To gain new insight into the role of B-cell autophagy, we generated two novel mouse models deficient for the autophagy-related gene (Atg)5, one from the outset pro-B cell stage (Atg5 f/ − Mb1 cre) and the other in mature B cells only (Atg5 f/ − CD21 cre). We show that autophagy is dispensable for pro-to pre-B cell transition, but necessary at a basal level to maintain normal numbers of peripheral B cells. It appears non-essential for B-cell activation under B-cell receptor stimulation but required for their survival after lipopolysaccharide stimulation that drives plasmablast differentiation and for specific IgM production after immunization. Results obtained using Atg5 f/ − CD21 cre × C57BL/6 lpr/lpr autoimmune-prone mice show that B-cell autophagy is involved in the maintenance of anti-nuclear antibody secretion, elevated number of long-lived plasma cells, and sustains IgG deposits in the kidneys. Thus, treatments specifically targeting autophagy might be beneficial in systemic autoimmune diseases. Cell Death and Differentiation (2016) 23, 853-864; doi:10.1038/cdd.2015 published online 20 November 2015 Macroautophagy is a catabolic process allowing the degradation of cytoplasmic material in double membrane vesicles, ultimately fusing with lysosomes. Macroautophagy, initially implicated in the generation of nutrients under metabolic stress, is known to have multiple roles, in different physiologic compartments, such as in vacuole trafficking, cell signalling, and cell death. Macroautophagy is deeply involved in the regulation of immunity.1 It has been shown that autophagy can regulate inflammation related to inflammasome activation and to type I interferon secretion. Moreover, it contributes to antigen presentation by both major histocompatibility complex (MHC) class I and class II molecules. 2Macroautophagy is also tightly linked to lymphocyte activation and survival. It has central roles in T-cell basal homeostasis, survival, and polarization.3 It is also involved in the regulation of T-cell signalling by downregulating the NF-κB pathway 4 and apoptosis processes through the procaspases 3 and 8 degradation. 5 Macroautophagy has additionally been described to regulate B-cell lineage, in particular during B-cell development. Thus, it has been shown that B cells generated from fetal liver chimaeras, with a complete deletion of the essential autophagy-related gene (Atg)5, exhibited a block at the proto pre-B stage transition.6,7 However, as the genetic deletion is systemic and occurs very early during development, the question remains over whether the developmental blockade could be due to defects resulting from early haematopoietic development. Indeed, macroautophagy has been shown to be fundamental to haematopoietic stem cell survival and renewal.8 Moreover, conditional deletion of Atg5 under the control of CD19 promoter expressed from the pre-B stage does not lead to major developmental breaks, except a decrease in B-1a B-cell population. 6 The contrast with results obtained with chimaeric mice could be due...

The design of multifunctional materials able to both selectively deliver a drug into cells in a targeted manner and display an enhanced propensity for biodegradation is an important goal. Here, graphene oxide (GO) is functionalized with the chemotactic peptide N-formyl-methionyl-leucylphenylalanine (fMLP) known to interact with the formyl peptide receptor, which is expressed in different cancer cells, including cervical carcinoma cells. This study highlights the ability of GOfMLP for targeted drug delivery and cancer cell killing and the subsequent degradation capacity of the hybrid. Biodegradation is assessed via Raman spectroscopy and transmission electron microscopy. The results show that GOfMLP is susceptible to faster myeloperoxidase-mediated degradation. The hybrid material, but not GO, is capable of inducing neutrophil degranulation with subsequent degradation, being the first study showing inducible neutrophil degradation by the nanomaterial itself with no prior activation of the cells. In addition, confocal imaging and flow cytometry using HeLa cells demonstrate that GOfMLP is able to deliver the chemotherapeutic agent doxorubicin faster into cells, inducing higher levels of apoptosis, when compared to nonfunctionalized GO. The results reveal that GOfMLP is a promising carrier able to efficiently deliver anticancer drugs, being endowed with the ability to induce its own biodegradation.

Studies of mice deficient for autophagy in T cells since thymic development, concluded that autophagy is integral to mature T cell homeostasis. Basal survival and functional impairments in vivo, limited the use of these models to delineate the role of autophagy during the immune response. We generated Atg5f/f distal Lck (dLck)-cre mice, with deletion of autophagy only at a mature stage. In this model, autophagy deficiency impacts CD8+ T cell survival but has no influence on CD4+ T cell number and short-term activation. Moreover, autophagy in T cells is dispensable during early humoral response but critical for long-term antibody production. Autophagy in CD4+ T cells is required to transfer humoral memory as shown by injection of antigen-experienced cells in naive mice. We also observed a selection of autophagy-competent cells in the CD4+ T cell memory compartment. We performed in vitro differentiation of memory CD4+ T cells, to better characterize autophagy-deficient memory cells. We identified mitochondrial and lipid load defects in differentiated memory CD4+ T cells, together with a compromised survival, without any collapse of energy production. We then propose that memory CD4+ T cells rely on autophagy for their survival to regulate toxic effects of mitochondrial activity and lipid overload.

The last decade has seen an increase in the application of graphene oxide (GO) in the biomedical field. GO has been successfully exploited for its ability to deliver many kinds of drugs into target cells. However, GO toxicity assessment is still controversial. Several studies demonstrated that GO protein coating is crucial to alleviate the material's toxicity. Besides, coronation leads to the formation of big agglomerates, reducing the cellular uptake of the material and thus its therapeutic efficiency. In this work, we propose a simple and efficient method based on rapid (Ultra-Turrax, UT) mixing to control protein corona formation. Using UT protocol, we were able to reduce GO agglomeration in the presence of proteins and to obtain stable GO dispersions in cell culture media. By labelling GO with luminescent nanoparticles (QDs), we studied the GO internalization kinetic and efficiency. Comparing the "classic" and the UT protocols, we found that the latter allows a faster and more efficient internalization both in macrophages and HeLa cells without affecting cell viability. We believe that the use of UT protocol will be interesting and suitable for the preparation of next generation GO-based drug delivery platforms. Recherche (ANR) through the LabEx project Chemistry of Complex Systems (ANR-10-LABX-0026_CSC).

ACTB: actin beta; ACTR2/3: ARP2/3 actin-related protein 2/3; APC: antigen-presenting cells; ATG: autophagy-related; BCR: B cell receptor; BECN1/Beclin 1: beclin 1, autophagy related; CDC42: cell division cycle 42; Cr2: complement receptor 2; CSFE: carboxyfluorescein succinimidyl ester; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; EEA1: early endosome antigen 1; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothyocyanate; GC: germinal center; GJA1/CX3: gap junction protein, alpha 1; Ig: immunoglobulin; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; LM: littermate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen activated protein kinase; MHC-II: major histocompatibility complex class II; MIIC: MHC class II compartment; OVAL: ovalbumin; PBS: phosphate-buffered saline; PCM1: pericentriolar material 1; PtdIns3K: phosphatidylinositol 3-kinase; PTPRC/CD45RB/B220; Protein tyrosine phosphatase, receptor type, C; SYK: spleen tyrosine kinase; TBS: Tris-buffered saline; TCR: T cell receptor; ULK1: unc-51 like kinase 1.

In the last decade, graphene-based materials have received increasing attention for both academic research and industrial uses. However, products containing graphene must meet the same standards for quality, safety and efficacy as products not containing nanomaterials. Our aim is to shed light on the toxicological characterization of few-layer graphene (FLG) dispersions. In the present study, graphene was easily dispersed in water using biocompatible riboflavin-5′-phosphate sodium salt (Rib). A highly concentrated FLG dispersion (G-Rib) was stable for months. G-Rib suspension was characterized by HR-TEM, Raman spectroscopy and ζ-potential. Our results showed that even at high concentration of G-Rib, cell survival was always above 85%. Then, we investigated the tissue distribution and toxic effects of G-Rib in mice up to 30 days after intravenous injections. Histological analysis of the tissues revealed hepatic accumulation and excretion through the kidneys. The biochemical and hematological parameters remained within the reference range showing no hematotoxicity. Finally, no signs of inflammation were detected in the cells isolated from the lymph nodes and spleen. The results of the study show that highly water dispersed FLG is a material with a very low toxic profile, which is one of the first requirements for the future development of biomedical applications.

Graphene-related materials (GRMs) are widely used in various applications due to their unique properties. A growing number of reports describe the impact of different carbon nanomaterials, including graphene oxide (GO), reduced GO (rGO), and carbon nanotubes (CNT), on immune cells, but there is still a very limited number of studies on graphene. In this work, we investigated the biological responses of few layer graphene (FLG) on mouse macrophages (bone marrow derived macrophages, BMDMs), which are part of the first line of defense in innate immunity. In particular, our paper describes our findings of short-term FLG treatment in BMDMs with a focus on observing material internalization and changes in general cell morphology. Subsequent investigation of cytotoxicity parameters showed that increasing doses of FLG did not hamper the viability of cells and did not trigger inflammatory responses. Basal level induced autophagic activity sufficed to maintain the cellular homeostasis of FLG treated cells. Our results shed light on the impact of FLG on primary macrophages and show that FLG does not elicit immunological responses leading to cell death.

Primary lymphocytes ensure their efficient function and maintenance through different mechanisms including autophagy. Few layer graphene does not affect either the viability and activation or the autophagic activity of B and T cells.