Autophagy is suppressed by low temperatures and is dispensable for cold acclimation in Arabidopsis.

Plants have evolved various mechanisms to adapt to the ever-changing external environment. Autophagy is one such mechanism and has been suggested to play a key role in responding to and adapting to abiotic stresses in plants. However, the role of autophagy in adaptation to cold and freezing stresses remains to be characterized in detail. Here, we investigated the role of autophagy in the low-temperature response of Arabidopsis using atg mutants. Both the atg5-1 and atg10-1 mutants exhibited normal freezing tolerance, regardless of cold acclimation. A comparison of fresh weights indicated that the difference in growth between the wild-type and atg plants under cold conditions was rather small compared with that under normal conditions. Analysis of COLD-REGULATED gene expression showed no significant differences between the atg mutants and wild type. Treatment with 3-methyladenine, an inhibitor of autophagy, did not impair the induction of COR15Apro::LUC expression upon exposure to low temperature. Evaluation of autophagic activity using transgenic plants expressing RBCS-mRFP demonstrated that autophagy was rarely induced by cold exposure, even in the dark. Taken together, these data suggest that autophagy is suppressed by low temperatures and is dispensable for cold acclimation and freezing tolerance in Arabidopsis.

Endonucleosis mediates internalization of cytoplasm into the nucleus.

Setd8 regulates transcription elongation, mitotic DNA condensation, DNA damage response and replication licensing. Here we show that, in mitogen-stimulated liver-specific Setd8-KO mice, most of the hepatocytes are eliminated by necrosis but a significant number of them survive via entering a stage exhibiting several senescence-related features. Setd8-deficient hepatocytes had enlarged nuclei, chromosomal hyperploidy and nuclear engulfments progressing to the formation of intranuclear vesicles surrounded by nuclear lamina. These vesicles contain glycogen, cytoplasmic proteins and even entire organelles. We term this process "endonucleosis". Intranuclear vesicles are absent in hepatocytes of Setd8/Atg5 knockout mice, suggesting that the process requires the function of the canonical autophagy machinery. Endonucleosis and hyperploidization are temporary, early events in the surviving Setd8-deficient cells. Larger vesicles break down into microvesicles over time and are eventually eliminated. The results reveal sequential events in cells with extensive DNA damage, which function as part of survival mechanisms to prevent necrotic death.

Full-length transcriptome sequencing of Arabidopsis plants provided new insights into the autophagic regulation of photosynthesis.

Autophagy is a highly conserved eukaryotic pathway and plays a crucial role in cell survival under stress conditions. Here, we applied a full-length transcriptome approach to study an Arabidopsis autophagy mutant (atg5-1) subjected to nitrogen-starvation, using Oxford Nanopore Technologies. A total of 39,033 transcripts were identified, including 11,356 new transcripts. In addition, alternative splicing (AS) events and lncRNAs were also detected between Col-0 (WT) and atg5-1. Differentially expressed transcript enrichment showed that autophagy upregulates the expression of many stress-responsive genes and inhibits the transcription of photosynthesis-associated genes. The qRT-PCR results showed that the expression patterns of photosynthesis-related genes in the atg5-1 differed under the conditions of nitrogen starvation and carbon starvation. Under nitrogen starvation treatment, many genes related to photosynthesis also exhibited AS. Chlorophyll fluorescence images revealed that the Fv/Fm and ΦPSII of old atg5-1 leaves were significantly reduced after nitrogen starvation treatment, but the Y(NPQ) indices were significantly increased compared to those of the WT plants. The results of qRT-PCR suggest that autophagy appears to be involved in the degradation of genes related to photodamage repair in PSII. Taken together, the full-length transcriptiome sequencing provide new insights into how new transcripts, lncRNAs and alternative splicing (AS) are involved in plant autophagy through full-length transcriptome sequencing and suggest a new potential link between autophagy and photosynthesis.

The role of Atg5 gene in tumorigenesis under autophagy deficiency conditions.

Autophagy is a self-recycling machinery to maintain cellular homeostasis by degrading harmful materials in the cell. Autophagy-related gene 5 (Atg5) is required for autophagosome maturation. However, the role of Atg5 in tumorigenesis under autophagy deficient conditions remains unclear. This study focused on the autophagy-independent role of Atg5 and the underlying mechanism in tumorigenesis. We demonstrated that knockout of autophagy-related genes including Atg5, Atg7, Atg9, and p62 in mouse embryonic fibroblast (MEF) cells consistently decreased cell proliferation and motility, implying that autophagy is required to maintain diverse cellular functions. An Atg7 knockout MEF (Atg7-/- MEF) cell line representing deprivation of autophagy function was used to clarify the role of Atg5 transgene in tumorigenesis. We found that Atg5-overexpressed Atg7-/-MEF (clone A) showed increased cell proliferation, colony formation, and migration under autophagy deficient conditions. Accordingly, rescuing the autophagy deficiency of clone A by overexpression of Atg7 gene shifts the role of Atg5 from pro-tumor to anti-tumor status, indicating the dual role of Atg5 in tumorigenesis. Notably, the xenograft mouse model showed that clone A of Atg5-overexpressed Atg7-/- MEF cells induced temporal tumor formation, but could not prolong further tumor growth. Finally, biomechanical analysis disclosed increased Wnt5a secretion and p-JNK expression along with decreased ß-catenin expression. In summary, Atg5 functions as a tumor suppressor to protect the cell under normal conditions. In contrast, Atg5 shifts to a pro-tumor status under autophagy deprivation conditions.

Effects of selegiline on neuronal autophagy involving α-synuclein secretion.

Cell-to-cell transmission of α-synuclein (α-syn) pathology underlies the spread of neurodegeneration in Parkinson's disease. α-Syn secretion is an important factor in the transmission of α-syn pathology. However, it is unclear how α-syn secretion is therapeutically modulated. Here, we investigated effects of monoamine oxidase (MAO)-B inhibitor selegiline on α-syn secretion. Treatment with selegiline promoted α-syn secretion in mouse primary cortical neuron cultures, and this increase was kept under glial cell-eliminated condition by Ara-C. Selegiline-induced α-syn secretion was blocked by cytosolic Ca2+ chelator BAPTA-AM in primary neurons. Selegiline-induced α-syn secretion was retained in MAOA siRNA knockdown, whereas it was abrogated by ATG5 knockdown in SH-SY5Y cells. Selegiline increased LC3-II generation with a reduction in intracellular p62/SQSTM1 levels in primary neurons. The increase in LC3-II generation was blocked by co-treatment with BAPTA-AM in primary neurons. Additionally, fractionation experiments showed that selegiline-induced α-syn secretion occurred in non-extracellular vesicle fractions of primary neurons and SH-SY5Y cells. Collectively, these findings show that selegiline promotes neuronal autophagy involving secretion of non-exosomal α-syn via a change of cytosolic Ca2+ levels.

ATG5-mediated keratinocyte ferroptosis promotes M1 polarization of macrophages to aggravate UVB-induced skin inflammation.

Autophagy participates in the regulation of ferroptosis. Among numerous autophagy-related genes (ATGs), ATG5 plays a pivotal role in ferroptosis. However, how ATG5-mediated ferroptosis functions in UVB-induced skin inflammation is still unclear. In this study, we unveil that the core ferroptosis inhibitor GPX4 is significantly decreased in human skin tissue exposed to sunlight. We report that ATG5 deletion in mouse keratinocytes strongly protects against UVB-induced keratinocyte ferroptosis and skin inflammation. Mechanistically, ATG5 promotes the autophagy-dependent degradation of GPX4 in UVB-exposed keratinocytes, which leads to UVB-induced keratinocyte ferroptosis. Furthermore, we find that IFN-γ secreted by ferroptotic keratinocytes facilitates the M1 polarization of macrophages, which results in the exacerbation of UVB-induced skin inflammation. Together, our data indicate that ATG5 exacerbates UVB-induced keratinocyte ferroptosis in the epidermis, which subsequently gives rise to the secretion of IFN-γ and M1 polarization. Our study provides novel evidence that targeting ATG5 may serve as a potential therapeutic strategy for the amelioration of UVB-caused skin damage.

Immunological synapse formation between T regulatory cells and cancer-associated fibroblasts promotes tumour development.

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA+ CAFs can form immunological synapses with Foxp3+ regulatory T cells (Tregs) in tumours. Notably, α-SMA+ CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA+ CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA+ CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA+ CAFs and Tregs in an autophagy-dependent manner.

Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn2+) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.

Autophagy-related protein 5 (ATG5) interacts with bone marrow stromal cell antigen 2 (BST2) to stimulate HBV replication through antagonizing the antiviral activity of BST2.

Hepatitis B virus (HBV) infection is a major global health burden with 820 000 deaths per year. In our previous study, we found that the knockdown of autophagy-related protein 5 (ATG5) significantly upregulated the interferon-stimulated genes (ISGs) expression to exert the anti-HCV effect. However, the regulation of ATG5 on HBV replication and its underlying mechanism remains unclear. In this study, we screened the altered expression of type I interferon (IFN-I) pathway genes using RT² Profiler™ PCR array following ATG5 knock-down and we found the bone marrow stromal cell antigen 2 (BST2) expression was significantly increased. We then verified the upregulation of BST2 by ATG5 knockdown using RT-qPCR and found that the knockdown of ATG5 activated the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway. ATG5 knockdown or BST2 overexpression decreased Hepatitis B core Antigen (HBcAg) protein, HBV DNA levels in cells and supernatants of HepAD38 and HBV-infected NTCP-HepG2. Knockdown of BST2 abrogated the anti-HBV effect of ATG5 knockdown. Furthermore, we found that ATG5 interacted with BST2, and further formed a ternary complex together with HBV-X (HBx). In conclusion, our finding indicates that ATG5 promotes HBV replication through decreasing BST2 expression and interacting with it directly to antagonize its antiviral function.

Hernandezine promotes cancer cell apoptosis and disrupts the lysosomal acidic environment and cathepsin D maturation.

Hernandezine (Her), a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum, is recognized for its range of biological activities inherent to this herbal medicine. Despite its notable properties, the anti-cancer effects of Her have remained largely unexplored. In this study, we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms. Furthermore, Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems, leading to LC3 lipidation. Our findings revealed that Her caused damage to the mitochondrial membrane, with the damaged mitochondria undergoing mitophagy, as evidenced by the elevated expression of mitophagy markers. Conversely, Her disrupted autophagic flux, demonstrated by the upregulation of p62 and accumulation of autolysosomes, as observed in the RFP-GFP-LC3 reporter assay. Initially, we determined that Her did not prevent the fusion of autophagosomes and lysosomes. However, it inhibited the maturation of cathepsin D and increased lysosomal pH, indicating an impairment of lysosomal function. The use of the early-stage autophagy inhibitor, 3-methyladenine (3-MA), did not suppress LC3II, suggesting that Her also induces noncanonical autophagy in autophagosome formation. The application of Bafilomycin A1, an inhibitor of noncanonical autophagy, diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her, thereby augmenting Her-induced cell death. These observations imply that while autophagy initially plays a protective role, the disruption of the autophagic process by Her promotes programmed cell death. This study provides the first evidence of Her's dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death. These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death, offering potential avenues for enhancing cancer prevention and therapeutic strategies.

Combining large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers for promoting protective melanosome autophagy via the PI3K/Akt/mTOR signalling pathway for the treatment of melasma.

Melasma is a common condition of hyperpigmented facial skin. Picosecond lasers are reported to be effective for the treatment of melasma. We aimed to identify the most effective therapeutic mode and elucidate the potential molecular mechanisms of picosecond lasers for the treatment of melasma. Female Kunming mice with melasma-like conditions were treated using four different picosecond laser modes. Concurrently, in vitro experiments were conducted to assess changes in melanin and autophagy in mouse melanoma B16-F10 cells treated with these laser modes. Changes in melanin in mouse skin were detected via Fontana-Masson staining, and melanin particles were evaluated in B16-F10 cells. Real-time polymerase chain reaction and western blotting were used to analyse the expression levels of melanosome and autophagy-related messenger ribonucleic acid (mRNA) and proteins. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers resulted insignificant decreases in melanin as well as in mRNA and protein expression of melanin-synthesizing enzymes (TYR, TRP-1 and MITF). This combination also led to increased expression of the autophagy-related proteins, Beclin1 and ATG5, with a marked decrease in p62 expression. Intervention with the PI3K activator, 740 Y-P, increased TYR, TRP-1, MITF, p-PI3K, p-AKT, p-mTOR and p62 expression but decreased the expression of LC3, ATG5 and Beclin1. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers proved more effective and safer. It inhibits melanin production, downregulates the PI3K/AKT/mTOR pathway, enhances melanocyte autophagy and accelerates melanin metabolism, thereby reducing melanin content.

[Silencing GmATG5 genes accelerated senescence and enhanced disease resistance in soybean].

Autophagy plays an essential role in recycling/re-utilizing nutrients and in adaptions to numerous stresses. However, the roles of autophagy in soybean have not been investigated extensively. In this study, a virus-induced gene silencing approach mediated by bean pod mottle virus (BPMV) was used to silence autophagy-related gene 5 (ATG5) genes in soybean (referred to as GmATG5). Our results showed that ATG8 proteins were massively accumulated in the dark-treated leaves of the GmATG5-silenced plants relative to the vector control plants (BPMV-0), indicating that autophagy pathway is impaired in the GmATG5-silenced plants. Consistent with the impaired autophagy, an accelerated senescence phenotype was observed on the leaves of the dark-treated GmATG5-silenced plants, which was not shown on the leaves of the dark-treated BPMV-0 plants. In addition, the accumulation levels of both reactive oxygen species (ROS) and salicylic acid (SA) were significantly induced in the GmATG5-silenced plants compared with that of the vector control plants (BPMV-0), indicating an activated immunity. Accordingly, the GmATG5-silenced plants exhibited significantly enhanced resistance against Pseudomonas syringae pv. glycinea (Psg) in comparison with the BPMV-0 plants. Nevertheless, the activated immunity observed in the GmATG5-silenced plant was independent of the activation of mitogen-activated protein kinase (MAPK).

Atg5 deficiency in macrophages protects against kidney fibrosis via the CCR6-CCL20 axis.

BACKGROUND: Autophagy is a lysosome-dependent degradation pathway that regulates macrophage activation, differentiation, and polarization. Autophagy related 5 (Atg5) is a key protein involved in phagocytic membrane elongation in autophagic vesicles that forms a complex with Atg12 and Atg16L1. Alterations in Atg5 are related to both acute and chronic kidney diseases in experimental models. However, the role of macrophage-expressed Atg5 in acute kidney injury remains unclear. METHODS: Using a myeloid cell-specific Atg5 knockout (MΦ atg5-/-) mouse, we established renal ischemia/reperfusion and unilateral ureteral obstruction models to evaluate the role of macrophage Atg5 in renal macrophage migration and fibrosis. RESULTS: Based on changes in the serum urea nitrogen and creatinine levels, Atg5 deletion had a minimal effect on renal function in the early stages after mild injury; however, MΦ atg5-/- mice had reduced renal fibrosis and reduced macrophage recruitment after 4 weeks of ischemia/reperfusion injury and 2 weeks of unilateral ureteral obstruction injury. Atg5 deficiency impaired the CCL20-CCR6 axis after severe ischemic kidneys. Chemotactic responses of bone marrow-derived monocytes (BMDMs) from MΦ atg5-/- mice to CCL20 were significantly attenuated compared with those of wild-type BMDMs, and this might be caused by the inhibition of PI3K, AKT, and ERK1/2 activation. CONCLUSIONS: Our data indicate that Atg5 deficiency decreased macrophage migration by impairing the CCL20-CCR6 axis and inhibited M2 polarization, thereby improving kidney fibrosis.

SS-31 inhibits the inflammatory response by increasing ATG5 and promoting autophagy in lipopolysaccharide-stimulated HepG2 cells.

SS-31 is a mitochondria-targeting short peptide. Recent studies have indicated its hepatoprotective effects. In our study, we investigated the impact of SS-31 on LPS-induced autophagy in HepG2 cells. The results obtained from a dual-fluorescence autophagy detection system revealed that SS-31 promotes the formation of autolysosomes and autophagosomes, thereby facilitating autophagic flux to a certain degree. Additionally, both ELISA and qPCR analyses provided further evidence that SS-31 safeguards HepG2 cells against inflammatory responses triggered by LPS through ATG5-dependent autophagy. In summary, our study demonstrates that SS-31 inhibits LPS-stimulated inflammation in HepG2 cells by upregulating ATG5-dependent autophagy.

Decreased intranuclear cardiac troponin I impairs cardiac autophagy through FOS/ATG5 in ageing hearts.

In our previous study, intranuclear cardiac troponin I (cTnI) may function as a co-factor of Yin Yang 1(YY1). Here, we aimed to explore the role of intranuclear cTnI in ageing hearts. Nuclear translocation of cTnI was demonstrated using Western blot and immunofluorescence. The potential nuclear localization sequences (NLSs) of cTnI were predicted by a web server and then verified in 293T cells by putative NLS-eGFP-GST and NLS-mutant transfection. The ratio of Nuclear cTnI/ Total cTnI (Nu/T) decreased significantly in ageing hearts, accompanied with ATG5-decline-related impaired cardiac autophagy. RNA sequencing was performed in cTnI knockout hearts. The differential expressed genes (DEGs) were analysed by overlapping with YY1 ChIP-sequencing data. cTnI gain and loss experiments in vitro determined those filtered DEGs' expression levels. A strong correlation was found between expression patterns cTnI and FOS. Using ChIP-q-PCR, we demonstrated that specific binding DNA sequences of cTnI were enriched in the FOS promoter -299 to -157 region. It was further verified that pcDNA3.1 (-)-cTnI could increase the promoter activity of FOS by using luciferase report assay. At last, we found that FOS can regulate the ATG5 (autophagy-related gene 5) gene by using a luciferase report assay. Taken together, our results indicate that decreased intranuclear cTnI in ageing hearts may cause impaired cardiac autophagy through the FOS/ATG5 pathway.

Maternal obesity: sex-specific in utero changes in fetal brain autophagy and mTOR.

OBJECTIVE: Maternal obesity affects 39.7% of reproductive-age women in the United States. Emerging research has suggested that in utero exposure to maternal obesity is associated with adverse neurodevelopmental outcomes, but knowledge of underlying mechanisms in human samples is lacking. METHODS: A matched case-control study was performed in women with singleton fetuses who were undergoing elective pregnancy termination at gestational ages 15 to 21 weeks. Maternal adiponectin levels from plasma were measured using ELISA kits. RNA was extracted from fetal brain tissue using RNeasy Mini Kit (QIAGEN). mRNA expression from ADIPOR1, ADIPOR2, MTOR, ATG5, ATG7, BECN1, and MAP1LC3B was quantified through the ΔΔCt method and using GAPDH as a housekeeping gene. RESULTS: We have identified transcription patterns associated with inhibition of autophagy in male fetal brain tissue exposed to maternal obesity (↑MTOR, ↓ATG5, ↓ATG7, and ↓MAP1LC3B), with female fetuses demonstrating either no change in transcription or nonsignificant changes associated with increased autophagy. There was significant downregulation of the autophagy-associated gene BECN1 in both male and female individuals who were exposed to obesity in utero. CONCLUSIONS: We present novel evidence suggesting that in utero exposure to maternal obesity in humans may significantly affect neurodevelopment, especially in male fetuses, through alterations in normal autophagy molecular mechanisms and with adiponectin as a potential mediator.

Indobufen alleviates ischemic stroke injury by regulating transcription factor NRF2 and inhibiting ATG5 expression.

BACKGROUND: Ischemic stroke (IS) is a detrimental neurological disease and IS lacks valuable methods to recover body function. Indobufen (IND) could alleviate IS. However, the possible mechanism remains undefined. METHODS: SH-SY5Y cells were cultured under the oxygen-glucose deprivation/reoxygenation (OGD/R) environment and then were treated with small interfering RNA (siRNA) of NRF2 and ATG5. The influence of various concentrations of IND (50 µM, 100 µM, 200 µM, and 400 µM) was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide. Levels of superoxide dismutase (SOD) and malonaldehyde (MDA) were examined by ELISA. Reactive oxygen species (ROS) production was determined by DCFH-DA staining. The protein levels of LC3II/LC3I, Beclin1, p62, NRF2, and ATG5 were detected by western blot. RESULTS: IND increased cell viability, while depressed the rate of apoptosis in SH-SY5Y cells of OGD/R environment. IND inhibited autophagy by suppressing the levels of LC3II/LC3I, Beclin1 protein, and increasing p62 protein expression in SH-SY5Y cells of OGD/R environment. IND limited the contents of ROS and MDA, while amplifying the activity of SOD in SH-SY5Y cells with OGD/R exposure. IND also promoted NRF2 expression in OGD/R environment. CONCLUSION: IND could inhibit autophagy, oxidative stress, and apoptosis in SH-SY5Y cells with OGD/R exposure, further alleviating IS injury by regulating transcription factor NRF2 and inhibiting ATG5 expression.

Serum autophagy-related gene 5 level in stroke patients: correlation with CD4+ T cells and cognition impairment during a 3-year follow-up.

Autophagy-related gene (ATG) 5 regulates blood lipids, chronic inflammation, CD4+ T-cell differentiation, and neuronal death and is involved in post-stroke cognitive impairment. This study aimed to explore the correlation of serum ATG5 with CD4+ T cells and cognition impairment in stroke patients. Peripheral blood was collected from 180 stroke patients for serum ATG5 and T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cell detection via enzyme-linked immunosorbent assays and flow cytometry. The Mini-Mental State Examination (MMSE) scale was completed at enrollment, year (Y)1, Y2, and Y3 in stroke patients. Serum ATG5 was also measured in 50 healthy controls (HCs). Serum ATG5 was elevated in stroke patients compared to HCs (P<0.001) and was positively correlated to Th2 cells (P=0.022), Th17 cells (P<0.001), and Th17/Treg ratio (P<0.001) in stroke patients but not correlated with Th1 cells, Th1/Th2 ratio, or Treg cells (all P>0.050). Serum ATG5 (P=0.037), Th1 cells (P=0.022), Th17 cells (P=0.002), and Th17/Treg ratio (P=0.018) were elevated in stroke patients with MMSE score-identified cognition impairment vs those without cognition impairment, whereas Th2 cells, Th1/Th2 ratio, and Treg cells were not different between them (all P>0.050). Importantly, serum ATG5 was negatively linked with MMSE score at enrollment (P=0.004), Y1 (P=0.002), Y2 (P=0.014), and Y3 (P=0.001); moreover, it was positively related to 2-year (P=0.024) and 3-year (P=0.012) MMSE score decline in stroke patients. Serum ATG5 was positively correlated with Th2 and Th17 cells and estimated cognitive function decline in stroke patients.

Autophagy-related 5 in acute ischemic stroke: Variation and linkage with neurofunction, and survival.

OBJECTIVE: Autophagy-related 5 (ATG5) facilitates the pathologic process of acute ischemic stroke (AIS) via multiple ways. This study aimed to identify the association of serum ATG5 with clinical outcomes in AIS patients. METHODS: Serum ATG5 from 280 AIS patients were detected at admission, Day (D)1, D3, D7, D30, and D90 after admission by enzyme-linked immunosorbent assay. The median (interquartile range) follow-up was 21.1 (5.9-43.9) months. Another 50 healthy controls (HCs) were also enrolled for serum ATG5 determination. RESULTS: ATG5 was elevated (p < 0.001) (vs. HCs), and positively correlated with hyperlipidemia (p = 0.016), and the national institutes of health stroke scale score (p = 0.001) in AIS patients. Interestingly, ATG5 was increased from admission to D1, but gradually decreased until D90 (p < 0.001). Besides, 85 (30.4%) and 195 (69.6%) AIS patients were assessed as modified Rankin Scale (mRS) >2 and mRS ≤2 at D90, respectively. ATG5 at admission, D1, D3, D30, and D90 was elevated in AIS patients with mRS >2 versus those with mRS ≤2 (all p < 0.050). ATG5 at admission, D1, D3, D7, D30, or D90 was elevated in relapsed (vs. non-relapsed) or died (vs. survived) AIS patients (all p < 0.050). Recurrence-free survival was shortened in AIS patients with high (≥52.0 ng/mL) ATG5 versus those with low (<52.0 ng/mL) ATG5 at admission, D3, D7, and D30 (all p < 0.050); overall survival was shorter in AIS patients with high (vs. low) ATG5 at D7 and D30 (both p < 0.050). INTERPRETATION: Serum ATG5 elevates at first, thereafter gradually declines, whose elevation associates with neurological dysfunction, recurrence, and death risk in AIS patients.

An autophagy program that promotes T cell egress from the lymph node controls responses to immune checkpoint blockade.

Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.