A Role for cis Interaction between the Inhibitory Ly49A receptor and MHC class I for natural killer cell education.

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.

Kaposi's sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis.

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.

Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing.

BACKGROUND: In contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata). RESULTS: Transmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse. CONCLUSIONS: Altogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.

The role of lysosomes in metabolic and autoimmune diseases.

Lysosomes are catabolic organelles that contribute to the degradation of intracellular constituents through autophagy and of extracellular components through endocytosis, phagocytosis and macropinocytosis. They also have roles in secretory mechanisms, the generation of extracellular vesicles and certain cell death pathways. These functions make lysosomes central organelles in cell homeostasis, metabolic regulation and responses to environment changes including nutrient stresses, endoplasmic reticulum stress and defects in proteostasis. Lysosomes also have important roles in inflammation, antigen presentation and the maintenance of long-lived immune cells. Their functions are tightly regulated by transcriptional modulation via TFEB and TFE3, as well as by major signalling pathways that lead to activation of mTORC1 and mTORC2, lysosome motility and fusion with other compartments. Lysosome dysfunction and alterations in autophagy processes have been identified in a wide variety of diseases, including autoimmune, metabolic and kidney diseases. Deregulation of autophagy can contribute to inflammation, and lysosomal defects in immune cells and/or kidney cells have been reported in inflammatory and autoimmune pathologies with kidney involvement. Defects in lysosomal activity have also been identified in several pathologies with disturbances in proteostasis, including autoimmune and metabolic diseases such as Parkinson disease, diabetes mellitus and lysosomal storage diseases. Targeting lysosomes is therefore a potential therapeutic strategy to regulate inflammation and metabolism in a variety of pathologies.

HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus.

BACKGROUND: The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells. OBJECTIVE: To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells. METHODS: P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers. RESULTS: P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux. CONCLUSION: The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.

Beyond Anti-viral Effects of Chloroquine/Hydroxychloroquine.

As the world is severely affected by COVID-19 pandemic, the use of chloroquine and hydroxychloroquine in prevention or for the treatment of patients is allowed in multiple countries but remained at the center of much controversy in recent days. This review describes the properties of chloroquine and hydroxychloroquine, and highlights not only their anti-viral effects but also their important immune-modulatory properties and their well-known use in autoimmune diseases, including systemic lupus and arthritis. Chloroquine appears to inhibit in vitro SARS virus' replication and to interfere with SARS-CoV2 receptor (ACE2). Chloroquine and hydroxychloroquine impede lysosomal activity and autophagy, leading to a decrease of antigen processing and presentation. They are also known to interfere with endosomal Toll-like receptors signaling and cytosolic sensors of nucleic acids, which result in a decreased cellular activation and thereby a lower type I interferons and inflammatory cytokine secretion. Given the antiviral and anti-inflammatory properties of chloroquine and hydroxychloroquine, there is a rational to use them against SARS-CoV2 infection. However, the anti-interferon properties of these molecules might be detrimental, and impaired host immune responses against the virus. This duality could explain the discrepancy with the recently published studies on CQ/HCQ treatment efficacy in COVID-19 patients. Moreover, although these treatments could be an interesting potential strategy to limit progression toward uncontrolled inflammation, they do not appear per se sufficiently potent to control the whole inflammatory process in COVID-19, and more targeted and/or potent therapies should be required at least in add-on.

ATG5 is required for B cell polarization and presentation of particulate antigens.

The involvement of macroautophagy/autophagy proteins in B-cell receptor (BCR) trafficking, although suspected, is not well understood. We show that ATG5 (autophagy related 5) contributes to BCR polarization after stimulation and internalization into LAMP1 (lysosomal-associated membrane protein 1)+ and major histocompatibility complex class II (MHC-II)+ compartments. BCR polarization is crucial in the context of immobilized antigen processing. Moreover, antigen presentation to cognate T cells is decreased in the absence of ATG5 when the model antigen OVAL/ovalbumin is provided in an immobilized form in contrast to the normal presentation of soluble OVAL. We further show that ATG5 is required for centrosome polarization and actin nucleation in the immune synapse area. This event is accompanied by an increased interaction between ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and the microtubule-organizing center-associated protein PCM1 (pericentriolar material 1). In the human B cell line BJAB, PCM1 is required for BCR polarization after stimulation. We thus propose that the ATG12 (autophagy related 12)-ATG5-ATG16L1 complex under BCR stimulation allows its interaction with PCM1 and consequently facilitates centrosome relocalization to the immune synapse, optimizing the presentation of particulate antigens. Abbreviations: 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.

Corrigendum: Lymphocyte Autophagy in Homeostasis, Activation, and Inflammatory Diseases.

[This corrects the article DOI: 10.3389/fimmu.2018.01801.].

Lymphocyte Autophagy in Homeostasis, Activation, and Inflammatory Diseases.

Autophagy is a catabolic mechanism, allowing the degradation of cytoplasmic content via lysosomal activity. Several forms of autophagy are described in mammals. Macroautophagy leads to integration of cytoplasmic portions into vesicles named autophagosomes that ultimately fuse with lysosomes. Chaperone-mediated autophagy is in contrast the direct translocation of protein in lysosomes. Macroautophagy is central to lymphocyte homeostasis. Although its role is controversial in lymphocyte development and in naive cell survival, it seems particularly involved in the maintenance of certain lymphocyte subtypes. Its importance in memory B and T cells biology has recently emerged. Moreover, some effector cells like plasma cells rely on autophagy for survival. Autophagy is central to glucose and lipid metabolism, and to the maintenance of organelles like mitochondria and endoplasmic reticulum. In addition macroautophagy, or individual components of its machinery, are also actors in antigen presentation by B cells, a crucial step to receive help from T cells, this crosstalk favoring their final differentiation into memory or plasma cells. Autophagy is deregulated in several autoimmune or autoinflammatory diseases like systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and Crohn's disease. Some treatments used in these pathologies impact autophagic activity, even if the causal link between autophagy regulation and the efficiency of the treatments has not yet been clearly established. In this review, we will first discuss the mechanisms linking autophagy to lymphocyte subtype survival and the signaling pathways involved. Finally, potential impacts of autophagy modulation in lymphocytes on the course of these diseases will be approached.

CD4 T cell autophagy is integral to memory maintenance.

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 Atg5 f/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.

[Autophagy in T and B cell homeostasis: recycling for sustainable growth]. / L'autophagie et l'homéostasie des lymphocytes T et B - Bien recycler pour un développement durable.

Macroautophagy often abbreviated by "autophagy" is an intracellular degradation mechanism linked to lysosomal activity. Autophagy is conserved from yeast to mammals and plays a role in the response to energetic stress and in organelle homeostasis. Autophagy is also involved in the regulation of immunity, in particular in the adaptive immune response, which involves B and T lymphocytes. It was indeed shown that autophagy impacts the development of B and T cells as well as the education of T cells in the thymus. Autophagy also modulates activation, survival and polarization of T cells. It plays a role in antigen presentation by B cells, and in their TLR-mediated activation, and thus likely in their initial activation. Finally, autophagy is required for the survival of memory lymphocytes and effector cells like antibody-producing plasma cells. Interestingly, autophagy is deregulated in several autoimmune pathologies. The modulation of this phenomenon could possibly lead to new treatments aiming at limiting lymphocyte activation driving these pathologies.

Capacity of myeloid and plasmacytoid dendritic cells especially at mature stage to express and secrete HLA-G molecules.

Human leukocyte antigen (HLA-G), a class Ib major histocompatibility complex molecule, is potentially relevant in the immune response through its various immune cell functions. Its expression noticed in some malignancies has also been shown on macrophages and dendritic cells (DC) in tumoral and inflammatory diseases. As DC constitute a key component in the immune response, this work aimed at assessing the expression of HLA-G at transcriptional and proteic levels during differentiation and maturation of the different DC subsets. We show that HLA-G transcription was induced during CD34+-derived DC differentiation and is associated with a cell-surface expression in half of cases and with a substantial secretion of soluble HLA-G in all cases. Results were very similar for monocyte-derived DC, but there was still a weak HLA-G cell-surface expression and a lower level of secretion. On the contrary, HLA-G transcription was weak in plasmacytoid DC without any HLA-G cell-surface expression and with a basal level of secretion. The mechanisms involved in HLA-G expression appear transcriptional and post-transcriptional. However, the amount of HLA-G transcripts and the expression of the protein are not related. HLA-G expression or secretion by DC may have negative consequences on the function of effective immune cells and also on DC themselves via the interaction with inhibitory receptors expressed by these cells. The capacity of DC to express or secrete HLA-G should be studied in the context of cellular therapy using DC in addition to its suppressive action in immune response.

Soluble HLA-G molecules are increased in lymphoproliferative disorders.

The immunomodulatory properties of soluble human leukocyte antigen G (sHLA-G) explain its potential interest in malignancies. HLA-G frequently transcribed in lymphoproliferative disorders is rarely expressed at cell surface. In this article, we will demonstrate that the plasmatic level of soluble HLA-G was significantly increased in 70% of B chronic lymphocytic leukemia, 53% of non-Hodgkin B lymphoma (B-NHL), and 45% of T-NHL. To explain this variable secretion, the HLA-G secreting cell was searched and was identified as tumoral T4 lymphocytes only in one patient with Sezary syndrome. To approach the mechanisms involved in sHLA-G secretion, the potential role of cytokines has been studied in vitro on T lymphomas. A significant increase of sHLA-G level is observed after activation by cytokines associated with a small increase in the quantity of transcripts using real-time polymerase chain reaction, suggesting an involvement of both transcriptional and post-transcriptional mechanisms. Western Blot analysis reveals no evident variation of the protein expression whatever the conditions, suggesting a continuous secretion and a low intracellular storage. The frequency of the sHLA-G secretion associated to its inhibiting role on T cells and natural killer cells during tumoral lymphoid malignancies suggests a potential role of these molecules as escape mechanism from antitumoral response.

Pharmacological regulators of autophagy and their link with modulators of lupus disease.

Autophagy is a central regulator of cell survival. It displays both anti- and pro-death roles that are decisive in the maintenance of cell homeostasis. Initially described in several eukaryotic cellular models as being induced under nutrient stress favouring survival by energy supply, autophagy was found later to display other decisive physiological roles, especially in the immune system. Thus, it is involved in antigen presentation and lymphocyte differentiation as well as in the balance regulating survival/death and activation of lymphocytes. Autophagy therefore appears to be central in the regulation of inflammation. The observation that autophagy is deregulated in systemic lupus erythematosus is recent. This discovery revives the programme dealing with the design and development of pharmacological autophagy regulators in the therapeutic context of lupus, a debilitating autoimmune disease that affects several million people in the world. A large number of molecules that positively and negatively regulate autophagy have been described, most of them with therapeutic indications in cancer and infection. Only a few, however, are effectively potent activators or inhibitors endowed with experimentally demonstrated selective properties. In this review article, we highlight the most relevant ones and summarize what we know regarding their mechanism of action. We emphasize the link between pharmacological regulators of autophagy and inducers or inhibitors of lupus disease and discuss the fundamental and pharmacological/therapeutic interest of this functional interplay.

Macroautophagy is deregulated in murine and human lupus T lymphocytes.

Macroautophagy was recently shown to regulate both lymphocyte biology and innate immunity. In this study we sought to determine whether a deregulation of autophagy was linked to the development of autoimmunity. Genome-wide association studies have pointed out nucleotide polymorphisms that can be associated with systemic lupus erythematosus, but the potential role of autophagy in the initiation and/or development of this syndrome is still unknown. Here, we provide first clues of macroautophagy deregulation in lupus. By the use of LC3 conversion assays and electron microscopy experiments, we observed that T cells from two distinct lupus-prone mouse models, i.e., MRL (lpr/lpr) and (NZB/NZW)F1, exhibit high loads of autophagic compartments compared with nonpathologic control CBA/J and BALB/c mice. Unlike normal mice, autophagy increases with age in murine lupus. In vivo lipopolysaccharide stimulation in CBA/J control mice efficiently activates T lymphocytes but fails to upregulate formation of autophagic compartments in these cells. This argues against a deregulation of autophagy in lupus T cells solely resulting from an acute inflammation injury. Autophagic vacuoles quantified by electron microscopy are also found to be significantly more frequent in T cells from lupus patients compared with healthy controls and patients with non-lupus autoimmune diseases. This elevated number of autophagic structures is not distributed homogeneously and appears to be more pronounced in certain T cells. These results suggest that autophagy could regulate the survival of autoreactive T cell during lupus, and could thus lead to design new therapeutic options for lupus.

A therapeutic peptide in lupus alters autophagic processes and stability of MHCII molecules in MRL/lpr B cells.

The P140 phosphopeptide encompassing residues 131-151 of the spliceosomal U1-70K snRNP protein displays protective properties in lupus patients and MRL/lpr mice. It increases peripheral blood lymphocyte apoptosis via a mechanism involving γδ T cells. After intravenous administration, P140 accumulates in the lungs and spleen. It binds both the HSC70/Hsp73 chaperone and MHC class II (MHCII) molecules, which colocalize in splenic MRL/lpr B cells. Expression of HSC70 and MHCII, which is increased in MRL/lpr splenic B cells, is diminished after P140 administration. P140 impairs refolding properties of HSC70 and alters expression of stable MHCII molecules in B lymphocytes. In MRL/lpr B cells, P140 increases the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulation of autophagic flux. Our study reveals a very unique property of P140 peptide that alters the autophagy pathway leading to a defect of endogenous (auto)antigen processing in MRL/lpr antigen-presenting B cells and a decrease of T cell priming and signaling.