Starvation-induced autophagy modulates spermatogenesis and sperm quality in Nile tilapia.

Spermatogenesis is a finely regulated process that involves the interaction of several cellular mechanisms to ensure the proper development and maturation of germ cells. This study assessed autophagy contribution and its relation to apoptosis in fish spermatogenesis during starvation. To that end, Nile tilapia males were subjected to 0 (control), 7, 14, 21, and 28 days of starvation to induce autophagy. Testes samples were obtained for analyses of spermatogenesis by histology, electron microscopy, immunohistochemistry, and western blotting. Sperm quality was assessed using a computer-assisted sperm analysis (CASA) system. Data indicated a significant reduction in gonadosomatic index, seminiferous tubule area, and spermatozoa proportion in fish subject to starvation compared to the control group. Immunoblotting revealed a reduction of Bcl2 and Beclin 1 associated with increased Bax and Caspase-3, mainly after 21 and 28 days of starvation. LC3 and P62 indicated reduced autophagic flux in these starvation times. Immunolabeling for autophagic and apoptotic proteins occurred in all development stages of the germ cells, but protein expression varied throughout starvation. Beclin 1 and Cathepsin D decreased while Bax and Caspase-3 increased in spermatocytes, spermatids, and spermatozoa after 21 and 28 days. Autophagic and lysosomal proteins colocalization indicated the fusion of autophagosomes with lysosomes and lysosomal degradation in spermatogenic cells. The CASA system indicated reduced sperm motility and velocity in animals subjected to 21 and 28 days of starvation. Altogether, the data support autophagy acting at different spermatogenesis stages in Nile tilapia, with decreased autophagy and increased apoptosis after 21 and 28 days of starvation, which results in a decrease in the spermatozoa number and sperm quality.

Autophagy as an innate immunity response against pathogens: a Tango dance.

Intracellular infections as well as changes in the cell nutritional environment are main events that trigger cellular stress responses. One crucial cell response to stress conditions is autophagy. During the last 30 years, several scenarios involving autophagy induction or inhibition over the course of an intracellular invasion by pathogens have been uncovered. In this review, we will present how this knowledge was gained by studying different microorganisms. We intend to discuss how the cell, via autophagy, tries to repel these attacks with the objective of destroying the intruder, but also how some pathogens have developed strategies to subvert this. These two fates can be compared with a Tango, a dance originated in Buenos Aires, Argentina, in which the partner dancers are in close connection. One of them is the leader, embracing and involving the partner, but the follower may respond escaping from the leader. This joint dance is indeed highly synchronized and controlled, perfectly reflecting the interaction between autophagy and microorganism.

Non-canonical Ca2+- Akt signaling pathway mediates the antiproteolytic effects induced by oxytocin receptor stimulation in skeletal muscle.

Although it has been previously demonstrated that oxytocin (OXT) receptor stimulation can control skeletal muscle mass in vivo, the intracellular mechanisms that mediate this effect are still poorly understood. Thus, rat oxidative skeletal muscles were isolated and incubated with OXT or WAY-267,464, a non-peptide selective OXT receptor (OXTR) agonist, in the presence or absence of atosiban (ATB), an OXTR antagonist, and overall proteolysis was evaluated. The results indicated that both OXT and WAY-267,464 suppressed muscle proteolysis, and this effect was blocked by the addition of ATB. Furthermore, the WAY-induced anti-catabolic action on protein metabolism did not involve the coupling between OXTR and Gαi since it was insensitive to pertussis toxin (PTX). The decrease in overall proteolysis induced by WAY was probably due to the inhibition of the autophagic/lysosomal system, as estimated by the decrease in LC3 (an autophagic/lysosomal marker), and was accompanied by an increase in the content of Ca2+-dependent protein kinase (PKC)-phosphorylated substrates, pSer473-Akt, and pSer256-FoxO1. Most of these effects were blocked by the inhibition of inositol triphosphate receptors (IP3R), which mediate Ca2+ release from the sarcoplasmic reticulum to the cytoplasm, and triciribine, an Akt inhibitor. Taken together, these findings indicate that the stimulation of OXTR directly induces skeletal muscle protein-sparing effects through a Gαq/IP3R/Ca2+-dependent pathway and crosstalk with Akt/FoxO1 signaling, which consequently decreases the expression of genes related to atrophy, such as LC3, as well as muscle proteolysis.

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKα and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

Ultrastructural and Molecular Evidence of Macroautophagy in Functioning PitNETs and Experimental Pituitary Tumors.

INTRODUCTION: Macroautophagy is a lysosome-mediated degradation process that controls the quality of cytoplasmic components and organelles, with its regulation depending on autophagy-related proteins (Atg) and with Beclin1/Atg6 and microtubule-associated protein light chain 3 (LC3/Atg8) being key players in the mammalian autophagy. As reports on this mechanism in the field of pituitary neuropathology and neuroendocrinology are scarce, our study analyzed the ultrastructural signs of macroautophagy and the expression of Beclin1 and LC3 proteins in human functioning PitNETs and in experimental pituitary tumors. METHODS: A group of humans functioning PitNETs and an experimental lactotroph model in rats of the F344 strain stimulated with estradiol benzoate (BE) were used. Ultrastructural and molecular evidence of the macroautophagic process was evaluated using different techniques. RESULTS: In functioning PitNETs cohort, 60% exhibited evidence of macroautophagy, with a significant difference found for Beclin1 and LC3 between macro- and micro-PitNETs (p < 0.05). In the experimental model, the expression of both Beclin1 and LC3 proteins was immunopositive in normal and tumoral glands when analyzed by immunofluorescence, Western blot, and immunohistochemistry. In the experimental model, protein expression was associated with increased glandular size and weight. CONCLUSIONS: Our study revealed evidence of macroautophagy at the pituitary level and the important role of Beclin1 and LC3 in the progression of functioning PitNETs, implying that this mechanism participate in regulating pituitary cell growth.

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/ LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKA and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

Participation of signaling proteins in sperm hyperactivation.

Sperm hyperactivation is described as a fast whip movement of the flagellum, an irregular trajectory, and an asymmetrically flagellum bend. This motility pattern is achieved during the passage of the sperm along the female genital tract. It helps the spermatozoa to cross through different viscous ambient fluids to finally reach the oocyte. Important signaling proteins are located in the sperm head and flagellum, and they all play an important role in the cascade that controls the sperm hyperactivation. The presence of HCO3- modulates the activity of the soluble adenylyl cyclase (sAC), leading to the production of cAMP. In turn, cAMP modulates the sperm-specific Na+/H+ exchanger (sNHE) and the t-complex protein 11 (TCP11) which play an essential role on the signaling pathway (cAMP/PKA and tyrosine phosphorylation) and sperm hypermotility. sNHE, cystic fibrosis transmembrane conductance regulator (CFTR), and voltage-gated proton channel (Hv) mainly contribute to the regulation of the intracellular pH (pHi) during capacitation. HCO3- entrance and the removal of H+ from the cytoplasm induces the alkalization of pHi, and this change will contribute to the activation of the cation channel of sperm (CatSper). Recently, it was described the participation on sperm motility and the regulation of calcium channels of an autophagy-related protein, the microtubule-associated protein light chain 3 (LC3). This review gathers important literature about the essential roles of sAC, sNHE, CFTR, Hv, and CatSper in the acquisition of sperm hyperactivation, and provides an integrated overview of recently described roles of TCP11 and LC3 on the sperm signaling pathway. Additionally, we provide insight into the infertility induced by the dysfunction of these critical proteins.

IL-6 secretion of CD4+ T cells stimulated by LC3-positive extracellular vesicles in human epithelial ovarian cancer.

BACKGROUND: Ovarian cancer (OC) as the most fatal gynecological malignancy worldwide, with epithelial ovarian cancer (EOC) being the predominant and most lethal form, poses a serious threat to human health. LC3-positive extracellular vesicles (LC3+ EVs) promote tumorigenesis by educating CD4+ T cells in a murine melanoma model. However, regulation of LC3+ EVs in human EOC remains largely unknown.  METHODS: Differential analysis of Rab8a, Hsp90α and Il6 expression was performed using GEPIA2. The number of LC3+ EVs and the frequency of Heat shock protein 90α+ LC3+ EVs (HSP90α+ LC3+ EVs) in the ascites of EOC patients were tested by flow cytometry. IL-6, IL-10, IFN-γ, IL-4 and TGF-ß were measured by ELISA. CD4+ T cells were isolated from peripheral blood of healthy human donors using MACS magnetic bead technology.  RESULTS: Higher Rab8a, Hsp90a and Il6 expression of cancer tissues compared with normal adjacent tissues in OC were found. The level of IL-6 was positively correlated with LC3+ EVs number, HSP90α+ LC3+ EVs percentage in the ascites, and ROMA index of the patient. In addition, elevated IL-6 production by CD4+ T cells induced by LC3+ EVs was observed, which was suppressed by anti-HSP90α or anti-TLR2.  CONCLUSIONS: LC3+ EVs level and HSP90α+ LC3+ EVs percentage were associated with elevated IL-6 in the ascites of EOC patients. HSP90α on LC3+ EVs from human EOC could stimulate CD4+ T cell production of IL-6 via TLR2.

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Regulation of total LC3 levels by angiotensin II in vascular smooth muscle cells.

Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II-induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co-chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT-qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II-mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co-administration with either losartan 1 µM (AT1R antagonist) or Y-27632 10 µM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

Down-regulation of autophagy proteins is associated with higher mTOR expression in the placenta of pregnant women with preeclampsia

Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged organelles maintaining cellular integrity. It seems to be essential for cell survival during stress, starvation, hypoxia, and consequently to the placenta implantation and development. Preeclampsia (PE) is a multisystemic disorder characterized by the onset of hypertension associated or not with proteinuria and other maternal complications. Considering that the placenta seems to play an important role in the pathogenesis of PE, the objective of the present study was to evaluate protein levels of light chain protein (LC3), beclin-1, and the mammalian target of rapamycin (mTOR) in the placenta of pregnant women with PE. Placental tissues collected from 20 women with PE and 20 normotensive (NT) pregnant women were evaluated for LC3, beclin-1, and mTOR expression by qPCR and immunohistochemistry. The mRNA for LC3 and beclin-1 were significantly lower, while mTOR gene expression was significantly higher in the placenta of pregnant women with PE than in the NT group. Placentas of PE women showed significantly decreased protein expression of LC3-II and beclin-1, whereas mTOR was significantly increased compared with the NT pregnant women. There was a negative correlation between protein expression of mTOR and LC3-II in the placental tissue of PE women. In conclusion, the results showed autophagy deficiency suggesting that failure in this degradation process may contribute to the pathogenesis of PE; however, new studies involving cross-talk between autophagy and inflammatory molecular mechanisms might help to better understand the autophagy process in this obstetric pathology.

Autophagy upregulation may explain inhibition of oral carcinoma in situ by photobiomodulation in vitro.

There is currently no clear understanding on the pathways involved in the process of cell inhibition by photobiomodulation (PBM). The present study evaluated the influence of PBM on the expression of autophagy markers in vitro in an in situ model of oral carcinoma. Oral squamous cell carcinoma (Cal27) and stromal fibroblasts (FG) cultures were used. The independent variables were 'cell type' (FG and CAL27) 'culture condition' (monocultures or co-cultures) and PBM (placebo and 36 J/cm2). The cultures were irradiated from a red LED source for mRNA expression and protein expression analyses. The autophagy markers evaluated were Beclin-1, LC3B and p62 as well as adjuvant markers (BAX Bcl-2, VEGF, CD105, CD34, PRDX1, PRDX4 and GRP78). The Cal27 cells upregulated the autophagy markers upon exposure to PBM both at the mRNA and protein expression levels, providing evidence to explain malignant cell inhibition by PBM.

PKCα Is Recruited to Staphylococcus aureus-Containing Phagosomes and Impairs Bacterial Replication by Inhibition of Autophagy.

Hijacking the autophagic machinery is a key mechanism through which invasive pathogens such as Staphylococcus aureus replicate in their host cells. We have previously demonstrated that the bacteria replicate in phagosomes labeled with the autophagic protein LC3, before escaping to the cytoplasm. Here, we show that the Ca2+-dependent PKCα binds to S. aureus-containing phagosomes and that α-hemolysin, secreted by S. aureus, promotes this recruitment of PKCα to phagosomal membranes. Interestingly, the presence of PKCα prevents the association of the autophagic protein LC3. Live cell imaging experiments using the PKC activity reporter CKAR reveal that treatment of cells with S. aureus culture supernatants containing staphylococcal secreted factors transiently activates PKC. Functional studies reveal that overexpression of PKCα causes a marked inhibition of bacterial replication. Taken together, our data identify enhancing PKCα activity as a potential approach to inhibit S. aureus replication in mammalian cells.

Leishmania (V.) braziliensis infection promotes macrophage autophagy by a LC3B-dependent and BECLIN1-independent mechanism.

Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phagocytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimination. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirming BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell.

Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Silencing of ATG6 and ATG8 promotes increased levels of triacylglycerol (TAG) in the fat body during prolonged starvation periods in the Chagas disease vector Rhodnius prolixus.

Rhodnius prolixus is an obligatorily hematophagous insect known as an important vector of Chagas disease. Autophagy is a conserved cellular mechanism that acts in response to nutrient starvation, where components of the cytoplasm are sequestered by a double membrane organelle, named autophagosome, which is targeted to fuse with the lysosome for degradation. Lipophagy is the process of lipid degradation by selective autophagy, where autophagosomes sequester lipid droplets and degrade triacylglycerol (TAG) generating free fatty acids for ß-oxidation. Here, two essential genes of the autophagic pathway, Atg6/Beclin1 (RpAtg6) and Atg8/LC3 (RpAtg8), were silenced and the storage of lipids during starvation in Rhodnius prolixus was monitored. We found that RNAi knockdown of both RpAtg6 and RpAtg8 resulted in higher levels of TAG in the fat body and the flight muscle, 24 days after the blood meal, as well as a larger average diameter of the lipid droplets in the fat body, as seen by Nile Red staining under the confocal fluorescence microscope. Silenced starved insects had lower survival rates when compared to control insects. Accordingly, when examined during the starvation period for monitored activity, silenced insects had lower spontaneous locomotor activity and lower forced flight rates. Furthermore, we found that some genes involved in lipid metabolism had their expression levels altered in silenced insects, such as the Brummer lipase (down regulated) and the adipokinetic hormone receptor (up regulated), suggesting that, as previously observed in mammalian models, the autophagy and neutral lipolysis machineries are interconnected at the transcriptional level. Altogether, our data indicate that autophagy in the fat body is important to allow insects to mobilize energy from lipid stores.

Increased Expression of Autophagy Protein LC3 in Two Patients With Progressing Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the western hemisphere. It is characterized by a clonal proliferation of a population of CD5+ B lymphocytes that accumulate in the secondary lymphoid tissues, bone marrow, and blood. Some CLL patients remain free of symptoms for decades, whereas others rapidly become symptomatic or develop high-risk disease. Studying autophagy, which may modulate key protein expression and cell survival, may be important to the search for novel prognostic factors and molecules. Here, we applied flow cytometry technology to simultaneously detect autophagy protein LC3B with classical phenotypical markers used for the identification of tumoral CLL B cell clones. We found that two patients with progressing CLL showed increased expression of the autophagy protein LC3B, in addition to positive expression of CD38 and ZAP70 and unmutated status of IGHV. Our data suggest that activation of autophagy flux may correlate with CLL progression even before Ibrutinib treatment.

Identification of Autophagy-related Proteins in Lungs From Hypersensitivity Pneumonitis Patients.

Autophagy has been involved in the pathogenesis of various lung diseases. However, it is not yet known whether autophagy plays a role in hypersensitivity pneumonitis (HP). HP is an interstitial lung disease resulting from exposure to a wide variety of antigens that provoke an exaggerated immune response in susceptible individuals. The aim of this study was to explore the localization of autophagy key proteins in lungs from HP patients and controls by immunohistochemistry and analyze their expression levels by immunoblot. Macrophages and epithelial cells were strongly positive for the autophagosome biomarker LC3B (microtubule-associated protein light chain 3 beta) in HP lungs compared with controls. A similar pattern was found for the autophagy receptor p62 and the enzyme ATG4B. Unexpectedly, nuclear p62 signal was also noticed in macrophages from HP lungs. Regarding ATG5 and ATG7 localization, we observed positive staining in neutrophils, vascular smooth muscle cells, and endothelial cells. Our findings provide for the first time evidence that proteins from the autophagy machinery are highly expressed in the lungs of HP patients and describe the specific cellular and subcellular localization of LC3B, p62, ATG4B, ATG5, and ATG7 in HP lungs.

Aggregatibacter Actinomycetemcomitans Induces Autophagy in Human Junctional Epithelium Keratinocytes.

The adverse environmental conditions found in the periodontium during periodontitis pathogenesis stimulate local autophagy responses, mainly due to a continuous inflammatory response against the dysbiotic subgingival microbiome. The junctional epithelium represents the main site of the initial interaction between the host and the dysbiotic biofilm. Here, we investigated the role of autophagy in junctional epithelium keratinocytes (JEKs) in response to Aggregatibacter actinomycetemcomitans or its purified lipopolysaccharides (LPS). Immunofluorescence confocal analysis revealed an extensive nuclear translocation of transcription factor EB (TFEB) and consequently, an increase in autophagy markers and LC3-turnover assessed by immunoblotting and qRT-PCR. Correspondingly, challenged JEKs showed a punctuate cytosolic profile of LC3 protein contrasting with the diffuse distribution observed in untreated controls. Three-dimensional reconstructions of confocal images displayed a close association between intracellular bacteria and LC3-positive vesicles. Similarly, a close association between autophagic vesicles and the protein p62 was observed in challenged JEKs, indicating that p62 is the main adapter protein recruited during A. actinomycetemcomitans infection. Finally, the pharmacological inhibition of autophagy significantly increased the number of bacteria-infected cells as well as their death, similar to treatment with LPS. Our results indicate that A. actinomycetemcomitans infection induces autophagy in JEKs, and this homeostatic process has a cytoprotective effect on the host cells during the early stages of infection.

Microglial autophagy is impaired by prolonged exposure to ß-amyloid peptides: evidence from experimental models and Alzheimer's disease patients.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of misfolded proteins, amyloid-ß (Aß) aggregates, and neuroinflammation in the brain. Microglial cells are key players in the context of AD, being capable of releasing cytokines in response to Aß and degrading aggregated proteins by mechanisms involving the ubiquitin-proteasome system and autophagy. Here, we present in vivo and in vitro evidence showing that microglial autophagy is affected during AD progression. PDAPPJ20 mice-murine model of AD-exhibited an accumulation of the autophagy receptor p62 and ubiquitin+ aggregates in Iba1+ microglial cells close to amyloid deposits in the hippocampus. Moreover, cultured microglial BV-2 cells showed an enhanced autophagic flux during a 2-h exposure to fibrillar Aß, which was decreased if the exposure was prolonged to 24 h, a condition analogous to the chronic exposure to Aß in the human pathology. The autophagic impairment was also associated with lysosomal damage, depicted by membrane permeabilization as shown by the presence of the acid hydrolase cathepsin-D in cytoplasm and altered LysoTracker staining. These results are compatible with microglial exhaustion caused by pro-inflammatory conditions and persistent exposure to aggregated Aß peptides. In addition, we found LC3-positive autophagic vesicles accumulated in phagocytic CD68+ microglia in human AD brain samples, suggesting defective autophagy in microglia of AD brain. Our results indicate that the capacity of microglia to degrade Aß and potentially other proteins through autophagy may be negatively affected as the disease progresses. Preserving autophagy in microglia thus emerges as a promising approach for treating AD. Graphical abstract.