Unconventional Activation of IRE1 Enhances Th17 Responses and Promotes Airway Neutrophilia.

Heightened unfolded protein responses (UPRs) are associated with the risk for asthma, including severe asthma. Treatment-refractory severe asthma manifests a neutrophilic phenotype with T helper (Th)17 responses. However, how UPRs participate in the deregulation of Th17 cells leading to neutrophilic asthma remains elusive. This study found that the UPR sensor IRE1 is induced in the murine lung with fungal asthma and is highly expressed in Th17 cells relative to naive CD4+ T cells. Cytokine (e.g., IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by both human and mouse Th17 cells. Ern1 (encoding IRE1) deficiency decreases the expression of endoplasmic reticulum stress factors and impairs the differentiation and cytokine secretion of Th17 cells. Genetic ablation of Ern1 leads to alleviated Th17 responses and airway neutrophilia in a fungal airway inflammation model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway in vivo and enhances Th17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPR-mediated secretory function of Th17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in Th17-biased TH2-low asthma.

The Autophagy, Inflammation and Metabolism Center international eSymposium - an early-career investigators' seminar series during the COVID-19 pandemic.

The Autophagy, Inflammation and Metabolism (AIM) Center organized a globally accessible, virtual eSymposium during the COVID-19 pandemic in 2020. The conference included presentations from scientific leaders, as well as a career discussion panel, and provided a much-needed platform for early-career investigators (ECIs) to showcase their research in autophagy. This Perspective summarizes the science presented by the ECIs during the event and discusses the lessons learned from a virtual meeting of this kind during the pandemic. The meeting was a learning experience for all involved, and the ECI participants herein offer their thoughts on the pros and cons of virtual meetings as a modality, either as standalone or hybrid events, with a view towards the post-pandemic world.

IRGM governs the core autophagy machinery to conduct antimicrobial defense.

IRGM, encoded by a uniquely human gene conferring risk for inflammatory diseases, affects autophagy through an unknown mechanism. Here, we show how IRGM controls autophagy. IRGM interacts with ULK1 and Beclin 1 and promotes their co-assembly thus governing the formation of autophagy initiation complexes. We further show that IRGM interacts with pattern recognition receptors including NOD2. IRGM, NOD2, and ATG16L1, all of which are Crohn's disease risk factors, form a molecular complex to modulate autophagic responses to microbial products. NOD2 enhances K63-linked polyubiquitination of IRGM, which is required for interactions of IRGM with the core autophagy factors and for microbial clearance. Thus, IRGM plays a direct role in organizing the core autophagy machinery to endow it with antimicrobial and anti-inflammatory functions.

A non-canonical role for the autophagy machinery in anti-retroviral signaling mediated by TRIM5α.

TRIM5α is a key cross-species barrier to retroviral infection, with certain TRIM5 alleles conferring increased risk of HIV-1 infection in humans. TRIM5α is best known as a species-specific restriction factor that directly inhibits the viral life cycle. Additionally, it is also a pattern-recognition receptor (PRR) that activates inflammatory signaling. How TRIM5α carries out its multi-faceted actions in antiviral defense remains incompletely understood. Here, we show that proteins required for autophagy, a cellular self-digestion pathway, play an important role in TRIM5α's function as a PRR. Genetic depletion of proteins involved in all stages of the autophagy pathway prevented TRIM5α-driven expression of NF-κB and AP1 responsive genes. One of these genes is the preeminent antiviral cytokine interferon ß (IFN-ß), whose TRIM5-dependent expression was lost in cells lacking the autophagy proteins ATG7, BECN1, and ULK1. Moreover, we found that the ability of TRIM5α to stimulate IFN-ß expression in response to recognition of a TRIM5α-restricted HIV-1 capsid mutant (P90A) was abrogated in cells lacking autophagy factors. Stimulation of human macrophage-like cells with the P90A virus protected them against subsequent infection with an otherwise resistant wild type HIV-1 in a manner requiring TRIM5α, BECN1, and ULK1. Mechanistically, TRIM5α was attenuated in its ability to activate the kinase TAK1 in autophagy deficient cells, and both BECN1 and ATG7 contributed to the assembly of TRIM5α-TAK1 complexes. These data demonstrate a non-canonical role for the autophagy machinery in assembling antiviral signaling complexes and in establishing a TRIM5α-dependent antiviral state.

TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation.

Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. Here, we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key proinflammatory cytokine, IL-1ß, induced autophagy leading to autophagic killing of mycobacteria in macrophages, and this IL-1ß activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.

TAK1 converts Sequestosome 1/p62 from an autophagy receptor to a signaling platform.

The protein p62/Sequestosome 1 (p62) has been described as a selective autophagy receptor and independently as a platform for pro-inflammatory and other intracellular signaling. How these seemingly disparate functional roles of p62 are coordinated has not been resolved. Here, we show that TAK1, a kinase involved in immune signaling, negatively regulates p62 action in autophagy. TAK1 reduces p62 localization to autophagosomes, dampening the autophagic degradation of both p62 and p62-directed autophagy substrates. TAK1 also relocalizes p62 into dynamic cytoplasmic bodies, a phenomenon that accompanies the stabilization of TAK1 complex components. On the other hand, p62 facilitates the assembly and activation of TAK1 complexes, suggesting a connection between p62's signaling functions and p62 body formation. Thus, TAK1 governs p62 action, switching it from an autophagy receptor to a signaling platform. This ability of TAK1 to disable p62 as an autophagy receptor may allow certain autophagic substrates to accumulate when needed for cellular functions.

Continual renewal and replication of persistent Leishmania major parasites in concomitantly immune hosts.

In most natural infections or after recovery, small numbers of Leishmania parasites remain indefinitely in the host. Persistent parasites play a vital role in protective immunity against disease pathology upon reinfection through the process of concomitant immunity, as well as in transmission and reactivation, yet are poorly understood. A key question is whether persistent parasites undergo replication, and we devised several approaches to probe the small numbers in persistent infections. We find two populations of persistent Leishmania major: one rapidly replicating, similar to parasites in acute infections, and another showing little evidence of replication. Persistent Leishmania were not found in "safe" immunoprivileged cell types, instead residing in macrophages and DCs, ∼60% of which expressed inducible nitric oxide synthase (iNOS). Remarkably, parasites within iNOS+ cells showed normal morphology and genome integrity and labeled comparably with BrdU to parasites within iNOS- cells, suggesting that these parasites may be unexpectedly resistant to NO. Nonetheless, because persistent parasite numbers remain roughly constant over time, their replication implies that ongoing destruction likewise occurs. Similar results were obtained with the attenuated lpg2- mutant, a convenient model that rapidly enters a persistent state without inducing pathology due to loss of the Golgi GDP mannose transporter. These data shed light on Leishmania persistence and concomitant immunity, suggesting a model wherein a parasite reservoir repopulates itself indefinitely, whereas some progeny are terminated in antigen-presenting cells, thereby stimulating immunity. This model may be relevant to understanding immunity to other persistent pathogen infections.

TRIM17 contributes to autophagy of midbodies while actively sparing other targets from degradation.

TRIM proteins contribute to selective autophagy, a process whereby cells target specific cargo for autophagic degradation. In a previously reported screen, TRIM17 acted as a prominent inhibitor of bulk autophagy, unlike the majority of TRIMs, which had positive roles. Nevertheless, TRIM17 showed biochemical hallmarks of autophagy-inducing TRIMs. To explain this paradox, here, we investigated how TRIM17 inhibits selective autophagic degradation of a subset of targets while promoting degradation of others. We traced the inhibitory function of TRIM17 to its actions on the anti-autophagy protein Mcl-1, which associates with and inactivates Beclin 1. TRIM17 expression stabilized Mcl-1-Beclin-1 complexes. Despite its ability to inhibit certain types of selective autophagy, TRIM17 promoted the removal of midbodies, remnants of the cell division machinery that are known autophagy targets. The selective loss of anti-autophagy Mcl-1 from TRIM17-Beclin-1 complexes at midbodies correlated with the ability of TRIM17 to promote midbody removal. This study further expands the roles of TRIMs in regulating selective autophagy by showing that a single TRIM can, depending upon a target, either positively or negatively regulate autophagy.

Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation.

Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis, but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M. tuberculosis infection of Atg5(fl/fl) LysM-Cre(+) mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5(fl/fl) LysM-Cre(+) mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M. tuberculosis growth and damaging inflammation.

Regulation of Mitochondria-Derived Immune Activation by 'Antiviral' TRIM Proteins.

Mitochondria are key orchestrators of antiviral responses that serve as platforms for the assembly and activation of innate immune-signaling complexes. In response to viral infection, mitochondria can be triggered to release immune-stimulatory molecules that can boost interferon production. These same molecules can be released by damaged mitochondria to induce pathogenic, antiviral-like immune responses in the absence of infection. This review explores how members of the tripartite motif-containing (TRIM) protein family, which are recognized for their roles in antiviral defense, regulate mitochondria-based innate immune activation. In antiviral defense, TRIMs are essential components of immune signal transduction pathways and function as directly acting viral restriction factors. TRIMs carry out conceptually similar activities when controlling immune activation related to mitochondria. First, they modulate immune-signaling pathways that can be activated by mitochondrial molecules. Second, they co-ordinate the direct removal of mitochondria and associated immune-activating factors through mitophagy. These insights broaden the scope of TRIM actions in innate immunity and may implicate TRIMs in diseases associated with mitochondria-derived inflammation.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

The retroviral restriction factor TRIM5/TRIM5α regulates mitochondrial quality control.

The protein TRIM5 is under intensive investigation related to its roles in antiviral defense, yet its underlying mechanisms of action remain elusive. In our study, we performed an unbiased identification of TRIM5-interacting partners and found proteins participating in a wide variety of cellular functions. We utilized this proteomics data set to uncover a role for TRIM5 in mitophagy, a mitochondrial quality control system that is impaired in multiple human diseases. Mitochondrial damage triggers the recruitment of TRIM5 to ER-mitochondria contact sites where TRIM5 colocalizes with markers of autophagosome biogenesis. Cells lacking TRIM5 are unable to carry out PRKN-dependent and PRKN-independent mitophagy pathways. TRIM5 knockout cells show reduced mitochondrial function and uncontrolled immune activation in response to mitochondrial damage; phenotypes consistent with a requirement for TRIM5 in mitophagy. Mechanistically, we found that TRIM5 is required for the recruitment of the autophagy initiation machinery to damaged mitochondria, where TRIM5 acts as a scaffold promoting interactions between protein markers of mitochondrial damage and the autophagy initiation machinery.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α. Mitochondrial damage triggers TRIM5α's auto-ubiquitination and its interaction with ubiquitin-binding autophagy adaptors including NDP52, optineurin, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1. TRIM5α with intact ubiquitination function is required for the proper accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Additionally, we show that TRIM5α can directly recruit autophagy initiation machinery to damaged mitochondria. Our data support a model in which TRIM5α provides a self-amplifying, mitochondria-localized, ubiquitin-based, assembly platform for TBK1 and mitophagy adaptors that is ultimately required to recruit the core autophagy machinery.

Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Neutrophilic Airway Inflammation.

Treatment-refractory severe asthma manifests a neutrophilic phenotype associated with TH17 responses. Heightened unfolded protein responses (UPRs) are associated with the risk of asthma, including severe asthma. However, how UPRs participate in the deregulation of TH17 cells leading to this type of asthma remains elusive. In this study, we investigated the role of the UPR sensor IRE1 in TH17 cell function and neutrophilic airway inflammation. We found that IRE1 is induced in fungal asthma and is highly expressed in TH17 cells relative to naïve CD4+ T cells. Cytokine (e.g. IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by TH17 cells. Ern1 (encoding IRE1)-deficiency decreases the expression of ER stress factors and impairs the differentiation and cytokine secretion of TH17 cells. Genetic ablation of Ern1 leads to alleviated TH17 responses and airway neutrophilia in a Candida albicans asthma model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway in vivo and enhances TH17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPRmediated secretory function of TH17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in TH17-biased TH2-low asthma.

Quantitative single-cell analysis of Leishmania major amastigote differentiation demonstrates variably extended expression of the lipophosphoglycan (LPG) virulence factor in different host cell types.

Immediately following their deposition into the mammalian host by an infected sand fly vector, Leishmania parasites encounter and are engulfed by a variety of cell types. From there, parasites may transit to other cell types, primarily macrophages or dendritic cells, where they replicate and induce pathology. During this time, Leishmania cells undergo a dramatic transformation from the motile non-replicating metacyclic stage to the non-motile replicative amastigote stage, a differentiative process that can be termed amastigogenesis. To follow this at the single cell level, we identified a suite of experimental 'landmarks' delineating different stages of amastigogenesis qualitatively or quantitatively, including new uses of amastigote-specific markers that showed interesting cellular localizations at the anterior or posterior ends. We compared amastigogenesis in synchronous infections of peritoneal and bone-marrow derived macrophages (PEM, BMM) or dendritic cells (BMDC). Overall, the marker suite expression showed an orderly transition post-infection with similar kinetics between host cell types, with the emergence of several amastigote traits within 12 hours, followed by parasite replication after 24 hours, with parasites in BMM or BMDC initiating DNA replication more slowly. Lipophosphoglycan (LPG) is a Leishmania virulence factor that facilitates metacyclic establishment in host cells but declines in amastigotes. Whereas LPG expression was lost by parasites within PEM by 48 hours, >40% of the parasites infecting BMM or BMDC retained metacyclic-level LPG expression at 72 hr. Thus L. major may prolong LPG expression in different intracellular environments, thereby extending its efficacy in promoting infectivity in situ and during cell-to-cell transfer of parasites expressing this key virulence factor.

Interactomic analysis reveals a homeostatic role for the HIV restriction factor TRIM5α in mitophagy.

The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.

Proteopathic tau primes and activates interleukin-1ß via myeloid-cell-specific MyD88- and NLRP3-ASC-inflammasome pathway.

Pathological hyperphosphorylation and aggregation of tau (pTau) and neuroinflammation, driven by interleukin-1ß (IL-1ß), are the major hallmarks of tauopathies. Here, we show that pTau primes and activates IL-1ß. First, RNA-sequence analysis suggests paired-helical filaments (PHFs) from human tauopathy brain primes nuclear factor κB (NF-κB), chemokine, and IL-1ß signaling clusters in human primary microglia. Treating microglia with pTau-containing neuronal media, exosomes, or PHFs causes IL-1ß activation, which is NLRP3, ASC, and caspase-1 dependent. Suppression of pTau or ASC reduces tau pathology and inflammasome activation in rTg4510 and hTau mice, respectively. Although the deletion of MyD88 prevents both IL-1ß expression and activation in the hTau mouse model of tauopathy, ASC deficiency in myeloid cells reduces pTau-induced IL-1ß activation and improves cognitive function in hTau mice. Finally, pTau burden co-exists with elevated IL-1ß and ASC in autopsy brains of human tauopathies. Together, our results suggest pTau activates IL-1ß via MyD88- and NLRP3-ASC-dependent pathways in myeloid cells, including microglia.

Migratory dermal dendritic cells act as rapid sensors of protozoan parasites.

Dendritic cells (DC), including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC) are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC) is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Galpha(i) protein-coupled receptor-dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.