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Evolution, role in inflammation, and redox control of leaderless secretory proteins

  • Roberto Sitia
    Correspondence
    For correspondence: Roberto Sitia; Anna Rubartelli
    Affiliations
    Division of Genetics and Cell Biology, Protein Transport and Secretion Unit, IRCCS Ospedale San Raffaele/Università Vita-Salute San Raffaele, Milan, Italy
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  • Anna Rubartelli
    Correspondence
    For correspondence: Roberto Sitia; Anna Rubartelli
    Affiliations
    Division of Genetics and Cell Biology, Protein Transport and Secretion Unit, IRCCS Ospedale San Raffaele/Università Vita-Salute San Raffaele, Milan, Italy

    Cell Biology Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy
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Open AccessPublished:April 24, 2020DOI:https://doi.org/10.1074/jbc.REV119.008907
      Members of the interleukin (IL)-1 family are key determinants of inflammation. Despite their role as intercellular mediators, most lack the leader peptide typically required for protein secretion. This lack is a characteristic of dozens of other proteins that are actively and selectively secreted from living cells independently of the classical endoplasmic reticulum-Golgi exocytic route. These proteins, termed leaderless secretory proteins (LLSPs), comprise proteins directly or indirectly involved in inflammation, including cytokines such as IL-1β and IL-18, growth factors such as fibroblast growth factor 2 (FGF2), redox enzymes such as thioredoxin, and proteins most expressed in the brain, some of which participate in the pathogenesis of neurodegenerative disorders. Despite much effort, motifs that promote LLSP secretion remain to be identified. In this review, we summarize the mechanisms and pathophysiological significance of the unconventional secretory pathways that cells use to release LLSPs. We place special emphasis on redox regulation and inflammation, with a focus on IL-1β, which is secreted after processing of its biologically inactive precursor pro-IL-1β in the cytosol. Although LLSP externalization remains poorly understood, some possible mechanisms have emerged. For example, a common feature of LLSP pathways is that they become more active in response to stress and that they involve several distinct excretion mechanisms, including direct plasma membrane translocation, lysosome exocytosis, exosome formation, membrane vesiculation, autophagy, and pyroptosis. Further investigations of unconventional secretory pathways for LLSP secretion may shed light on their evolution and could help advance therapeutic avenues for managing pathological conditions, such as diseases arising from inflammation.

      Introduction

      How secreted proteins are selected from the crowded interior of cells is a key question in biology. In the early 1950s, by integrating both morphological and biochemical findings, George Palade along with other researchers established the role of the endoplasmic reticulum (ER) and the Golgi apparatus in the transport and release of secreted proteins (
      • Palade G.E.
      Structure and function at the cellular level.
      ). Günter Blobe, César Milstein, and others further demonstrated that an N-terminal stretch of hydrophobic amino acids (called signal peptide or leader sequence) is characteristic of proteins destined to enter the ER and begin their voyage toward the extracellular space (
      • Milstein C.
      • Brownlee G.G.
      • Harrison T.M.
      • Mathews M.B.
      A possible precursor of immunoglobulin light chains.
      ,
      • Blobel G.
      • Dobberstein B.
      Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.
      ). Thus, when in 1984 Charles Dinarello's laboratory cloned the gene of the soluble inflammatory cytokine interleukin-1β (IL-1β) (
      • Auron P.E.
      • Webb A.C.
      • Rosenwasser L.J.
      • Mucci S.F.
      • Rich A.
      • Wolff S.M.
      • Dinarello C.A.
      Nucleotide sequence of human monocyte interleukin 1 precursor cDNA.
      ), the absence of a signal peptide in this protein raised an intriguing question for cell biologists. How can a protein devoid of a secretory signal find its way out of the cell? At first, given its proinflammatory role, it was thought that IL-1β is released by dying cells at sites of injury or infection. However, many lines of evidence argued against this hypothesis. Intrigued by this problem, we and others investigated IL-1β secretion in human monocytes and observed the following.
      (i) The release of IL-1β is selective: upon being triggered by lipopolysaccharide (LPS), human monocytes release mature IL-1β, but little if any of other abundant proteins, as are released upon necrosis (
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      ).
      (ii) Monocytes accumulate the biologically inactive precursor pro-IL-1β (33 kDa) in the cytosol and secrete active IL-1β (17 kDa). Dying cells release only pro-IL-1β, implying that its processing requires living cells and is coupled to secretion.
      (iii) Drugs that block ER-to-Golgi traffic (e.g. brefeldin A) do not inhibit IL-1β secretion (
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      ).
      (iv) Human IL-1β lacks post-translational modifications that occur along the ER-Golgi route. However, glycosylated IL-1β is secreted when it is appended with a leader sequence (
      • Baldari C.
      • Murray J.A.
      • Ghiara P.
      • Cesareni G.
      • Galeotti C.L.
      A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1β in Saccharomyces cerevisiae.
      ).
      Together, these observations implied the existence of a novel pathway for IL-1β secretion (
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      ) and possibly for other proteins devoid of a signal peptide (
      • Cooper D.N.
      • Barondes S.H.
      Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism.
      ). The existence of secretory pathways alternative to the ER-Golgi route had been shown in yeast for a-mating factor (
      • Kuchler K.
      • Sterne R.E.
      • Thorner J.
      Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells.
      ). The family of leaderless secretory proteins (LLSPs) grew 2 years later with the discovery of the secretion of the cytosolic oxidoreductase thioredoxin (Trx) (
      • Rubartelli A.
      • Bajetto A.
      • Allavena G.
      • Wollman E.
      • Sitia R.
      Secretion of Trx by normal and neoplastic cells through a leaderless secretory pathway.
      ) and basic fibroblast growth factor (bFGF, now FGF2) (
      • Mignatti P.
      • Morimoto T.
      • Rifkin D.B.
      Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex.
      ), prompting us to introduce the term “leaderless secretion” (
      • Rubartelli A.
      • Bajetto A.
      • Allavena G.
      • Wollman E.
      • Sitia R.
      Secretion of Trx by normal and neoplastic cells through a leaderless secretory pathway.
      ). Many LLSPs were later found (Table 1). More recently, the term “unconventional protein secretion” (UPS) was introduced to include the transport of some transmembrane proteins that are translocated co-translationally into the ER but bypass the Golgi to reach the plasma membrane (Fig. 1A). Readers are referred to a special issue of Seminars in Cell and Developmental Biology entirely devoted to the UPS (
      ).
      Table 1Cellular functions and locations of leaderless secretory proteins
      ProteinExtracellular functionIntracellular localization and functionReferences
      Cytokines
       IL-1αMediator of inflammation and immune responseNucleus, regulation of transcription
      • Gross O.
      • Yazdi A.S.
      • Thomas C.J.
      • Masin M.
      • Heinz L.X.
      • Guarda G.
      • Quadroni M.
      • Drexler S.K.
      • Tschopp J.
      Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.
      ,
      • Keller M.
      • Rüegg A.
      • Werner S.
      • Beer H.D.
      Active caspase-1 is a regulator of unconventional protein secretion.
      (see also Ref.
      • Carta S.
      • Lavieri R.
      • Rubartelli A.
      Different members of the IL-1 family come out in different ways: DAMPs vs. cytokines?.
      and references therein)
       IL-1βMediator of inflammation and immune responseCytosol, function ND
      ND, not determined.
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      (see also Ref.
      • Carta S.
      • Lavieri R.
      • Rubartelli A.
      Different members of the IL-1 family come out in different ways: DAMPs vs. cytokines?.
      and references therein
       IL-18Mediator of inflammation and immune responseCytosol, function ND
      • Carta S.
      • Lavieri R.
      • Rubartelli A.
      Different members of the IL-1 family come out in different ways: DAMPs vs. cytokines?.
      (and references therein),
      • Gardella S.
      • Andrei C.
      • Poggi A.
      • Zocchi M.R.
      • Rubartelli A.
      Control of interleukin-18 secretion by dendritic cells: role of calcium influxes.
       IL-33Mediator of inflammation and immune responseNucleus, function ND
      • Kakkar R.
      • Hei H.
      • Dobner S.
      • Lee R.T.
      Interleukin 33 as a mechanically responsive cytokine secreted by living cells.
      (see also Ref.
      • Carta S.
      • Lavieri R.
      • Rubartelli A.
      Different members of the IL-1 family come out in different ways: DAMPs vs. cytokines?.
      and references therein)
       IL-37Inhibitor of innate immunityCytosol and nucleus, inhibitor of innate immunity
      • Bulau A.M.
      • Nold M.F.
      • Li S.
      • Nold-Petry C.A.
      • Fink M.
      • Mansell A.
      • Schwerd T.
      • Hong J.
      • Rubartelli A.
      • Dinarello C.A.
      • Bufler P.
      Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses.
       Caspase-1Extracellular processing of pro-IL-1?Cytosol, IL-1β, and IL-18 convertase; mediator of secretion of other LLSPs
      • Keller M.
      • Rüegg A.
      • Werner S.
      • Beer H.D.
      Active caspase-1 is a regulator of unconventional protein secretion.
      ,
      • Gattorno M.
      • Tassi S.
      • Carta S.
      • Delfino L.
      • Ferlito F.
      • Pelagatti M.A.
      • D'Osualdo A.
      • Buoncompagni A.
      • Alpigiani M.G.
      • Alessio M.
      • Martini A.
      • Rubartelli A.
      Pattern of interleukin-1β secretion in response to lipopolysaccharide and ATP before and after interleukin-1 blockade in patients with CIAS1 mutations.
       FGF-1/acidic FGFGrowth, survival, and differentiating factorNucleus, anti-apoptotic, neurotrophic factor
      • Prudovsky I.
      • Tarantini F.
      • Landriscina M.
      • Neivandt D.
      • Soldi R.
      • Kirov A.
      • Small D.
      • Kathir K.M.
      • Rajalingam D.
      • Kumar T.K.
      Secretion without Golgi.
      (and references therein)
       FGF-2/basic FGFGrowth, survival, and differentiating factorNuclear, anti-apoptotic factor
      • Mignatti P.
      • Morimoto T.
      • Rifkin D.B.
      Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex.
      (see also Ref.
      • Steringer J.P.
      • Nickel W.
      A direct gateway into the extracellular space: unconventional secretion of FGF2 through self-sustained plasma membrane pores.
      and references therein)
       Annexin A1, A2, A4, A5Modulators of coagulation and anti-inflammatory mediator; soluble or bound to the outer leaflet of the plasma membraneCytosol: membrane trafficking and organization; nucleus: pro-apoptotic
      • Popa S.J.
      • Stewart S.E.
      • Moreau K.
      Unconventional secretion of annexins and galectins.
      (and references therein)
       Members of the galectin protein familyMediator of innate immunityCytosol, pro-apoptotic
      • Cooper D.N.
      • Barondes S.H.
      Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism.
      (see also Ref.
      • Popa S.J.
      • Stewart S.E.
      • Moreau K.
      Unconventional secretion of annexins and galectins.
      and references therein)
       Members of the S100 protein family (S100A8, S100A9)Antimicrobial and chemotactic activitiesCytosol, EF-hand domain–containing Ca2+-binding proteins
      • Rammes A.
      • Roth J.
      • Goebeler M.
      • Klempt M.
      • Hartmann M.
      • Sorg C.
      Myeloid-related protein (MRP) 8 and MRP14, calcium-binding proteins of the S100 family, are secreted by activated monocytes via a novel, tubulin-dependent pathway.
       MIF-1Pro-inflammatory mediatorCytosol, function ND
      • Flieger O.
      • Engling A.
      • Bucala R.
      • Lue H.
      • Nickel W.
      • Bernhagen J.
      Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter.
      ,
      • Lee J.P.
      • Foote A.
      • Fan H.
      • Peral de Castro C.
      • Lang T.
      • Jones S.A.
      • Gavrilescu N.
      • Mills K.H.
      • Leech M.
      • Morand E.F.
      • Harris J.
      Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages.
       Endothelial monocyte–activating polypeptide (EMAP)-2Pro-inflammatory mediator with anti-angiogenic propertiesCytosol, protein translation (tRNA multisynthetase complex)
      • Wakasugi K.
      • Schimmel P.
      Two distinct cytokines released from a human aminoacyl-tRNA synthetase.
       HMGB1Mediator of inflammation and immune responseNucleus, transcriptional regulation, DNA replication and repair, and nucleosome assembly
      • Gardella S.
      • Andrei C.
      • Ferrera D.
      • Lotti L.V.
      • Torrisi M.R.
      • Bianchi M.E.
      • Rubartelli A.
      The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway.
      (see also Ref.
      • Andersson U.
      • Tracey K.J.
      HMGB1 is a therapeutic target for sterile inflammation and infection.
      and references therein)
       Phosphoglucose isomerase/autocrine motility factor (PGI/AMF)Inducer of cell migrationCytosol, glycolytic enzyme (phosphoglucose isomerase)
      • Niinaka Y.
      • Paku S.
      • Haga A.
      • Watanabe H.
      • Raz A.
      Expression and secretion of neuroleukin/phosphohexose isomerase/maturation factor as autocrine motility factor by tumor cells.
      Redox enzymes
       TrxCytokine-like inflammatory mediator, possibly through thiol-disulfide exchangeCytosol, oxidoreductase
      • Rubartelli A.
      • Bajetto A.
      • Allavena G.
      • Wollman E.
      • Sitia R.
      Secretion of Trx by normal and neoplastic cells through a leaderless secretory pathway.
       Trx reductase (TrxR)Regeneration of oxidized Trx?Cytosol, Trx reductase
      • Söderberg A.
      • Sahaf B.
      • Rosén A.
      Trx 2, a redox-active selenoprotein, is secreted by normal and neoplastic cells: presence in human plasma.
       Peroxiredoxin (PRDX)1, PRDX2Cytokine-like inflammatory mediator, possibly through peroxidation reactionsCytosol, peroxidases
      • Checconi P.
      • Salzano S.
      • Bowler L.
      • Mullen L.
      • Mengozzi M.
      • Hanschmann E.M.
      • Lillig C.H.
      • Sgarbanti R.
      • Panella S.
      • Nencioni L.
      • Palamara A.T.
      • Ghezzi P.
      Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress.
      ,
      • Salzano S.
      • Checconi P.
      • Hanschmann E.M.
      • Lillig C.H.
      • Bowler L.D.
      • Chan P.
      • Vaudry D.
      • Mengozzi M.
      • Coppo L.
      • Sacre S.
      • Atkuri K.R.
      • Sahaf B.
      • Herzenberg L.A.
      • Herzenberg L.A.
      • Mullen L.
      • Ghezzi P.
      Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal.
       SOD-1Prion-like properties, involved in familial ALSCytosol; converts superoxide radicals to molecular oxygen and hydrogen peroxide
      • Münch C.
      • O'Brien J.
      • Bertolotti A.
      Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells.
      ,
      • Mondola P.
      • Annella T.
      • Santillo M.
      • Santangelo F.
      Evidence for secretion of cytosolic CuZn superoxide dismutase by HEPG2 cells and human fibroblast.
      Brain proteins
       APE1/Ref-1Cytokine-like anti-inflammatory mediator, through thiol-disulfide exchangeNucleus, antioxidant
      • Lee Y.R.
      • Joo H.K.
      • Lee E.O.
      • Cho H.S.
      • Choi S.
      • Kim C.S.
      • Jeon B.H.
      ATP binding cassette transporter A1 is involved in extracellular secretion of acetylated APE1/Ref-1.
      ,
      • Park M.S.
      • Choi S.
      • Lee Y.R.
      • Joo H.K.
      • Kang G.
      • Kim C.S.
      • Kim S.J.
      • Lee S.D.
      • Jeon B.H.
      Secreted APE1/Ref-1 inhibits TNF-α-stimulated endothelial inflammation via thiol-disulfide exchange in TNF receptor.
       CNTFCytokine, member of the IL-6 family; neuroprotective, anti-inflammatoryCytosol, function ND
      • Jones S.A.
      • Jenkins B.J.
      Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer.
      ,
      • Sendtner M.
      • Stöckli K.A.
      • Thoenen H.
      Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration.
       TauInterneuronal signaling, trans-synaptic transmissionCytosol, stabilization of microtubules
      • Pooler A.M.
      • Phillips E.C.
      • Lau D.H.
      • Noble W.
      • Hanger D.P.
      Physiological release of endogenous tau is stimulated by neuronal activity.
      (see also Ref.
      • Pernègre C.
      • Duquette A.
      • Leclerc N.
      Tau secretion: good and bad for neurons.
      and references therein)
       α-SynucleinInternalized by neurons, modulator of neuronal deathPresynaptic vesicle, functioning as modulator of synaptic vesicle mobility
      • Lee H.J.
      • Patel S.
      • Lee S.J.
      Intravesicular localization and exocytosis of α-synuclein and its aggregates.
      • Emmanouilidou E.
      • Melachroinou K.
      • Roumeliotis T.
      • Garbis S.D.
      • Ntzouni M.
      • Margaritis L.H.
      • Stefanis L.
      • Vekrellis K.
      Cell-produced α-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
      • Rodriguez L.
      • Marano M.M.
      • Tandon A.
      Import and export of misfolded α-synuclein.
       Engrailed 2Secreted by tumor cells, modifies tumor microenvironmentNucleus, homeodomain-containing transcription factor functioning in early development
      • Maizel A.
      • Tassetto M.
      • Filhol O.
      • Cochet C.
      • Prochiantz A.
      • Joliot A.
      Engrailed homeoprotein secretion is a regulated process.
      ,
      • Punia N.
      • Primon M.
      • Simpson G.R.
      • Pandha H.S.
      • Morgan R.
      Membrane insertion and secretion of the Engrailed-2 (EN2) transcription factor by prostate cancer cells may induce antiviral activity in the stroma.
      Others
       TransglutaminaseCross-linking enzyme with pleiotropic activity; regulates the cellular interactions with the extracellular matrixCytosol, nucleus, mitochondria; cross-linking enzyme with pleiotropic activity; NF-KB activator
      • Lorand L.
      • Graham R.M.
      Transglutaminases: crosslinking enzymes with pleiotropic functions.
      ,
      • Zemskov E.A.
      • Mikhailenko I.
      • Hsia R.C.
      • Zaritskaya L.
      • Belkin A.M.
      Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes.
       Insulin-degrading enzyme (IDE)Zn2+ peptidase, amyloid peptide degradationCytosol, Zn2+ peptidase
      • Tamboli I.Y.
      • Barth E.
      • Christian L.
      • Siepmann M.
      • Kumar S.
      • Singh S.
      • Tolksdorf K.
      • Heneka M.T.
      • Lütjohann D.
      • Wunderlich P.
      • Walter J.
      Statins promote the degradation of extracellular amyloid-peptide by microglia via stimulation of exosome-associated insulin-degrading enzyme (IDE) secretion.
      a ND, not determined.
      Figure thumbnail gr1
      Figure 1Different protein secretion routes. A, the left section of the panel depicts the classical secretory pathway. Secretory and membrane proteins endowed with a signal peptide are co-translationally translocated into the ER and transported to the Golgi and downstream organelles of the exocytic pathway. Secretory vesicles eventually fuse with the plasma membrane, releasing secretory proteins into the extracellular environment and exposing membrane-bound proteins (
      • Palade G.E.
      Structure and function at the cellular level.
      • Milstein C.
      • Brownlee G.G.
      • Harrison T.M.
      • Mathews M.B.
      A possible precursor of immunoglobulin light chains.
      • Blobel G.
      • Dobberstein B.
      Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.
      ). Routes different from Classical exocytosis grouped under the name Unconventional protein secretion (UPS) (
      ) are shown on the right. A few leader sequence–bearing transmembrane proteins translocate co-translationally into the ER but bypass the Golgi en route to the plasma membrane. Many cytosolic proteins lacking the secretory signal peptide (i.e. LLSPs) are secreted via direct translocation through the plasma membrane (pore-mediated) or via intracellular vesicles that fuse with the membrane. Some LLSPs can be released via exosomes or microvesicles. B, DAMPs are released upon rupture of the cell membrane (
      • Vénéreau E.
      • Ceriotti C.
      • Bianchi M.E.
      DAMPs from cell death to new life.
      ). LLSPs instead selectively leave living cells via membrane translocation or via vesicles that pinch off the cell membrane (
      • Rubartelli A.
      • Sitia R.
      Secretion of mammalian proteins that lack a signal sequence.
      ). The latter mechanism requires selective accumulation at the membrane that will give rise to the vesicle. Translocation may occur at the plasma membrane or into intracellular vesicles, such as secretory lysosomes. Likewise, pinching off may occur directly at the plasma membrane, followed by shedding of microvesicles, or indirectly via multivesicular bodies that release exosomes. Upon translocation, LLSPs are immediately accessible, whereas, after pinching off, they are protected by a membrane until the vesicles break up.
      In this review, we summarize the current state of knowledge of the mechanistic features of LLSP secretion by mammalian cells and highlight the interplays among LLSPs, redox mechanisms, and inflammation, focusing on the prototypic cytokine, IL-1β.

      Key open questions concerning LLSPs

      How are LLSPs recognized?

      Despite numerous efforts (
      • Bendtsen J.D.
      • Jensen L.J.
      • Blom N.
      • Von Heijne G.
      • Brunak S.
      Feature-based prediction of non-classical and leaderless protein secretion.
      ,
      • Garg A.
      • Raghava G.P.
      A machine learning based method for the prediction of secretory proteins using amino acid composition, their order and similarity-search.
      ,
      • Kandaswamy K.K.
      • Pugalenthi G.
      • Hartmann E.
      • Kalies K.U.
      • Möller S.
      • Suganthan P.N.
      • Martinetz T.
      SPRED: a machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes.
      ,
      • Huang W.-L.
      Ranking gene ontology terms for predicting non-classical secretory proteins in eukaryotes and prokaryotes.
      ), the signals or motifs shared among LLSPs that mediate their secretion remained unidentified for many years. Sequence analyses revealed two features associated with LLSP subsets (Table 1) (
      • Bendtsen J.D.
      • Jensen L.J.
      • Blom N.
      • Von Heijne G.
      • Brunak S.
      Feature-based prediction of non-classical and leaderless protein secretion.
      ): (i) basic amino acid stretches in FGF1 and FGF2, IL-1α, high-mobility group box 1 (HMGB1), histone 4, and engrailed-2 (i.e. in LLSPs with a nuclear localization) and (ii) the redox-active CXXC in oxidoredoxins (e.g. Trx and macrophage migration–inhibitory factor (MIF)). In FGF1 (
      • Graziani I.
      • Bagalá C.
      • Duarte M.
      • Soldi R.
      • Kolev V.
      • Tarantini F.
      • Kumar T.K.
      • Doyle A.
      • Neivandt D.
      • Yu C.
      • Maciag T.
      • Prudovsky I.
      Release of FGF1 and p40 synaptotagmin 1 correlates with their membrane destabilizing ability.
      ) and FGF2 (
      • Zehe C.
      • Engling A.
      • Wegehingel S.
      • Schäfer T.
      • Nickel W.
      Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2.
      ), the basic stretch is indeed required for their secretion. In other LLSPs, however, the role of the basic sequence in secretion has not been addressed. Moreover, many redox enzymes carrying the CXXC motif are not secreted, and mutations of the redox active site in Trx do not block its export (
      • Tanudji M.
      • Hevi S.
      • Chuck S.L.
      The nonclassic secretion of Trx is not sensitive to redox state.
      ). Very recently, a study by Zhang et al. (
      • Zhang M.
      • Liu L.
      • Lin X.
      • Wang Y.
      • Li Y.
      • Guo Q.
      • Li S.
      • Sun Y.
      • Tao X.
      • Zhang D.
      • Lv X.
      • Zheng L.
      • Ge L.
      A translocation pathway for vesicle-mediated unconventional protein secretion.
      ) proposed that a motif present on various IL-1 family members and other LLSPs drives the secretion of these proteins and showed that this motif is sufficient to direct a leaderless cargo to the route of secretion.

      How do LLSPs exit from living cells?

      To exit cells without jeopardizing the cells' integrity, LLSPs must either translocate through a membrane or localize to selected membrane areas that pinch off as vesicles (
      • Rubartelli A.
      • Sitia R.
      Secretion of mammalian proteins that lack a signal sequence.
      ) (Fig. 1B). Translocation can involve the plasma membrane or intracellular vesicles, such as secretory lysosomes, that eventually fuse with the plasma membrane. Likewise, vesicular mechanisms can exploit release of microvesicles from the plasma membrane or of exosomes after exocytosis of multivesicular bodies. Additional or different routes are used by different cell types (
      ). For instance, autophagy has been proposed for secretion of the acyl CoA-binding protein Acb1 in yeast (
      • Duran J.M.
      • Anjard C.
      • Stefan C.
      • Loomis W.F.
      • Malhotra V.
      Unconventional secretion of Acb1 is mediated by autophagosomes.
      ) and IL-1β in mammals (
      • Dupont N.
      • Jiang S.
      • Pilli M.
      • Ornatowski W.
      • Bhattacharya D.
      • Deretic V.
      Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β.
      ) (Fig. 2A). However, the role of autophagy remains controversial, as findings from other studies suggest that autophagy prevents IL-1β secretion (
      • Saitoh T.
      • Fujita N.
      • Jang M.H.
      • Uematsu S.
      • Yang B.G.
      • Satoh T.
      • Omori H.
      • Noda T.
      • Yamamoto N.
      • Komatsu M.
      • Tanaka K.
      • Kawai T.
      • Tsujimura T.
      • Takeuchi O.
      • Yoshimori T.
      • Akira S.
      Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production.
      ,
      • Harris J.
      • Hartman M.
      • Roche C.
      • Zeng S.G.
      • O'Shea A.
      • Sharp F.A.
      • Lambe E.M.
      • Creagh E.M.
      • Golenbock D.T.
      • Tschopp J.
      • Kornfeld H.
      • Fitzgerald K.A.
      • Lavelle E.C.
      Autophagy controls IL-1β secretion by targeting pro-IL-1β for degradation.
      ,
      • Zhou R.
      • Yazdi A.S.
      • Menu P.
      • Tschopp J.
      A role for mitochondria in NLRP3 inflammasome activation.
      ) and that autophagic failure promotes α-synuclein release (
      • Lee H.-J.
      • Cho E.-D.
      • Lee K.W.
      • Kim J.-H.
      • Cho S.-G.
      • Lee S.-J.
      Autophagic failure promotes the exocytosis and intercellular transfer of α-synuclein.
      ).
      Figure thumbnail gr2
      Figure 2LLSPs that translocate into autophagic or endolysosomal vesicles can be partially degraded. A, through still largely undefined mechanisms, LLSPs may end up in secretory lysosomes or else in the lumen or the intermembrane space of double-membrane autophagosomes (thick arrows). Both vesicle types can fuse with the cell membrane and release their contents into the extracellular space. If they fuse with lysosomes, however, their contents are degraded. B, cytosolic proteins may translocate to late endocytic vesicles following recognition by HSP chaperones. In MAPS, misfolded substrates are first recognized by HSP70 and enriched at the ER surface by USP19 (
      • Lee J.G.
      • Takahama S.
      • Zhang G.
      • Tomarev S.I.
      • Ye Y.
      Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.
      ,
      • Lee J.
      • Xu Y.
      • Zhang T.
      • Cui L.
      • Saidi L.
      • Ye Y.
      Secretion of misfolded cytosolic proteins from mammalian cells is independent of chaperone-mediated autophagy.
      ). They are then transferred to late endosomes by DNAJC5 for translocation that is followed by exocytosis. C, in CMA, HSC70 recognizes substrates with KFERQ-like motifs and targets them to lysosomes for LAMP2a-dependent translocation (
      • Dice J.F.
      • Chiang H.L.
      • Spencer E.P.
      • Backer J.M.
      Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7–11 as the essential pentapeptide.
      ,
      • Cuervo A.M.
      • Dice J.F.
      A receptor for the selective uptake and degradation of proteins by lysosomes.
      ). Although the “classical” CMA is not part of the pathways for LLSP secretion, it has many features common to other UPS pathways. Interestingly, some CMA cargoes are not degraded but are secreted upon fusion with the plasma membrane (
      • Zhou D.
      • Li P.
      • Lin Y.
      • Lott J.M.
      • Hislop A.D.
      • Canaday D.H.
      • Brutkiewicz R.R.
      • Blum J.S.
      Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens.
      ).
      In yeast, nutrient starvation favors the appearance of Acb1-containing vesicles that lack the double membrane typical of autophagosomes (
      • Cruz-Garcia D.
      • Curwin A.J.
      • Popoff J.F.
      • Bruns C.
      • Duran J.M.
      • Malhotra V.
      Remodeling of secretory compartments creates CUPS during nutrient starvation.
      ). These structures, belonging to the so-called compartment for UPS (CUPS) are then released as exosomes (
      • Cruz-Garcia D.
      • Curwin A.J.
      • Popoff J.F.
      • Bruns C.
      • Duran J.M.
      • Malhotra V.
      Remodeling of secretory compartments creates CUPS during nutrient starvation.
      ). The relationships among LLSP-containing mammalian autophagic vesicles (
      • Dupont N.
      • Jiang S.
      • Pilli M.
      • Ornatowski W.
      • Bhattacharya D.
      • Deretic V.
      Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β.
      ), secretory lysosomes (
      • Andrei C.
      • Dazzi C.
      • Lotti L.
      • Torrisi M.R.
      • Chimini G.
      • Rubartelli A.
      The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles.
      ,
      • Gardella S.
      • Andrei C.
      • Lotti L.V.
      • Poggi A.
      • Torrisi M.R.
      • Zocchi M.R.
      • Rubartelli A.
      CD8+ T lymphocytes induce polarized exocytosis of secretory lysosomes by dendritic cells with release of interleukin-1β and cathepsin D.
      ,
      • Gardella S.
      • Andrei C.
      • Ferrera D.
      • Lotti L.V.
      • Torrisi M.R.
      • Bianchi M.E.
      • Rubartelli A.
      The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway.
      ), and yeast CUPS remain to be investigated.
      An interesting secretion route is the so-called misfolding-associated protein secretion (MAPS) pathway (
      • Lee J.G.
      • Takahama S.
      • Zhang G.
      • Tomarev S.I.
      • Ye Y.
      Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.
      ), which mediates the release of potentially cytotoxic polypeptides such as α-synuclein and Tau and other cytosolic misfolded proteins. These proteins are delivered to late endosomes that, like secretory lysosomes, eventually fuse with the plasma membrane, releasing their contents (
      • Lee J.G.
      • Takahama S.
      • Zhang G.
      • Tomarev S.I.
      • Ye Y.
      Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.
      ,
      • Fontaine S.N.
      • Zheng D.
      • Sabbagh J.J.
      • Martin M.D.
      • Chaput D.
      • Darling A.
      • Trotter J.H.
      • Stothert A.R.
      • Nordhues B.A.
      • Lussier A.
      • Baker J.
      • Shelton L.
      • Kahn M.
      • Blair L.J.
      • Stevens Jr., S.M.
      • Dickey C.A.
      DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.
      ) (Fig. 2B).

      How many unconventional LLSP secretory pathways are there?

      In some cases, a single LLSP can exploit more than one secretion pathway (
      • Pernègre C.
      • Duquette A.
      • Leclerc N.
      Tau secretion: good and bad for neurons.
      ) (Table 1). For instance, Tau can use MAPS but can also directly translocate through the plasma membrane (
      • Merezhko M.
      • Brunello C.A.
      • Yan X.
      • Vihinen H.
      • Jokitalo E.
      • Uronen R.L.
      • Huttunen H.J.
      Secretion of Tau via an unconventional non-vesicular mechanism.
      ,
      • Katsinelos T.
      • Zeitler M.
      • Dimou E.
      • Karakatsani A.
      • Muller H.M.
      • Nachman E.
      • Steringer J.P.
      • Ruiz de Almodovar C.
      • Nickel W.
      • Jahn T.R.
      Unconventional secretion mediates the trans-cellular spreading of tau.
      ) or be released via endo-lysosomal vesicles or exosomes (
      • Pernègre C.
      • Duquette A.
      • Leclerc N.
      Tau secretion: good and bad for neurons.
      ). Multiple mechanisms have been described also for IL-1β secretion and have possible implications in tuning inflammation (see below).
      A common feature of the LLSP pathways is their induction by stress. Our initial observation that stress promotes IL-1β secretion (
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      ) has gained strong support. Lysosome exocytosis, exosome formation, membrane vesiculation, autophagy, and pyroptosis are all tightly regulated by stresses, especially redox stress (
      • Harris J.
      • Hartman M.
      • Roche C.
      • Zeng S.G.
      • O'Shea A.
      • Sharp F.A.
      • Lambe E.M.
      • Creagh E.M.
      • Golenbock D.T.
      • Tschopp J.
      • Kornfeld H.
      • Fitzgerald K.A.
      • Lavelle E.C.
      Autophagy controls IL-1β secretion by targeting pro-IL-1β for degradation.
      ,
      • Scott R.E.
      • Perkins R.G.
      • Zschunke M.A.
      • Hoerl B.J.
      • Maercklein P.B.
      Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization.
      ,
      • Benedikter B.J.
      • Volgers C.
      • van Eijck P.H.
      • Wouters E.F.M.
      • Savelkoul P.H.M.
      • Reynaert N.L.
      • Haenen G.R.M.M.
      • Rohde G.G.
      • Weseler A.R.
      • Stassen F.R.M.
      Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants.
      ,
      • Li X.
      • Gulbins E.
      • Zhang Y.
      Oxidative stress triggers Ca-dependent lysosome trafficking and activation of acid sphingomyelinase.
      ,
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ).
      Although LLSP externalization remains poorly understood, some plausible mechanisms have emerged (Fig. 3). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a phospholipid enriched at the inner leaflet of the plasma membrane, is required for the translocation and secretion of HIV-1 Tat (
      • Rayne F.
      • Debaisieux S.
      • Yezid H.
      • Lin Y.L.
      • Mettling C.
      • Konate K.
      • Chazal N.
      • Arold S.T.
      • Pugnière M.
      • Sanchez F.
      • Bonhoure A.
      • Briant L.
      • Loret E.
      • Roy C.
      • Beaumelle B.
      Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.
      ) and FGF2 (
      • Steringer J.P.
      • Bleicken S.
      • Andreas H.
      • Zacherl S.
      • Laussmann M.
      • Temmerman K.
      • Contreras F.X.
      • Bharat T.A.
      • Lechner J.
      • Müller H.M.
      • Briggs J.A.
      • García-Sáez A.J.
      • Nickel W.
      Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion.
      ). Both proteins accumulate at the plasma membrane via binding to PI(4,5)P2. This interaction induces oligomerization and translocation, possibly through lipidic pores. Heparan sulfate proteoglycans at the cell surface are required for FGF2 secretion (
      • Zehe C.
      • Engling A.
      • Wegehingel S.
      • Schäfer T.
      • Nickel W.
      Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2.
      ), likely acting as a pulling ratchet (Fig. 3A).
      Figure thumbnail gr3
      Figure 3Secretion through membrane pores. A, direct translocation of LLSPs across the plasma membrane through lipidic pores has been described for HIV-Tat (
      • Rayne F.
      • Debaisieux S.
      • Yezid H.
      • Lin Y.L.
      • Mettling C.
      • Konate K.
      • Chazal N.
      • Arold S.T.
      • Pugnière M.
      • Sanchez F.
      • Bonhoure A.
      • Briant L.
      • Loret E.
      • Roy C.
      • Beaumelle B.
      Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.
      ) and FGF (
      • Steringer J.P.
      • Bleicken S.
      • Andreas H.
      • Zacherl S.
      • Laussmann M.
      • Temmerman K.
      • Contreras F.X.
      • Bharat T.A.
      • Lechner J.
      • Müller H.M.
      • Briggs J.A.
      • García-Sáez A.J.
      • Nickel W.
      Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion.
      ). These proteins interact with a phosphoinositide, PI(4,5)P2, at the inner leaflet and with heparan sulfates at the outer leaflet of the plasma membrane. PI(4,5)P2 induces oligomerization of FGF2 that is phosphorylated by Tec kinase and promotes formation of a lipidic membrane pore through which the protein is externalized. B, LLSPs can be directly translocated across the plasma membrane through proteinaceous pores. Activated caspase-1 or -4 can cleave GSDMD, generating an N-terminal domain that oligomerizes and forms pores in the plasma membrane (
      • Kayagaki N.
      • Stowe I.B.
      • Lee B.L.
      • O'Rourke K.
      • Anderson K.
      • Warming S.
      • Cuellar T.
      • Haley B.
      • Roose-Girma M.
      • Phung Q.T.
      • Liu P.S.
      • Lill J.R.
      • Li H.
      • Wu J.
      • Kummerfeld S.
      • et al.
      Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • Zhuang Y.
      • Cai T.
      • Wang F.
      • Shao F.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      • He W.T.
      • Wan H.
      • Hu L.
      • Chen P.
      • Wang X.
      • Huang Z.
      • Yang Z.H.
      • Zhong C.Q.
      • Han J.
      Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.
      ). PI(4,5)P2 favors membrane insertion, whereas cholesterol inhibits it (
      • He W.T.
      • Wan H.
      • Hu L.
      • Chen P.
      • Wang X.
      • Huang Z.
      • Yang Z.H.
      • Zhong C.Q.
      • Han J.
      Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.
      ). Initially, the pore size allows passage of small proteins, such as the mature form of IL-1β or IL-18, but not of larger cytoplasmic molecules. However, ion imbalances cause cell swelling and eventually membrane rupture, pyroptosis, and release of DAMPs and larger complexes (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Mulvihill E.
      • Sborgi L.
      • Mari S.A.
      • Pfreundschuh M.
      • Hiller S.
      • Müller D.J.
      Mechanism of membrane pore formation by human gasdermin-D.
      ,
      • Russo H.M.
      • Rathkey J.
      • Boyd-Tressler A.
      • Katsnelson M.A.
      • Abbott D.W.
      • Dubyak G.R.
      Active caspase-1 induces plasma membrane pores that precede pyroptotic lysis and are blocked by lanthanides.
      ).
      Proteinaceous pores can be formed by gasdermin D (GSDMD) (
      • Kayagaki N.
      • Stowe I.B.
      • Lee B.L.
      • O'Rourke K.
      • Anderson K.
      • Warming S.
      • Cuellar T.
      • Haley B.
      • Roose-Girma M.
      • Phung Q.T.
      • Liu P.S.
      • Lill J.R.
      • Li H.
      • Wu J.
      • Kummerfeld S.
      • et al.
      Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.
      ,
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • Zhuang Y.
      • Cai T.
      • Wang F.
      • Shao F.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      ) (Fig. 3B). Upon activation of inflammatory caspase-4 (caspase-11 in mice) or caspase-1 by intracellular LPS or intense extracellular inflammatory stimuli (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Kayagaki N.
      • Stowe I.B.
      • Lee B.L.
      • O'Rourke K.
      • Anderson K.
      • Warming S.
      • Cuellar T.
      • Haley B.
      • Roose-Girma M.
      • Phung Q.T.
      • Liu P.S.
      • Lill J.R.
      • Li H.
      • Wu J.
      • Kummerfeld S.
      • et al.
      Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.
      ), GSDMD undergoes proteolytic cleavage. The unleashed N-terminal domains insert into the plasma membrane lipids, oligomerize, and form pores that mediate the exit of small cytokines, such as IL-1β and IL-18, before pyroptotic cell death (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • He W.T.
      • Wan H.
      • Hu L.
      • Chen P.
      • Wang X.
      • Huang Z.
      • Yang Z.H.
      • Zhong C.Q.
      • Han J.
      Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.
      ,
      • Mulvihill E.
      • Sborgi L.
      • Mari S.A.
      • Pfreundschuh M.
      • Hiller S.
      • Müller D.J.
      Mechanism of membrane pore formation by human gasdermin-D.
      ). PI(4,5)P2 increases and cholesterol reduces membrane insertion of GSDMD (
      • Russo H.M.
      • Rathkey J.
      • Boyd-Tressler A.
      • Katsnelson M.A.
      • Abbott D.W.
      • Dubyak G.R.
      Active caspase-1 induces plasma membrane pores that precede pyroptotic lysis and are blocked by lanthanides.
      ).
      ABC subfamily A member 1 (ABCA1) is responsible for cholesterol efflux and has been implicated in the release of IL-1β (
      • Hamon Y.
      • Luciani M.F.
      • Becq F.
      • Verrier B.
      • Rubartelli A.
      • Chimini G.
      Interleukin-1β secretion is impaired by inhibitors of the Atp binding cassette transporter, ABC1.
      ), MIF-1 (
      • Flieger O.
      • Engling A.
      • Bucala R.
      • Lue H.
      • Nickel W.
      • Bernhagen J.
      Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter.
      ), and apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1), a nuclear factor controlling the responses to oxidative stress (
      • Lee Y.R.
      • Joo H.K.
      • Lee E.O.
      • Cho H.S.
      • Choi S.
      • Kim C.S.
      • Jeon B.H.
      ATP binding cassette transporter A1 is involved in extracellular secretion of acetylated APE1/Ref-1.
      ). Interestingly, ABCA1 is a mammalian homolog of yeast STE6 (
      • Dean M.
      • Hamon Y.
      • Chimini G.
      The human ATP-binding cassette (ABC) transporter superfamily.
      ) required for secretion of the leaderless protein a-factor (
      • Kuchler K.
      • Sterne R.E.
      • Thorner J.
      Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells.
      ).
      LLSPs secreted through exosomes or microvesicles (Fig. 2) or autophagic vesicles (Fig. 3A) are likely to accumulate where the membrane bends to form the vesicles with no need for translocation. However, KFERQ-like motifs have been proposed for IL-1β translocation into vesicle intermediates in autophagy-mediated secretion (
      • Zhang M.
      • Kenny S.J.
      • Ge L.
      • Xu K.
      • Schekman R.
      Translocation of interleukin-1β into a vesicle intermediate in autophagy-mediated secretion.
      ). These motifs mediate the stress-dependent translocation of cytosolic proteins into lysosomes through lysosomal-associated membrane protein 2A (LAMP2A), in a process named chaperone-mediated autophagy (CMA) (
      • Dice J.F.
      • Chiang H.L.
      • Spencer E.P.
      • Backer J.M.
      Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7–11 as the essential pentapeptide.
      ,
      • Cuervo A.M.
      • Dice J.F.
      A receptor for the selective uptake and degradation of proteins by lysosomes.
      ). Although the main fate of these proteins is their degradation, some CMA cargoes may be externalized intact, including cytoplasmic antigens destined for major histocompatibility complex class II presentation (
      • Zhou D.
      • Li P.
      • Lin Y.
      • Lott J.M.
      • Hislop A.D.
      • Canaday D.H.
      • Brutkiewicz R.R.
      • Blum J.S.
      Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens.
      ). Thus, it is tempting to speculate that this mechanism might mediate secretion of other LLSPs containing KFERQ-like motifs. Different signals may then trigger degradation or secretion of proteins that reach lysosomes via CMA (Fig. 4B), a feature that, in the case of leaderless cytokines, can allow for tuning of inflammation.
      Figure thumbnail gr4
      Figure 4Redox-dependent control of inflammation. A, in all cell types, Trx reduces protein disulfide bonds by interchange reactions (
      • Holmgren A.
      Enzymatic reduction-oxidation of protein disulfides by Trx.
      ). Its reductive power is restored by Trx reductase in a reaction that depends on NADPH. B, owing to the presence of Trx and other systems, thiols in the cytosol are mostly reduced (SH>S-S). In contrast, disulfides predominate outside the cell (S-S>SH). LLSPs and DAMPs are externalized with reduced cysteines, switching or losing their bioactivity upon oxidation that occurs in a time- and distance-dependent manner in the oxidizing extracellular milieu (
      • Rubartelli A.
      • Lotze M.T.
      Inside, outside, upside down: damage-associated molecular-pattern molecules (DAMPs) and redox.
      ). Thus, the levels of Trx, other reductases, and NADPH in the extracellular space control the strength and duration of LLSP and DAMP activities (
      • Rubartelli A.
      • Lotze M.T.
      Inside, outside, upside down: damage-associated molecular-pattern molecules (DAMPs) and redox.
      ,
      • Zanoni I.
      • Tan Y.
      • Di Gioia M.
      • Broggi A.
      • Ruan J.
      • Shi J.
      • Donado C.A.
      • Shao F.
      • Wu H.
      • Springstead J.R.
      • Kagan J.C.
      An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells.
      ). Especially during stress, Trx, Trx reductase, glutaredoxins, peroxiredoxins, SOD-1, APE1/Ref-1, and MIF-1 are secreted through unconventional pathways, impacting the outcome of inflammatory responses (
      • Rubartelli A.
      • Sitia R.
      Stress as an intercellular signal: the emergence of stress-associated molecular patterns (SAMP).
      ) via thiol-disulfide exchange reactions (
      • Schwertassek U.
      • Balmer Y.
      • Gutscher M.
      • Weingarten L.
      • Preuss M.
      • Engelhard J.
      • Winkler M.
      • Dick T.P.
      Selective redox regulation of cytokine receptor signaling by extracellular Trx-1.
      ). Extracellular LLSP redoxins reduce regulatory disulfides, modulating the activity of target receptors (
      • Schwertassek U.
      • Balmer Y.
      • Gutscher M.
      • Weingarten L.
      • Preuss M.
      • Engelhard J.
      • Winkler M.
      • Dick T.P.
      Selective redox regulation of cytokine receptor signaling by extracellular Trx-1.
      ,
      • Matthias L.J.
      • Yam P.T.
      • Jiang X.M.
      • Vandegraaff N.
      • Li P.
      • Poumbourios P.
      • Donoghue N.
      • Hogg P.J.
      Disulfide exchange in domain 2 of CD4 is required for entry of HIV-1.
      ,
      • Xu S.Z.
      • Sukumar P.
      • Zeng F.
      • Li J.
      • Jairaman A.
      • English A.
      • Naylor J.
      • Ciurtin C.
      • Majeed Y.
      • Milligan C.J.
      • Bahnasi Y.M.
      • Al-Shawaf E.
      • Porter K.E.
      • Jiang L.H.
      • Emery P.
      • et al.
      TRPC channel activation by extracellular Trx.
      ) as well as of soluble ligands secreted through the classical exocytic route. For instance, IL-4 is inactivated when a disulfide is reduced by Trx (
      • Plugis N.M.
      • Weng N.
      • Zhao Q.
      • Palanski B.A.
      • Maecker H.T.
      • Habtezion A.
      • Khosla C.
      Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular Trx.
      ).
      As depicted in Fig. 2 (B and C), CMA and MAPS have similar characteristics. However, in CMA, heat shock cognate 71-kDa protein (HSC70) targets substrates carrying suitable motifs to LAMP2A for lysosomal translocation (Fig. 2B). In contrast, the deubiquitinase USP19 recruits misfolded proteins at the ER surface for the subsequent, DNAJC5-dependent translocation into late endosomes (
      • Lee J.
      • Xu Y.
      • Zhang T.
      • Cui L.
      • Saidi L.
      • Ye Y.
      Secretion of misfolded cytosolic proteins from mammalian cells is independent of chaperone-mediated autophagy.
      ) in MAPS (Fig. 3C).
      Yet another pathway similar to CMA has been recently reported to mediate the translocation of leaderless proteins from the cytosol into intracellular vesicles (
      • Zhang M.
      • Liu L.
      • Lin X.
      • Wang Y.
      • Li Y.
      • Guo Q.
      • Li S.
      • Sun Y.
      • Tao X.
      • Zhang D.
      • Lv X.
      • Zheng L.
      • Ge L.
      A translocation pathway for vesicle-mediated unconventional protein secretion.
      ). The transmembrane protein TMED10/TMP21 is proposed to directly facilitate the translocation of a broad spectrum of LLSPs into the ER-Golgi intermediate compartment (ERGIC). The process is enhanced by chaperones of the HSP90 family (
      • Zhang M.
      • Liu L.
      • Lin X.
      • Wang Y.
      • Li Y.
      • Guo Q.
      • Li S.
      • Sun Y.
      • Tao X.
      • Zhang D.
      • Lv X.
      • Zheng L.
      • Ge L.
      A translocation pathway for vesicle-mediated unconventional protein secretion.
      ). It remains to be investigated whether and how this pathway is related to the previously described pathways involving autophagosomes (
      • Dupont N.
      • Jiang S.
      • Pilli M.
      • Ornatowski W.
      • Bhattacharya D.
      • Deretic V.
      Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β.
      ), multivesicular bodies, and secretory endolysosomes (
      • Andrei C.
      • Dazzi C.
      • Lotti L.
      • Torrisi M.R.
      • Chimini G.
      • Rubartelli A.
      The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles.
      ,
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Zhang M.
      • Kenny S.J.
      • Ge L.
      • Xu K.
      • Schekman R.
      Translocation of interleukin-1β into a vesicle intermediate in autophagy-mediated secretion.
      ).

      How did different pathways for LLSP externalization evolve?

      An intriguing possibility is that UPS pathways initially served to remove mutated, overexpressed, or aged proteins that are potentially toxic to cells lacking efficient proteolytic mechanisms. Examples of such proteins have been documented in yeast and in mammalian cells that overexpress rhodanese or GFP (
      • Sloan I.S.
      • Horowitz P.M.
      • Chirgwin J.M.
      Rapid secretion by a non classical pathway of overexpressed mammalian mitochondrial rhodanese.
      ,
      • Cleves A.E.
      • Cooper D.N.
      • Barondes S.H.
      • Kelly R.B.
      A new pathway for protein export in Saccharomyces cerevisiae.
      ,
      • Tanudji M.
      • Hevi S.
      • Chuck S.L.
      Improperly folded green fluorescent protein is secreted via a non-classical pathway.
      ). Such pathways are advantageous for unicellular organisms, providing a mechanism allowing the expulsion of toxic molecules, but they could be detrimental in metazoans. Indeed, MAPS (
      • Lee J.G.
      • Takahama S.
      • Zhang G.
      • Tomarev S.I.
      • Ye Y.
      Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.
      ,
      • Lee J.
      • Xu Y.
      • Zhang T.
      • Cui L.
      • Saidi L.
      • Ye Y.
      Secretion of misfolded cytosolic proteins from mammalian cells is independent of chaperone-mediated autophagy.
      ) can mediate the release of protein aggregates that contribute to the pathogenesis of human neurogenerative disease, such as Tau and α-synuclein aggregates (
      • Fontaine S.N.
      • Zheng D.
      • Sabbagh J.J.
      • Martin M.D.
      • Chaput D.
      • Darling A.
      • Trotter J.H.
      • Stothert A.R.
      • Nordhues B.A.
      • Lussier A.
      • Baker J.
      • Shelton L.
      • Kahn M.
      • Blair L.J.
      • Stevens Jr., S.M.
      • Dickey C.A.
      DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.
      ). LLSP molecules fulfilling a defined intracellular function (e.g. FGF2, annexins, galectins, and redoxins) might have been expelled from stressed cells as a primitive mechanism of protein down-regulation. Extruded proteins that can activate cell-surface receptors (e.g. IL-1 and FGF family members), enter vicinal cells (e.g. FGF2 and engrailed-2) (
      • Wiedłocha A.
      • Sørensen V.
      Signaling, internalization, and intracellular activity of fibroblast growth factor.
      ,
      • Maizel A.
      • Tassetto M.
      • Filhol O.
      • Cochet C.
      • Prochiantz A.
      • Joliot A.
      Engrailed homeoprotein secretion is a regulated process.
      ), or modulate the bioactivity of receptors or ligands (e.g. Trx) (
      • Schwertassek U.
      • Balmer Y.
      • Gutscher M.
      • Weingarten L.
      • Preuss M.
      • Engelhard J.
      • Winkler M.
      • Dick T.P.
      Selective redox regulation of cytokine receptor signaling by extracellular Trx-1.
      ) might have been selected as players in intercellular communications. More recently, viral proteins such as HIV-Tat (
      • Debaisieux S.
      • Rayne F.
      • Yezid H.
      • Beaumelle B.
      The ins and outs of HIV-1 Tat.
      ) have been reported to exploit the same mechanisms. Secreted HIV-Tat interacts with membrane receptors and enters vicinal cells, contributing to both nonimmune and immune dysfunctions in AIDS.
      During evolution, some LLSPs, such as IL-1β, lost their intracellular function, whereas others retained key functions. For example, IL-1α is a transcriptional regulator inside the cell and functions as a pro-inflammatory cytokine when externalized. Trx and other LLSPs play similar intra- and extracellular roles (Table 1).

      Why should LLSPs lack a signal sequence?

      Additional regulatory possibilities or different stress sensitivity could make a particular secretion path better-suited than the classical secretory route involving leader peptides, especially for cargoes impacting inflammation. Transit along the ER-Golgi route may result in posttranslational modifications that interfere with a secreted protein's activity and function. For example, when dispatched to the ER, IL-1β undergoes N-glycosylation and loses bioactivity (
      • Livi G.P.
      • Lillquist J.S.
      • Miles L.M.
      • Ferrara A.
      • Sathe G.M.
      • Simon P.L.
      • Meyers C.A.
      • Gorman J.A.
      • Young P.R.
      Secretion of N-glycosylated interleukin-1β in Saccharomyces cerevisiae using a leader peptide from Candida albicans: effect of N-linked glycosylation on biological activity.
      ). Galectin and other LLSPs with lectin activity might be trapped in secretory organelles containing abundant glycoproteins, glycolipids, and polysaccharides. Formation of disulfides, which increase the stability of classical secretory proteins, may change the conformation of LLSPs and impair their function. Remarkably, avoiding classical exocytosis may prevent dangerous autocrine circuits, as is the case for FGF2: expression of a chimeric signal peptide-containing FGF2—but not of leaderless FGF2—constitutively activates FGF receptors and induces cell transformation (
      • Yayon A.
      • Klagsbrun M.
      Autocrine transformation by chimeric signal peptide-basic fibroblast growth factor: reversal by suramin.
      ).

      LLSP or DAMP?

      Damage-associated molecular patterns (DAMPs) are molecules with defined intracellular functions that, when released upon loss of membrane integrity, sustain sterile inflammation (
      • Lotze M.T.
      • Zeh H.J.
      • Rubartelli A.
      • Sparvero L.J.
      • Amoscato A.A.
      • Washburn N.R.
      • Devera M.E.
      • Liang X.
      • Tör M.
      • Billiar T.
      The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity.
      ). Unlike DAMPs, which are passively released, LLSPs undergo active and regulated secretion. In this respect, IL-1β is a prototypic LLSP, active only if secreted upon processing of inactive pro-IL-1β precursors. In between lie proteins such as HMGB1, IL-1α, IL-33, and IL-37 (reviewed in Ref.
      • Carta S.
      • Lavieri R.
      • Rubartelli A.
      Different members of the IL-1 family come out in different ways: DAMPs vs. cytokines?.
      ). Being active in their unprocessed form, they act as DAMP when released by injured or dying cells (
      • Scaffidi P.
      • Misteli T.
      • Bianchi M.E.
      Release of chromatin protein HMGB1 by necrotic cells triggers inflammation.
      ). However, they can also be actively secreted by living cells, especially upon stress (
      • Gardella S.
      • Andrei C.
      • Ferrera D.
      • Lotti L.V.
      • Torrisi M.R.
      • Bianchi M.E.
      • Rubartelli A.
      The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway.
      ,
      • Gross O.
      • Yazdi A.S.
      • Thomas C.J.
      • Masin M.
      • Heinz L.X.
      • Guarda G.
      • Quadroni M.
      • Drexler S.K.
      • Tschopp J.
      Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.
      ,
      • Kakkar R.
      • Hei H.
      • Dobner S.
      • Lee R.T.
      Interleukin 33 as a mechanically responsive cytokine secreted by living cells.
      ,
      • Bulau A.M.
      • Nold M.F.
      • Li S.
      • Nold-Petry C.A.
      • Fink M.
      • Mansell A.
      • Schwerd T.
      • Hong J.
      • Rubartelli A.
      • Dinarello C.A.
      • Bufler P.
      Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses.
      ,
      • Wang H.
      • Yang H.
      • Tracey K.J.
      Extracellular role of HMGB1 in inflammation and sepsis.
      ).
      The capability of cells to release pro-inflammatory signals before losing membrane integrity provides survival advantages presumably selected during the evolution of cellular defenses. To that end, the selective release of LLSPs does not entail DAMP efflux and is key for limiting inflammatory responses.
      LLSPs can be classified into cytokines, redox enzymes, and brain proteins (Table 1), three groups that largely overlap and impact inflammation. Cytokines are direct inflammatory mediators, whereas redoxins determine the duration and intensity of the responses (see Fig. 4 and next paragraph). The third group comprises members with clear inflammatory roles, including ciliary neurotrophic factor (CNTF), a cytokine with tissue-reparative properties (
      • Jones S.A.
      • Jenkins B.J.
      Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer.
      ), and APE1/Ref-1, a redoxin that inhibits tumor necrosis factor α (TNFα)-mediated inflammation (
      • Park M.S.
      • Choi S.
      • Lee Y.R.
      • Joo H.K.
      • Kang G.
      • Kim C.S.
      • Kim S.J.
      • Lee S.D.
      • Jeon B.H.
      Secreted APE1/Ref-1 inhibits TNF-α-stimulated endothelial inflammation via thiol-disulfide exchange in TNF receptor.
      ). Moreover, the LLSPs Tau, α-synuclein, and superoxide dismutase 1 (SOD-1), when secreted, cause neurodegenerative disorders with relevant inflammatory components (
      • Fontaine S.N.
      • Zheng D.
      • Sabbagh J.J.
      • Martin M.D.
      • Chaput D.
      • Darling A.
      • Trotter J.H.
      • Stothert A.R.
      • Nordhues B.A.
      • Lussier A.
      • Baker J.
      • Shelton L.
      • Kahn M.
      • Blair L.J.
      • Stevens Jr., S.M.
      • Dickey C.A.
      DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.
      ,
      • Merezhko M.
      • Brunello C.A.
      • Yan X.
      • Vihinen H.
      • Jokitalo E.
      • Uronen R.L.
      • Huttunen H.J.
      Secretion of Tau via an unconventional non-vesicular mechanism.
      ,
      • Goedert M.
      Neurodegeneration. Alzheimer's and Parkinson's diseases: the prion concept in relation to assembled Aβ, tau, and α-synuclein.
      ,
      • Münch C.
      • O'Brien J.
      • Bertolotti A.
      Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells.
      ,
      • Xu Y.
      • Cui L.
      • Dibello A.
      • Wang L.
      • Lee J.
      • Saidi L.
      • Lee J.G.
      • Ye Y.
      DNAJC5 facilitates USP19-dependent unconventional secretion of misfolded cytosolic proteins.
      ,
      • Zhang Q.S.
      • Heng Y.
      • Yuan Y.H.
      • Chen N.H.
      Pathological α-synuclein exacerbates the progression of Parkinson's disease through microglial activation.
      ,
      • Perea J.R.
      • Ávila J.
      • Bolós M.
      Dephosphorylated rather than hyperphosphorylated Tau triggers a pro-inflammatory profile in microglia through the p38 MAPK pathway.
      ).

      LLSPs, redox regulation, and inflammation

      Inflammation is tightly linked to redox remodeling. Stressed cells activate responses that entail reactive oxygen species (ROS) production (
      • Naviaux R.K.
      Oxidative shielding or oxidative stress?.
      ). ROS control many steps in inflammatory responses, from the anti-pathogen oxidative burst to wound healing (
      • Lorenzen I.
      • Mullen L.
      • Bekeschus S.
      • Hanschmann E.M.
      Redox regulation of inflammatory processes is enzymatically controlled.
      ). ROS production is balanced with antioxidant responses that can affect the extracellular redox poise (
      • Castellani P.
      • Balza E.
      • Rubartelli A.
      Inflammation, DAMPs, tumor development, and progression: a vicious circle orchestrated by redox signaling.
      ) (Fig. 4). The extracellular space is normally oxidizing, with high cystine/cysteine ratios, and both soluble and membrane-bound proteins externalized through the classical pathway contain many stabilizing disulfide bonds. In contrast, the cysteines in cytosolic proteins (including LLSPs) are mainly in the reduced state (Fig. 4B). Secreted small thiols and redox enzymes can alter the extracellular redox state, thereby modulating inflammation (
      • Yi M.C.
      • Khosla C.
      Thiol-disulfide exchange reactions in the mammalian extracellular environment.
      ), blood clotting (
      • Sharda A.
      • Furie B.
      Regulatory role of thiol isomerases in thrombus formation.
      ), and virus infection (
      • Matthias L.J.
      • Yam P.T.
      • Jiang X.M.
      • Vandegraaff N.
      • Li P.
      • Poumbourios P.
      • Donoghue N.
      • Hogg P.J.
      Disulfide exchange in domain 2 of CD4 is required for entry of HIV-1.
      ). Some secreted redox enzymes normally reside in the ER (e.g. protein-disulfide isomerase, P5, peroxiredoxin 4, and ERp44). Others, such as Trx (
      • Rayne F.
      • Debaisieux S.
      • Yezid H.
      • Lin Y.L.
      • Mettling C.
      • Konate K.
      • Chazal N.
      • Arold S.T.
      • Pugnière M.
      • Sanchez F.
      • Bonhoure A.
      • Briant L.
      • Loret E.
      • Roy C.
      • Beaumelle B.
      Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.
      ) (Fig. 4), Trx reductase, and peroxiredoxins 1 and 2 (
      • Checconi P.
      • Salzano S.
      • Bowler L.
      • Mullen L.
      • Mengozzi M.
      • Hanschmann E.M.
      • Lillig C.H.
      • Sgarbanti R.
      • Panella S.
      • Nencioni L.
      • Palamara A.T.
      • Ghezzi P.
      Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress.
      ), are LLSPs. Stress-associated molecular patterns (SAMPs) are present on redox modulators released from stressed cells and influence inflammatory responses (
      • Rubartelli A.
      • Sitia R.
      Stress as an intercellular signal: the emergence of stress-associated molecular patterns (SAMP).
      ). SAMPs such as cysteine are key, for instance, in antigen presentation, when dendritic cells release cysteine to feed T cells, which lack transporters for oxidized cystine, the molecular form of the amino acid that predominates extracellularly. Cysteine feeding allows antigen-specific T cell proliferation. Upon robust stimulation, dendritic cells also secrete Trx that maintains cysteines in their reduced form, prolonging T cell feeding (
      • Angelini G.
      • Gardella S.
      • Ardy M.
      • Ciriolo M.R.
      • Filomeni G.
      • Di Trapani G.
      • Clarke F.
      • Sitia R.
      • Rubartelli A.
      Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation.
      ). Also ROS-activated monocytes (
      • Rubartelli A.
      • Bajetto A.
      • Allavena G.
      • Wollman E.
      • Sitia R.
      Secretion of Trx by normal and neoplastic cells through a leaderless secretory pathway.
      ,
      • Tassi S.
      • Carta S.
      • Vené R.
      • Delfino L.
      • Caorsi R.
      • Martini A.
      • Gattorno M.
      • Rubartelli A.
      Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1β secretion.
      ) and B lymphocytes (
      • Rubartelli A.
      • Bajetto A.
      • Allavena G.
      • Wollman E.
      • Sitia R.
      Secretion of Trx by normal and neoplastic cells through a leaderless secretory pathway.
      ,
      • Vené R.
      • Delfino L.
      • Castellani P.
      • Balza E.
      • Bertolotti M.
      • Sitia R.
      • Rubartelli A.
      Redox remodeling allows and controls B-cell activation and differentiation.
      ) secrete Trx. Of note, serum Trx is in the low nanomolar range in healthy individuals but is dramatically increased in diseases having a strong inflammatory component (reviewed in Ref.
      • Yi M.C.
      • Khosla C.
      Thiol-disulfide exchange reactions in the mammalian extracellular environment.
      ). Secreted redoxins impact inflammatory circuits, by reshuffling disulfides in ligand and receptor proteins (
      • Schwertassek U.
      • Balmer Y.
      • Gutscher M.
      • Weingarten L.
      • Preuss M.
      • Engelhard J.
      • Winkler M.
      • Dick T.P.
      Selective redox regulation of cytokine receptor signaling by extracellular Trx-1.
      ) (Fig. 4B). For instance, reduction of an allosteric disulfide in CD4 facilitates HIV entry into T cells (
      • Matthias L.J.
      • Yam P.T.
      • Jiang X.M.
      • Vandegraaff N.
      • Li P.
      • Poumbourios P.
      • Donoghue N.
      • Hogg P.J.
      Disulfide exchange in domain 2 of CD4 is required for entry of HIV-1.
      ). Similarly, Trx-dependent reduction of disulfides in the extracellular domains of TRPC5 (
      • Xu S.Z.
      • Sukumar P.
      • Zeng F.
      • Li J.
      • Jairaman A.
      • English A.
      • Naylor J.
      • Ciurtin C.
      • Majeed Y.
      • Milligan C.J.
      • Bahnasi Y.M.
      • Al-Shawaf E.
      • Porter K.E.
      • Jiang L.H.
      • Emery P.
      • et al.
      TRPC channel activation by extracellular Trx.
      ) and CD30 (
      • Schwertassek U.
      • Balmer Y.
      • Gutscher M.
      • Weingarten L.
      • Preuss M.
      • Engelhard J.
      • Winkler M.
      • Dick T.P.
      Selective redox regulation of cytokine receptor signaling by extracellular Trx-1.
      ) regulates their activities. Secreted APE1/Ref-1, a nuclear redoxin with cytokine-like activity, modifies the redox state of TNFα receptors, thereby inhibiting TNFα–mediated inflammation in endothelial cells (
      • Park M.S.
      • Choi S.
      • Lee Y.R.
      • Joo H.K.
      • Kang G.
      • Kim C.S.
      • Kim S.J.
      • Lee S.D.
      • Jeon B.H.
      Secreted APE1/Ref-1 inhibits TNF-α-stimulated endothelial inflammation via thiol-disulfide exchange in TNF receptor.
      ).
      When released in the extracellular microenvironment, LLSPs and DAMPs may undergo cysteine oxidation, a modification that often alters their bioactivities (
      • Rubartelli A.
      • Lotze M.T.
      Inside, outside, upside down: damage-associated molecular-pattern molecules (DAMPs) and redox.
      ). A classic example is HMGB1, which, upon oxidation of its three cysteines, shifts from chemo-attractant to cytokine inducer and, ultimately, to regenerative factor (
      • Vénéreau E.
      • Ceriotti C.
      • Bianchi M.E.
      DAMPs from cell death to new life.
      ). However, the presence of extracellular glutaredoxin, Trx, or other SAMPs may counteract HMGB1 oxidation, prolonging its chemokine activity (
      • Rubartelli A.
      • Lotze M.T.
      Inside, outside, upside down: damage-associated molecular-pattern molecules (DAMPs) and redox.
      ). Accordingly, higher Trx activity, which correlates with aggressive rheumatoid arthritis, causes accumulation of reduced HMGB1-CXCL12 complexes in the synovial fluid. Such complexes are transient in healthy individuals, because the fast HMGB1 oxidation in the absence of inflammation-induced SAMPs impairs their stability (
      • Cecchinato V.
      • D'Agostino G.
      • Raeli L.
      • Nerviani A.
      • Schiraldi M.
      • Danelon G.
      • Manzo A.
      • Thelen M.
      • Ciurea A.
      • Bianchi M.E.
      • Rubartelli A.
      • Pitzalis C.
      • Uguccioni M.
      Redox-mediated mechanisms fuel monocyte responses to CXCL12/HMGB1 in active rheumatoid arthritis.
      ). Also, tissue transglutaminase 2 (TG2), which can deamidate gluten-derived peptides, is rapidly oxidized in the extracellular milieu but can be reactivated by Trx. Interferon-γ, a key cytokine in celiac disease, induces Trx secretion, supporting a pathogenic circuit (
      • Jin X.
      • Stamnaes J.
      • Klöck C.
      • DiRaimondo T.R.
      • Sollid L.M.
      • Khosla C.
      Activation of extracellular transglutaminase 2 by Trx.
      ).

      A feverish topic: IL-1β secretion

      Models for IL-1β externalization

      As fever is one of the few afflictions that affect all human beings, the cloning of the gene encoding IL-1β, previously called the “endogenous pyrogen” (
      • Auron P.E.
      • Webb A.C.
      • Rosenwasser L.J.
      • Mucci S.F.
      • Rich A.
      • Wolff S.M.
      • Dinarello C.A.
      Nucleotide sequence of human monocyte interleukin 1 precursor cDNA.
      ), generated huge interest in the biomedical research community. In the following years, a number of models explaining how this leaderless protein is secreted were suggested. The first model proposed that IL-1β is secreted through secretory lysosomes that fuse with the plasma membrane (
      • Andrei C.
      • Dazzi C.
      • Lotti L.
      • Torrisi M.R.
      • Chimini G.
      • Rubartelli A.
      The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles.
      ,
      • Gardella S.
      • Andrei C.
      • Lotti L.V.
      • Poggi A.
      • Torrisi M.R.
      • Zocchi M.R.
      • Rubartelli A.
      CD8+ T lymphocytes induce polarized exocytosis of secretory lysosomes by dendritic cells with release of interleukin-1β and cathepsin D.
      ,
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Andrei C.
      • Margiocco P.
      • Poggi A.
      • Lotti L.V.
      • Torrisi M.R.
      • Rubartelli A.
      Phospholipases C and A2 control lysosome-mediated IL-1β secretion: implications for inflammatory processes.
      ). This model implicates translocation across the endolysosomal membrane.
      Other vesicular pathways were then proposed. Solid evidence indicates that autophagic vesicles mediate protein release (
      • Dupont N.
      • Jiang S.
      • Pilli M.
      • Ornatowski W.
      • Bhattacharya D.
      • Deretic V.
      Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β.
      ) but has also shown that inhibition of autophagic degradation promotes IL-1β secretion (
      • Saitoh T.
      • Fujita N.
      • Jang M.H.
      • Uematsu S.
      • Yang B.G.
      • Satoh T.
      • Omori H.
      • Noda T.
      • Yamamoto N.
      • Komatsu M.
      • Tanaka K.
      • Kawai T.
      • Tsujimura T.
      • Takeuchi O.
      • Yoshimori T.
      • Akira S.
      Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production.
      ,
      • Harris J.
      • Hartman M.
      • Roche C.
      • Zeng S.G.
      • O'Shea A.
      • Sharp F.A.
      • Lambe E.M.
      • Creagh E.M.
      • Golenbock D.T.
      • Tschopp J.
      • Kornfeld H.
      • Fitzgerald K.A.
      • Lavelle E.C.
      Autophagy controls IL-1β secretion by targeting pro-IL-1β for degradation.
      ,
      • Zhou R.
      • Yazdi A.S.
      • Menu P.
      • Tschopp J.
      A role for mitochondria in NLRP3 inflammasome activation.
      ). Microvesicles (
      • MacKenzie A.
      • Wilson H.L.
      • Kiss-Toth E.
      • Dower S.K.
      • North R.A.
      • Surprenant A.
      Rapid secretion of interleukin-1β by microvesicle shedding.
      ) and exosomes (
      • Qu Y.
      • Franchi L.
      • Nunez G.
      • Dubyak G.R.
      Nonclassical IL-1β secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.
      ) are also suggested as carriers of IL-1β, raising the question of how pro-IL-1β is recognized and included in these vesicles.
      The identities, functions, and interrelationships of these vesicles remain to be clarified. From a functional viewpoint, a unifying explanation could be that pro-IL-1β–containing vesicles can fuse either with the plasma membrane or with degradation-competent lysosomes, depending on the type of signal received. In this scenario, lysosomal degradation could be a way to halt IL-1β release from cells exposed to weak or ambiguous pro-inflammatory signals.
      Recently, the Schekman laboratory reconstructed in vitro translocation of pro-IL-1β into LAMP2A-expressing vesicles. This translocation requires heat shock protein 90 (HSP90) and pentapeptide motifs in IL-1β that are loosely related to KFERQ (
      • Zhang M.
      • Kenny S.J.
      • Ge L.
      • Xu K.
      • Schekman R.
      Translocation of interleukin-1β into a vesicle intermediate in autophagy-mediated secretion.
      ). Interestingly, caspase-1 contains a similar motif, and IL-1β–containing lysosomes are positive for LAMP2A (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ), suggesting that secretion of IL-1β and caspase-1 (and possibly other LLSPs) through secretory lysosomes may represent a variant of CMA (Fig. 2C).
      As detailed above, IL-1β can be secreted also by direct translocation through GSDMD pores at the plasma membrane (Fig. 3B) that allow passive release of cytosolic proteins in a size-dependent manner (
      • Kayagaki N.
      • Stowe I.B.
      • Lee B.L.
      • O'Rourke K.
      • Anderson K.
      • Warming S.
      • Cuellar T.
      • Haley B.
      • Roose-Girma M.
      • Phung Q.T.
      • Liu P.S.
      • Lill J.R.
      • Li H.
      • Wu J.
      • Kummerfeld S.
      • et al.
      Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.
      ,
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • Zhuang Y.
      • Cai T.
      • Wang F.
      • Shao F.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      ,
      • He W.T.
      • Wan H.
      • Hu L.
      • Chen P.
      • Wang X.
      • Huang Z.
      • Yang Z.H.
      • Zhong C.Q.
      • Han J.
      Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.
      ,
      • Russo H.M.
      • Rathkey J.
      • Boyd-Tressler A.
      • Katsnelson M.A.
      • Abbott D.W.
      • Dubyak G.R.
      Active caspase-1 induces plasma membrane pores that precede pyroptotic lysis and are blocked by lanthanides.
      ,
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ).

      The mystery of IL-1β processing

      As noted above, IL-1β is produced as an inactive precursor, pro-IL-1β, that requires processing by caspase-1 (
      • Cerretti D.P.
      • Kozlosky C.J.
      • Mosley B.
      • Nelson N.
      • Van Ness K.
      • Greenstreet T.A.
      • March C.J.
      • Kronheim S.R.
      • Druck T.
      • Cannizzaro L.A.
      Molecular cloning of the interleukin-1 beta converting enzyme.
      ). This enzyme, also called IL-1–converting enzyme (ICE) is activated by the inflammasome (
      • Martinon F.
      • Burns K.
      • Tschopp J.
      The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β.
      ). Human monocytes activated by inflammatory stimuli such as Toll-like receptor (TLR) agonists contain abundant pro-IL-1β but little if any processed molecules. The reverse is true in the extracellular space (
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      ,
      • Cruz-Garcia D.
      • Curwin A.J.
      • Popoff J.F.
      • Bruns C.
      • Duran J.M.
      • Malhotra V.
      Remodeling of secretory compartments creates CUPS during nutrient starvation.
      ), implying that pro-IL-1β cleavage is coupled to exocytosis or takes place extracellularly, immediately upon release. During efficient pore-mediated secretion (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ), only traces of pro-IL-1β and lactate dehydrogenase are found extracellularly, suggesting that the size of the pore restricts pro-IL-1β and lactate dehydrogenase egress, until pyroptosis releases DAMPs and other large molecules (
      • Kovacs S.B.
      • Miao E.A.
      Gasdermins: effectors of pyroptosis.
      ). A tempting possibility is that active ICE is recruited near pores, ensuring the preferential release of bioactive IL-1β.
      It is unclear how human monocytes activate IL-1β processing during the fusion of secretory lysosomes with the plasma membrane. Lysosomes at the plasma membrane have a higher pH than the juxtanuclear ones (
      • Johnson D.E.
      • Ostrowski P.
      • Jaumouillé V.
      • Grinstein S.
      The position of lysosomes within the cell determines their luminal pH.
      ). It is hence possible that this pH shift couples vesicular exocytosis (
      • Sundler R.
      Lysosomal and cytosolic pH as regulators of exocytosis in mousemacrophages.
      ) and pro-IL-1β processing.

      Physiologic significance of different secretory mechanisms for IL-1β

      Why this redundancy in transport mechanisms? Are all of these pathways physiologically relevant? A first technical constraint in providing definitive answers is that only a few cell types, mainly myelomonocytic cells and microglia, are professional IL-1β–secreting cells. These cells are short-lived and difficult to manipulate genetically. Thus, most studies have aimed at reconstituting the IL-1β secretory pathways in vitro in cell lines that do not normally secrete IL-1β. However, discrepant results can be generated by the use of different model systems (
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      ,
      • Andrei C.
      • Dazzi C.
      • Lotti L.
      • Torrisi M.R.
      • Chimini G.
      • Rubartelli A.
      The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles.
      ,
      • Gardella S.
      • Andrei C.
      • Costigliolo S.
      • Olcese L.
      • Zocchi M.R.
      • Rubartelli A.
      Secretion of bioactive interleukin-1β by dendritic cells is modulated by interaction with antigen specific T cells.
      ,
      • Carta S.
      • Tassi S.
      • Pettinati I.
      • Delfino L.
      • Dinarello C.A.
      • Rubartelli A.
      The rate of interleukin-1β secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering.
      ,
      • Mariathasan S.
      • Weiss D.S.
      • Newton K.
      • McBride J.
      • O'Rourke K.
      • Roose-Girma M.
      • Lee W.P.
      • Weinrauch Y.
      • Monack D.M.
      • Dixit V.M.
      Cryopyrin activates the inflammasome in response to toxins and ATP.
      ,
      • Netea M.G.
      • Nold-Petry C.A.
      • Nold M.F.
      • Joosten L.A.
      • Opitz P.
      • van der Meer J.H.
      • van de Veerdonk F.L.
      • Ferwerda G.
      • Heinhuis B.
      • Devesa I.
      • Funk C.J.
      • Mason R.J.
      • Kullberg B.J.
      • Rubartelli A.
      • van der Meer J.W.
      • Dinarello C.A.
      Differential requirement for the activation of the inflammasome for processing and release of IL-1β in monocytes and macrophages.
      ,
      • Zanoni I.
      • Tan Y.
      • Di Gioia M.
      • Broggi A.
      • Ruan J.
      • Shi J.
      • Donado C.A.
      • Shao F.
      • Wu H.
      • Springstead J.R.
      • Kagan J.C.
      An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells.
      ). For example, secretion of mature IL-1β by mouse macrophages and human monocytic cell lines requires two signals (Fig. 5). Signal 1 (i.e. TLR agonists) triggers pro-IL-1β synthesis; signal 2 (extracellular ATP (
      • Mariathasan S.
      • Weiss D.S.
      • Newton K.
      • McBride J.
      • O'Rourke K.
      • Roose-Girma M.
      • Lee W.P.
      • Weinrauch Y.
      • Monack D.M.
      • Dixit V.M.
      Cryopyrin activates the inflammasome in response to toxins and ATP.
      ,
      • Netea M.G.
      • Nold-Petry C.A.
      • Nold M.F.
      • Joosten L.A.
      • Opitz P.
      • van der Meer J.H.
      • van de Veerdonk F.L.
      • Ferwerda G.
      • Heinhuis B.
      • Devesa I.
      • Funk C.J.
      • Mason R.J.
      • Kullberg B.J.
      • Rubartelli A.
      • van der Meer J.W.
      • Dinarello C.A.
      Differential requirement for the activation of the inflammasome for processing and release of IL-1β in monocytes and macrophages.
      ) or other triggers (
      • Dostert C.
      • Pétrilli V.
      • Van Bruggen R.
      • Steele C.
      • Mossman B.T.
      • Tschopp J.
      Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica.
      )) is needed for inflammasome activation and IL-1β secretion. By contrast, signal 2 is dispensable in primary human monocytes (Fig. 5), which are true professional IL-1β–secreting cells (
      • Rubartelli A.
      • Cozzolino F.
      • Talio M.
      • Sitia R.
      A novel secretory pathway for interleukin-1β, a protein lacking a signal sequence.
      ,
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Piccini A.
      • Carta S.
      • Tassi S.
      • Lasiglié D.
      • Fossati G.
      • Rubartelli A.
      ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way.
      ). These differences reflect mainly differences in stress sensitivity and redox defenses among these cells (
      • Carta S.
      • Tassi S.
      • Pettinati I.
      • Delfino L.
      • Dinarello C.A.
      • Rubartelli A.
      The rate of interleukin-1β secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering.
      ). In monocytes that are stress-hypersensitive, TLR agonists induce accumulation of ROS that enhance pro-IL-1β biosynthesis and trigger release of endogenous ATP. Autocrine activation of purinergic P2X7 receptors (
      • Piccini A.
      • Carta S.
      • Tassi S.
      • Lasiglié D.
      • Fossati G.
      • Rubartelli A.
      ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way.
      ,
      • Carta S.
      • Penco F.
      • Lavieri R.
      • Martini A.
      • Dinarello C.A.
      • Gattorno M.
      • Rubartelli A.
      Cell stress increases ATP release in NLRP3 inflammasome-mediated autoinflammatory diseases, resulting in cytokine imbalance.
      ) causes further ROS production, K+ efflux, Ca2+ entry, inflammasome activation, and IL-1β secretion (
      • Di Virgilio F.
      Liaisons dangereuses: P2X(7) and the inflammasome.
      ). The strength and mechanism of administration of the two different signals also impact the rate and mode of secretion. TLR4 activation by extracellular LPS induces mild secretion through secretory lysosomes (
      • Andrei C.
      • Dazzi C.
      • Lotti L.
      • Torrisi M.R.
      • Chimini G.
      • Rubartelli A.
      The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles.
      ,
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Andrei C.
      • Margiocco P.
      • Poggi A.
      • Lotti L.V.
      • Torrisi M.R.
      • Rubartelli A.
      Phospholipases C and A2 control lysosome-mediated IL-1β secretion: implications for inflammatory processes.
      ). However, when injected intracellularly, LPS leads to massive IL-1β release via GSDMD pores (
      • Kayagaki N.
      • Stowe I.B.
      • Lee B.L.
      • O'Rourke K.
      • Anderson K.
      • Warming S.
      • Cuellar T.
      • Haley B.
      • Roose-Girma M.
      • Phung Q.T.
      • Liu P.S.
      • Lill J.R.
      • Li H.
      • Wu J.
      • Kummerfeld S.
      • et al.
      Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.
      ,
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • Zhuang Y.
      • Cai T.
      • Wang F.
      • Shao F.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      ,
      • He W.T.
      • Wan H.
      • Hu L.
      • Chen P.
      • Wang X.
      • Huang Z.
      • Yang Z.H.
      • Zhong C.Q.
      • Han J.
      Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.
      ). Likewise, the simultaneous activation of multiple TLR agonists results in a switch from lysosome-mediated to pore-mediated secretion (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ). Intercellular circuits are also important: release of ATP and other DAMPs by injured vicinal cells could sustain inflammasome activation and IL-1β secretion. Thus, the intensity and type of the pro-inflammatory stimuli, and the context in which they are perceived by monocytes, dictate pathway selection and impact the efficacy of IL-1β antagonistic therapies (
      • Dinarello C.A.
      • Simon A.
      • van der Meer J.W.
      Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases.
      ). For instance, use of neutralizing antibodies or of soluble decoy receptors to intercept IL-1β would be less active against microvesicles or exosomes than reagents blocking IL-1 receptor on target cells.
      Figure thumbnail gr5
      Figure 5Different cell types have different redox sensitivity that determines the requirements for IL-1β secretion. In murine macrophages and myelomonocytic cell lines (A), two signals are required for secretion of mature IL-1β. Signal 1 induces pro-IL-1β and ROS production and leads to release of only low ATP levels that are unable to productively activate P2X7 receptors. Therefore, exogenous ATP (signal 2) is required to activate P2X7 receptors and downstream pathways, leading to IL-1β processing and secretion. In human monocytes instead (B), a single signal (i.e. TLR activation) induces pro-IL-1β that triggers the release of ATP at levels sufficient to autocrinally activate P2X7 receptors, inflammasomes, and eventually abundant secretion of mature IL-1β.

      Stress dependence of IL-1β secretion

      As alluded to in the foregoing, ROS-dependent events control IL-1β release at many key steps, including transcription via redox-sensitive NF-κB, processing by inflammasomes (
      • Martinon F.
      Signaling by ROS drives inflammasome activation.
      ), secretion (
      • Tassi S.
      • Carta S.
      • Vené R.
      • Delfino L.
      • Caorsi R.
      • Martini A.
      • Gattorno M.
      • Rubartelli A.
      Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1β secretion.
      ,
      • Lavieri R.
      • Piccioli P.
      • Carta S.
      • Delfino L.
      • Castellani P.
      • Rubartelli A.
      TLR costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production.
      ,
      • Tassi S.
      • Carta S.
      • Vené R.
      • Delfino L.
      • Ciriolo M.R.
      • Rubartelli A.
      Pathogen-induced interleukin-1β processing and secretion is regulated by a biphasic redox response.
      ), lysosome exocytosis (
      • Li X.
      • Gulbins E.
      • Zhang Y.
      Oxidative stress triggers Ca-dependent lysosome trafficking and activation of acid sphingomyelinase.
      ), GSDMD pore formation (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ), and autophagy (
      • Harris J.
      • Hartman M.
      • Roche C.
      • Zeng S.G.
      • O'Shea A.
      • Sharp F.A.
      • Lambe E.M.
      • Creagh E.M.
      • Golenbock D.T.
      • Tschopp J.
      • Kornfeld H.
      • Fitzgerald K.A.
      • Lavelle E.C.
      Autophagy controls IL-1β secretion by targeting pro-IL-1β for degradation.
      ,
      • Zhou R.
      • Yazdi A.S.
      • Menu P.
      • Tschopp J.
      A role for mitochondria in NLRP3 inflammasome activation.
      ).
      The level of stress may then influence the choice of a pathway or the switch from one to another. A remarkable example is how monocytes adapt IL-1β secretion to environmental cues, shifting from a vesicular to a pore-dependent path. This decision depends on the strength of stimulation. If only TLR4 signaling is triggered, pro-IL-1β translocates to LAMP2A+ intracellular lysosomes that eventually fuse with the plasma membrane, thereby releasing IL-1β (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ). Interestingly, inhibitors of lysosomal proteases increase overall IL-1β secretion (
      • Andrei C.
      • Dazzi C.
      • Lotti L.
      • Torrisi M.R.
      • Chimini G.
      • Rubartelli A.
      The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles.
      ), implying that, in the absence of inhibitors, some of the 33-kDa precursors are normally degraded in lysosomes. It is conceivable the there is a ratio between degradation and secretion that changes with the intensity and duration of the inflammatory stimulus. Thus, the lysosomal pathway mediates IL-1β secretion but also offers a point of return by allowing degradation to prevail over secretion when IL-1β secretion is no longer required. Furthermore, upon mild stimulation, monocytes die by undergoing apoptosis, limiting inflammation (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ). However, when a strong pro-inflammatory stimulus is provided, stress increases, resulting in increased ROS production, ATP release, and P2X7R-mediated signaling (
      • Lavieri R.
      • Piccioli P.
      • Carta S.
      • Delfino L.
      • Castellani P.
      • Rubartelli A.
      TLR costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production.
      ), which eventually activate GSDMD cleavage (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ). The result of this cascade of events is that IL-1β secretion rates increase and, importantly, monocytes undergo pyroptosis, releasing additional DAMPs and hence extending inflammation. Specific inhibition of ROS production prevents this cascade, highlighting the redox sensitivity of its many steps (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Tassi S.
      • Carta S.
      • Vené R.
      • Delfino L.
      • Caorsi R.
      • Martini A.
      • Gattorno M.
      • Rubartelli A.
      Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1β secretion.
      ).
      Venturing into pathophysiological implications, we surmise that secretory lysosome-mediated mechanisms dominate in conditions of low pathogen load or small sterile injury, inducing a self-limiting redox response and allowing a controlled release of IL-1β to restore homeostasis. However, in severe conditions like multiple infections or severe trauma, GSDMD/pyroptosis-mediated secretion is activated. The explosive secretion through pores may initially benefit the host, but a vicious circle of release of IL-1β and additional DAMPs may soon start, possibly leading to cell death, as in sepsis and shock (
      • Sitia R.
      • Rubartelli A.
      The unconventional secretion of IL-1β: handling a dangerous weapon to optimize inflammatory responses.
      ).

      Which way to take? How pore-mediated IL-1β secretion predominates in cryopyrin-associated periodic syndromes

      Cryopyrin-associated periodic syndromes (CAPS) are a group of severe autoinflammatory diseases linked to gain-of-function mutations in the inflammasome component NLRP3. Their devastating inflammatory manifestations are largely dependent on dysregulated IL-1β secretion due to inflammasome hyperactivation (
      • Manthiram K.
      • Zhou Q.
      • Aksentijevich I.
      • Kastner D.L.
      The monogenic autoinflammatory diseases define new pathways in human innate immunity and inflammation.
      ). Monocytes from individuals with CAPS are chronically stressed, possibly because of the presence of the mutant NLRP3, and display ROS levels that are constitutively higher than in unstressed monocytes (
      • Tassi S.
      • Carta S.
      • Vené R.
      • Delfino L.
      • Caorsi R.
      • Martini A.
      • Gattorno M.
      • Rubartelli A.
      Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1β secretion.
      ,
      • Carta S.
      • Penco F.
      • Lavieri R.
      • Martini A.
      • Dinarello C.A.
      • Gattorno M.
      • Rubartelli A.
      Cell stress increases ATP release in NLRP3 inflammasome-mediated autoinflammatory diseases, resulting in cytokine imbalance.
      ,
      • Carta S.
      • Tassi S.
      • Delfino L.
      • Omenetti A.
      • Raffa S.
      • Torrisi M.R.
      • Martini A.
      • Gattorno M.
      • Rubartelli A.
      Deficient production of IL-1 receptor antagonist and IL-6 coupled to oxidative stress in cryopyrin-associated periodic syndrome monocytes.
      ). These features lower the threshold for inflammasome activation. As a consequence, low doses of LPS that are unable to activate and induce IL-1β secretion in healthy monocytes elicit exaggerated ATP release, inflammasome activation, and accelerated and abundant IL-1β secretion through GSDMD pores (
      • Sitia R.
      • Rubartelli A.
      The unconventional secretion of IL-1β: handling a dangerous weapon to optimize inflammatory responses.
      ), followed by pyroptosis in CAPS monocytes (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ). This phenotype is similar to that observed in healthy monocytes upon stimulation with three TLR agonists (
      • Semino C.
      • Carta S.
      • Gattorno M.
      • Sitia R.
      • Rubartelli A.
      Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.
      ,
      • Lavieri R.
      • Piccioli P.
      • Carta S.
      • Delfino L.
      • Castellani P.
      • Rubartelli A.
      TLR costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production.
      ). The activation of the GSDMD, IL-1β hypersecretion, and the subsequent release of DAMPs through pyroptosis in CAPS monocytes exposed to minor stimuli may explain the recurrent episodes of severe systemic inflammatory attacks in the absence of recognizable triggers (
      • Manthiram K.
      • Zhou Q.
      • Aksentijevich I.
      • Kastner D.L.
      The monogenic autoinflammatory diseases define new pathways in human innate immunity and inflammation.
      ).
      In summary, in human monocytes, mild stimuli normally activate lysosome-dependent IL-1β secretion. Pore-mediated secretion is induced only when mild stimulation is matched by stress, either “acquired” following additional stimulation or “constitutive,” due to genetic alterations or metabolic disturbances. In constitutively stressed cells, such as monocytes from individuals with CAPS, even a mild stimulus can activate the pyroptotic pathway of secretion, with explosive release of IL-1β and possibly other DAMPs that further amply inflammation.

      Concluding remarks

      For their growth and survival, multicellular organisms depend on proper signaling among all of their components. Ligands and receptors must be released in appropriate quantities and at the right time and location. Thus, it is not surprising that multiple secretory pathways have evolved for optimal precision and timing of intercellular dialogues. These pathways include those involving LLSPs, and many open questions remain in the wide-ranging field of UPS. First, we do not have a complete picture of how LLSPs are recognized in the crowded cytosol. The export pathways have been characterized for only a few of them. In most cases, it remains largely undefined how their translocation across the plasma membrane or the membrane of intermediate vesicles occurs. A general question still awaiting an answer concerns the reason for additional pathways, given the many mechanisms that can regulate classical protein secretion. Are all unconventional pathways aimed at secreting LLSPs, or do some of them have a different primary role? This question is relevant in light of the hypothesis that some secretion pathways have evolved as mechanisms that protect cells against toxic or dysfunctional proteins and that some secreted proteins were then coopted in danger-signaling circuits. The identification of more LLSPs that exploit unconventional pathways might enable identification of the signals (e.g. protein sequence, conformation, or posttranslational modifications) and molecular machineries that permit their selection and secretion. Often, the characterization of a given mechanism helps to answer wider questions and spur important practical applications. Further dissection of the unconventional secretory pathways may shed light on their evolution and could help to identify key therapeutic avenues to manage many pathological conditions, such as dysregulated inflammation.

      Acknowledgments

      We are grateful to Simone Cenci and Giulio Cavalli for critically reading the manuscript and for valuable comments. We also thank Claudia Semino and Sonia Carta for helpful discussion, Gianmario Sambuceti for suggestions and support, and Marco Dalla Torre for help in editing the manuscript. We apologize to all of the colleagues whose excellent studies could not be cited because of space limitations.

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