underfeeding has been increasingly recognized as a

strategy to promote a positive clinical outcome. In a large randomized trial, a total of 894 critically ill patients were randomized into 2 groups: those receiving standard enteral feeding, and those subjected to permissive underfeeding. The incidence of renal replacement therapy of the latter group was significantly lower.74 These findings suggested that maintaining a moderate starvation state to sustain autophagy activity might reduce sepsis morbidity. Therapeutic activation of autophagy appears to be protective against septic insults.75,76 Sinomenine, a natural herbal extract, improves survival in septic mice by increased conversion of LC3-I to LC3-II in the liver and lungs.18 A green tea extract, epigallocatechin-3-gallate, also reduces expression of HMGB1 in LPS-challenged murine macrophages by BECN1-dependent autophagy.22 BECN1 is essential for carbon monoxide inhalation therapy in septic mice. Conversely, knocking out Becn1 renders the mice more susceptible to septic injury and unresponsive to carbon monoxide therapy.10 A helminth product, ES-62, also downregulates TLR2- and TLR4-driven inflammatory responses by enhancing MYD88-dependent autophagosome formation.21 The mechanisms of protective effect of autophagy activation in sepsis remain to be elucidated. Further pharmacokinetic/pharmacodynamic studies of the potential therapeutic agents may reveal which organs are protected. This can be helpful in developing organ-targeted therapy to sustain vital organ function over the course of sepsis. Most of the studies to date are largely in vitro experiments and animal models, limiting their translational value. Clinical implications Autophagy plays a protective role in sepsis partially by preserving the integrity of mitochondria, preventing apoptosis and promoting antigen presentation.5,29,40 However, numerous studies have indicated autophagy suppression at the late stage of the disease. The reduced autophagy activity is associated with organ failure.11,36,37,57 One of the reasons for reduced autophagy activity could be impaired fusion of autophagosomes with lysosomes.37 This indicates that modulating the later stage of autophagy at the appropriate time of sepsis may be a promising therapeutic strategy. Determination of the etiology contributing to the impairment (i.e. exacerbation due to a negative factor or inhibition of a positive one) will help identify target molecules for further investigations. Concluding remarks and perspectives Collectively, both cell and animal models suggest that autophagy is induced in the early stage of sepsis. This appears to involve predominantly the liver and the heart. Further studies may be useful to determine other types of cells that respond to sepsis-induced autophagy. In addition, the association between ATG gene polymorphisms and sepsis morbidity and mortality may lead to further studies using targeted capture sequencing of relevant genetic loci to determine their prognostic value. The late-stage exhaustion of autophagic activity is associated with inflammatory dysregulation, histological changes, mitochondrial dysfunction and apoptosis. Experimental induction of autophagy has increased survival rate among septic animals.

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The feasibility of using autophagy activators as sepsis therapy is exemplified by successfully minimized organ injury and mortality using a variety of therapeutic agents in septic animals, but their effects on late-stage autophagy (i.e., autophagosome-lysosome fusion and lysosome-dependent degradation) remain to be examined. Reverse of late stage autophagy may be a good therapeutic strategy. However, an investigation of a biomarker for the diagnosis of autophagic impairment in vivo will be essential to guide further trials on administrating autophagy inducers at the appropriate time-point. Although it requires further investigations to translate these animal experiments into clinical practice, these data suggest that the use of autophagy modulators would be a promising strategy for minimizing sepsis-associated health care burden. However, the mechanisms underlying autophagy exhaustion after transient induction has to be elucidated before implementation. The use of siRNA may provide some clues in the molecular pathway involved in the process. Currently, the autophagy kinetics beyond 48 h remain under investigated. Understanding of autophagy regulation at this later stage of sepsis may better reflect physiological and molecular determinants of mortality in the immunosuppressive phase of sepsis.

NFE2L2 NFKB NLRP3 NOS2 PARK2 PIK3C3 PROC Rubcn SFTPA1 SQSTM1 TNF TLR VPS

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nuclear factor, erythroid derived 2, like 2 nuclear factor of kappa light polypeptide gene enhancer in B cells NLR family, pyrin domain containing 3 nitric oxide synthase 2, inducible Parkinson disease (autosomal recessive, juvenile) 2, parkin phosphoinositide-3-kinase, class 3 protein C; RAB7, RAB7, member RAS oncogene family RUN domain and cysteine-rich domain containing, Beclin 1-interacting, protein surfactant associated protein A1 sequestosome 1 tumor necrosis factor toll- like receptor vaculolar protein sorting

Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

Funding Abbreviations AIM2 ATF3 ATG BAX BCL2 BECN1 BNIP3

absent in melanoma 2 activating transcription factor 3 autophagy related BCL2-associated X protein B-cell CLL/lymphoma 2 Beclin 1, autophagy related Bcl2/adenovirus E1B 19kDa interacting protein 3 BUN blood urea nitrogen CAMK4 calcium/calmodulin-dependent protein kinase IV CASP3 caspase 3 CAT catalase CLP cecal ligation and puncture CST3 cystatin C CYCS cytochrome C, somatic ER endoplasmic reticulum FADD Fas (TNFRSF6)-associated via death domain GAPDH glyceraldegyde-3-phosphate dehydrogenase GSK3B glycogen synthase kinase 3 b HSPA5 heat shock protein 5 IL1B interleukin 1 b; IL18, interleukin 18 IRF1 interferon regulatory factor 1 IRGM immunity-related GTPase family M LAMP1 lysosomal-associated membrane protein 1 LPS lipopolysaccharide MAP1LC3/LC3 microtubule-associated protein 1 light chain 3 MAPK mitogen-activated protein kinase MHC major histocompatibility complex MTOR mechanistic target of rapamycin (serine/threonine kinase) MYD88 myeloid differentiation primary response gene 88

This work was supported by Hong Kong Research Grant Council-General Research Fund (464212, 24115815) and Food and Health BureauCommissioned Research on Control of Infectious Diseases (CU-15-B2) and -Health and Medical Research Fund (15140132).

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