Search Results (22)

Tumor-associated macrophages (TAMs) play a pivotal role in cancer regulation by influencing tumor growth, metastasis, and the immune microenvironment. By providing low doses and ultra-low doses (ULD) of immune regulators to the organism, micro-immunotherapy (MI) medicines (MIM) could be seen as valuable adjuvant drugs in the context of a wide range of pathological conditions, including cancers. Thus, these MIM could target TAMs, affecting their phenotype and activities. In this study, the anti-tumor and the immune-stimulatory effects of four capsules out of the ten composing the Labo’life’s MIM 2LC1^® (2LC1-1, 2LC1-6, 2LC1-7, and 2LC1-8), as well as the specific nucleic acid (SNA^®) sequence SNA-MYC present at ULD in this medicine have been evaluated in vitro, in several cancer models, and in human monocyte-derived macrophages. Our results showed that the tested MI formulations increased the tumor cell death of spheroids from HCT-116 colon cancer cells, while reducing the spheroid volume. Moreover, the treatments impaired the clonogenic capabilities of two cancer cell lines from epithelial origin, the LNCaP prostate cancer and the MCF-7 breast cancer cells. Interestingly, ULD of the SNA-MYC shared similar anti-cancer capabilities in those models, and it led to a significant reduction in the expression of C-MYC when evaluated in a model of human M2 macrophages. In the same model, the MI formulations also increased the expression of CD86 and HLA-DR, two markers of M1 anti-tumor macrophages. In addition, the tested items modulated the secretion of a panel of chemokines related to macrophage activity and immune cell recruitment. Finally, our results showed that 2LC1-8 increased the phagocytosis capabilities of human monocyte-derived macrophages, thus possibly contributing to sustaining the immune functions of M1, which are crucial in the context of cancer. Even if more research is needed to uncover their exact mechanism of action, these results suggest that the tested capsules of 2LC1 as well as ULD of SNA-MYC display both anti-tumor and immune-enhancing effects. Full article

Phosphatidylcholine cytidine transferase α (CCTα) is a key rate-limiting enzyme in the CDP–choline pathway, the primary pathway for phosphatidylcholine (PC) synthesis in mammals. This study investigated the role of CCTα in lipid droplet (LD) formation, phospholipid synthesis, LD fusion, and lipophagy in bovine mammary epithelial cells (BMECs) through CCTα gene knockout (CCT-KO) and overexpression (CCT-OE). CCTα mRNA expression was significantly increased in bovine mammary gland tissue after lactation. In BMECs, CCTα was transferred from the nucleus to the endoplasmic reticulum and localized on LD surfaces in the presence of linoleic acid. Compared with normal BMECs (NC), CCTα knockout (CCT-KO) cells had significantly greater LD diameters (1.53 μm vs. 1.68 μm, p < 0.05), lower proportions of small LDs (<1 µm; 11.39% vs. 5.42%), and higher proportions of large LDs (>3 µm; 0.67% vs. 2.88%). In contrast, CCTα overexpression (CCT-OE) decreased the diameter of LDs to 1.18 μm (p < 0.01), increased the proportion of small LDs to 35.48%, and decreased the proportion of large LDs to 0.24%. CCTα knockout significantly decreased the PC content and the ratio of PC to PE, whereas CCTα overexpression increased the PC content and the ratio of PC to phosphatidyl ethanolamine (PE) (p < 0.05). The lipidomics analysis indicated that PC synthesis was significantly influenced by CCTα gene expression. Live cell observations showed that CCTα knockout promoted the fusion of small LDs into large LDs. In cells with CCT α overexpression, the expression of the microtubule-associated protein 1 light chain 3 (LC3) protein and the number of lysosomes was elevated, and the lysosomal phagocytosis of LDs was observed through transmission electron microscopy, thus indicating that CCTα overexpression enhanced lipophagy. In conclusion, these results suggest that CCTα plays a role in regulating LD formation by influencing PC synthesis, LD fusion, and lipophagy in BMECs. Full article

Lysosome-associated membrane glycoproteins (LAMPs), including lysosomal membrane protein 1 (Lamp1) and lysosomal membrane protein 2 (Lamp2), are involved in phagocytosis, chaperone-mediated autophagy (CMA), and other pathways that interact with lysosomal activity. However, the role of Lamp2 in teleosts has not been clarified. In this study, we investigated the functions of lamp2 genes during Vibrio vulnificus infection. We achieved subcellular localization of the lamp2 gene at the cellular level and performed overexpression and RNA interference experiments followed by Lipopolysaccharides (LPS) stimulation to probe the expression changes of related genes. Ultrapathology analysis of the head-kidney revealed an increase in lysosomes and the formation of autophagosomal vesicles after V. vulnificus infection, suggesting that lysosomes bind to autophagosomes. The lamp2 gene, encoding 401 amino acids in Cynoglossus semilaevis, was constitutively expressed in all examined tissues of healthy half-smooth tongue sole, with the highest expression in blood. A challenge test was conducted to assess the response of half-smooth tongue sole (Cynoglossus semilaevis) to different concentrations of V. vulnificus. The results showed that the relative expression of lamp2 and its related genes—lc3, rab7, vamp8, atg14, stx17, snap29, ctsb, and ctsd—varied with time and concentration in the gill, spleen, head-kidney, blood, liver, and gut tissues. From the results of lamp2 gene overexpression and RNA interference experiments, it is hypothesized that lamp2 positively regulates lc3, rab7, vamp8, snap29, and stx17, and negatively regulates ctsd and ctsb. Our findings provide new primary data for the function of lamp2 gene in the half-smooth tongue sole., particularly its role in regulating the immune response against V. vulnificus. Full article

Neutrophils, the most abundant circulating white blood cells, are essential for the initial immune response to infection and injury. Emerging research reveals a dualistic function of neutrophils in cancer, where they can promote or inhibit tumor progression. This dichotomy is influenced by the tumor microenvironment, with neutrophils capable of remodeling the extracellular matrix, promoting angiogenesis, or alternatively inducing cancer cell death and enhancing immune responses. An intriguing yet poorly understood aspect of neutrophil–cancer interactions is the phenomenon of neutrophil engulfment by cancer cells, which has been observed across various cancers. This process, potentially mediated by LC3-associated phagocytosis (LAP), raises questions about whether it serves as a mechanism for immune evasion or contributes to tumor cell death through pathways like ferroptosis. This review examines current knowledge on neutrophil development, their roles in cancer, and the mechanisms of LAP in neutrophil engulfment by tumor cells. We discuss how manipulating LAP impacts cancer progression and may represent a therapeutic strategy. We also explore neutrophils’ potential as delivery vehicles for cancer therapeutic agents. Understanding the complex functions of tumor-associated neutrophils (TANs) and the molecular mechanisms underlying LAP in cancer may open new avenues for effective therapeutic interventions and mitigate potential risks. Full article

(1) Background: Macrophagic myofasciitis (MMF) is an inflammatory histopathological lesion demonstrating long-term biopersistence of vaccine-derived aluminum adjuvants within muscular phagocytic cells. Affected patients suffer from widespread myalgia and severe fatigue consistent with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a poorly understood disorder suspected to result from chronic immune stimulation by infectious and inorganic particles. (2) Methods: In this study we determined the immuno-metabolic properties of MMF phagocytic cells compared to controls, at rest and upon exposure to aluminum oxyhydroxide adjuvant, with or without adsorbed antigens, using protein quantification and an oxygen consumption assay. (3) Results: MMF and control cells similarly internalized the adjuvant and vaccine but MMF cells specifically expressed Rubicon and Nox2, two molecules unique to the LC3-associated phagocytosis (LAP) machinery, a non-canonical autophagic pathway able to downregulate canonical autophagy. MMF cells exhibited an altered inflammatory secretome, producing more pain-inducing CXC chemokines and less TNF-α than controls, consistent with chronic myalgia and exhaustion of the immune system previously documented in ME/CFS. MMF cells exhibited mitochondrial metabolism dysfunction, with exacerbated reaction to adjuvanted vaccine, contrasting with limited spare respiratory capacity and marked proton leak weakening energy production. (4) Conclusions: MMF phagocytes seemingly use LAP to handle aluminum oxyhydroxide vaccine particles, secrete pain-inducing molecules, and exhibit exacerbated metabolic reaction to the vaccine with limited capacity to respond to ongoing energetic requests. Full article

LC3b (Map1lc3b) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b to promote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes, such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells, utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis, and neuroprotection. In a mouse model of retinal lipid steatosis-mice lacking LC3b (LC3b^−/−), we observed increased lipid deposition, metabolic dysregulation, and enhanced inflammation. Herein, we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT and LC3b^−/− mice revealed 1533 DEGs, with ~73% upregulated and 27% downregulated. Enriched gene ontology (GO) terms included inflammatory response (upregulated DEGs), fatty acid metabolism, and vascular transport (downregulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with a potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology. Full article

Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy. Full article

Recent studies have highlighted the importance of autophagy and particularly non-canonical autophagy in the development and progression of acute pancreatitis (a frequent disease with considerable morbidity and significant mortality). An important early event in the development of acute pancreatitis is the intrapancreatic activation of trypsinogen, (i.e., formation of trypsin) leading to the autodigestion of the organ. Another prominent phenomenon associated with the initiation of this disease is vacuolisation and specifically the formation of giant endocytic vacuoles in pancreatic acinar cells. These organelles develop in acinar cells exposed to several inducers of acute pancreatitis (including taurolithocholic acid and high concentrations of secretagogues cholecystokinin and acetylcholine). Notably, early trypsinogen activation occurs in the endocytic vacuoles. These trypsinogen-activating organelles undergo activation, long-distance trafficking, and non-canonical autophagy. In this review, we will discuss the role of autophagy in acute pancreatitis and particularly focus on the recently discovered LAP-like non-canonical autophagy (LNCA) of endocytic vacuoles. Full article

LC3-associated phagocytosis (LAP) is a noncanonical autophagy process reported in recent years and is one of the effective mechanisms of host defense against bacterial infection. During LAP, bacteria are recognized by pattern recognition receptors (PRRs), enter the body, and then recruit LC3 onto a single-membrane phagosome to form a LAPosome. LC3 conjugation can promote the fusion of the LAPosomes with lysosomes, resulting in their maturation into phagolysosomes, which can effectively kill the identified pathogens. However, to survive in host cells, bacteria have also evolved strategies to evade killing by LAP. In this review, we summarized the mechanism of LAP in resistance to bacterial infection and the ways in which bacteria escape LAP. We aim to provide new clues for developing novel therapeutic strategies for bacterial infectious diseases. Full article

Infectious diseases are a burden for aquaculture. Antigen processing and presentation (APP) to the immune effector cells that fight pathogens is key in the adaptive immune response. At the core of the adaptive immunity that appeared in lower vertebrates during evolution are the variable genes encoding the major histocompatibility complex (MHC). MHC class I molecules mainly present peptides processed in the cytosol by the proteasome and transported to the cell surface of all cells through secretory compartments. Professional antigen-presenting cells (pAPC) also express MHC class II molecules, which normally present peptides processed from exogenous antigens through lysosomal pathways. Autophagy is an intracellular self-degradation process that is conserved in all eukaryotes and is induced by starvation to contribute to cellular homeostasis. Self-digestion during autophagy mainly occurs by the fusion of autophagosomes, which engulf portions of cytosol and fuse with lysosomes (macroautophagy) or assisted by chaperones (chaperone-mediated autophagy, CMA) that deliver proteins to lysosomes. Thus, during self-degradation, antigens can be processed to be presented by the MHC to immune effector cells, thus, linking autophagy to APP. This review is focused on the essential components of the APP that are conserved in teleost fish and the increasing evidence related to the modulation of APP and autophagy during pathogen infection. Full article

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. Full article

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen persisting in phagosomes that has the ability to escape host immune surveillance causing tuberculosis (TB). Lipoarabinomannan (LAM), as a glycolipid, is one of the complex outermost components of the mycobacterial cell envelope and plays a critical role in modulating host responses during M. tb infection. Different species within the Mycobacterium genus exhibit distinct LAM structures and elicit diverse innate immune responses. However, little is known about the mechanisms. In this study, we first constructed a LAM-truncated mutant with fewer arabinofuranose (Araf) residues named M. sm-ΔM_6387 (Mycobacterium smegmatis arabinosyltransferase EmbC gene knockout strain). It exhibited some prominent cell wall defects, including tardiness of mycobacterial migration, loss of acid-fast staining, and increased cell wall permeability. Within alveolar epithelial cells (A549) infected by M. sm-ΔM_6387, the uptake rate was lower, phagosomes with bacterial degradation appeared, and microtubule-associated protein light chain 3 (LC3) recruitment was enhanced compared to wild type Mycobacterium smegmatis (M. smegmatis). We further confirmed that the variability in the removal capability of M. sm-ΔM_6387 resulted from host cell responses rather than the changes in the mycobacterial cell envelope. Moreover, we found that M. sm-ΔM_6387 or its glycolipid extracts significantly induced expression changes in some genes related to innate immune responses, including Toll-like receptor 2 (TLR2), class A scavenger receptor (SR-A), Rubicon, LC3, tumor necrosis factor alpha (TNF-α), Bcl-2, and Bax. Therefore, our studies suggest that nonpathogenic M. smegmatis can deposit LC3 on phagosomal membranes, and the decrease in the quantity of Araf residues for LAM molecules not only impacts mycobacterial cell wall integrity but also enhances host defense responses against the intracellular pathogens and decreases phagocytosis of host cells. Full article

Timely and efficient elimination of apoptotic substrates, continuously produced during one’s lifespan, is a vital need for all tissues of the body. This task is achieved by cells endowed with phagocytic activity. In blood-separated tissues such as the retina, the testis and the ovaries, the resident cells of epithelial origin as retinal pigmented epithelial cells (RPE), testis Sertoli cells and ovarian granulosa cells (GC) provide phagocytic cleaning of apoptotic cells and cell membranes. Disruption of this process leads to functional ablation as blindness in the retina and compromised fertility in males and females. To ensure the efficient elimination of apoptotic substrates, RPE, Sertoli cells and GC combine various mechanisms allowing maintenance of tissue homeostasis and avoiding acute inflammation, tissue disorganization and functional ablation. In tight cooperation with other phagocytosis receptors, MERTK—a member of the TAM family of receptor tyrosine kinases (RTK)—plays a pivotal role in apoptotic substrate cleaning from the retina, the testis and the ovaries through unconventional autophagy-assisted phagocytosis process LAP (LC3-associated phagocytosis). In this review, we focus on the interplay between TAM RTKs, autophagy-related proteins, LAP, and Toll-like receptors (TLR), as well as the regulatory mechanisms allowing these components to sustain tissue homeostasis and prevent functional ablation of the retina, the testis and the ovaries. Full article

Modeling human infectious diseases using the early life stages of zebrafish provides unprecedented opportunities for visualizing and studying the interaction between pathogens and phagocytic cells of the innate immune system. Intracellular pathogens use phagocytes or other host cells, like gut epithelial cells, as a replication niche. The intracellular growth of these pathogens can be counteracted by host defense mechanisms that rely on the autophagy machinery. In recent years, zebrafish embryo infection models have provided in vivo evidence for the significance of the autophagic defenses and these models are now being used to explore autophagy as a therapeutic target. In line with studies in mammalian models, research in zebrafish has shown that selective autophagy mediated by ubiquitin receptors, such as p62, is important for host resistance against several bacterial pathogens, including Shigella flexneri, Mycobacterium marinum, and Staphylococcus aureus. Furthermore, an autophagy related process, Lc3-associated phagocytosis (LAP), proved host beneficial in the case of Salmonella Typhimurium infection but host detrimental in the case of S. aureus infection, where LAP delivers the pathogen to a replication niche. These studies provide valuable information for developing novel therapeutic strategies aimed at directing the autophagy machinery towards bacterial degradation. Full article

Modern medicine is challenged by several potentially severe fungal pathogens such as Aspergillus fumigatus, Candida albicans, or Histoplasma capsulatum. Though not all fungal pathogens have evolved as primary pathogens, opportunistic pathogens can still cause fatal infections in immuno-compromised patients. After infection with these fungi, the ingestion and clearance by innate immune cells is an important part of the host immune response. Innate immune cells utilize two different autophagic pathways, the canonical pathway and the non-canonical pathway, also called microtubule-associated protein 1A/1B-light chain 3 (LC3) -associated pathway (LAP), to clear fungal pathogens from the intracellular environment. The outcome of autophagy-related host immune responses depends on the pathogen and cell type. Therefore, the understanding of underlying molecular mechanisms of autophagy is crucial for the development and improvement of antifungal therapies. One of those molecular mechanisms is the interaction of the transcription-factor hypoxia-inducible factor 1α (HIF-1α) with the autophagic immune response. During this review, we will focus on a comprehensive overview of the role of autophagy and HIF-1α on the outcome of fungal infections. Full article