SummaryMacrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

BackgroundBesides its anti-inflammatory effects, cinnamaldehyde has been reported to have anti-carcinogenic activity. Here, we investigated its impact on immune cells.MethodsActivation of nuclear factor-κB by cinnamaldehyde (0–10 µg/ml) alone or in combination with lipopolysaccharide was assessed in THP1XBlue human monocytic cell line and in human peripheral blood mononuclear cells (PBMCs). Proliferation and secretion of cytokines (IL10 and TNFα) was determined in primary immune cells and the human cell lines (THP1, Jurkat E6-1 and Raji cell lines) stimulated with cinnamaldehyde alone or in conjunction with lipopolysaccharide. Nitric oxide was determined in mouse RAW264.7 cells. Moreover, different treated PBMCs were stained for CD3, CD20 and AnnexinV.ResultsLow concentrations (up to 1 µg/ml) of cinnamaldehyde resulted in a slight increase in nuclar factor-kB activation, whereas higher concentrations led to a dose-dependent decrease of nuclear factor-kB activation (up to 50%) in lipopolysachharide-stimulated THP1 cells and PBMCs. Accordingly, nitric oxide, interleukin 10 secretion as well as cell proliferation were reduced in lipopolysachharide-stimulated RAW264.7 cells, PBMCs and THP1, Raji and Jurkat-E6 immune cells in the presence of cinnamaldehyde in a concentration-dependent manner. Flow cytometric analysis of PBMCs revealed that CD3+ were more affected than CD20+ cells to apopotosis by cinnamaldehyde.ConclusionWe attribute the anti-inflammatory properties of cinnamaldehyde to its ability to block nuclear factor-κB activation in immune cells. Treatment with cinnamaldehyde led to inhibition of cell viability, proliferation and induced apoptosis in a dose-dependent manner in primary and immortalized immune cells. Therefore, despite its described anti-carcinogenic property, treatment with cinnamaldehyde in cancer patients might be contraindicated due to its ability to inhibit immune cell activation.

Studying the relationship between virus infection and cellular response is paradigmatic for our understanding of how perturbation changes biological systems. Immune response, in this context is a complex yet evolutionarily adapted and robust cellular change, and is experimentally amenable to molecular analysis. To visualize the full cellular response to virus infection, we performed temporal transcriptomics, proteomics, and phosphoproteomics analysis of vesicular stomatitis virus (VSV)-infected mouse macrophages. This enabled the understanding of how infection-induced changes in host gene and protein expression are coordinated with post-translational modifications by cells in time to best measure and control the infection process. The vast and complex molecular changes measured could be decomposed in a limited number of clusters within each category (transcripts, proteins, and protein phosphorylation) each with own kinetic parameter and characteristic pathways/processes, suggesting multiple regulatory options in the overall sensing and homeostatic program. Altogether, the data underscored a prevalent executive function to phosphorylation. Resolution of the molecular events affecting the RIG-I pathway, central to viral recognition, reveals that phosphorylation of the key innate immunity adaptor mitochondrial antiviral-signaling protein (MAVS) on S328/S330 is necessary for activation of type-I interferon and nuclear factor κ B (NFκB) pathways. To further understand the hierarchical relationships, we analyzed kinase–substrate relationships and found RAF1 and, to a lesser extent, ARAF to be inhibiting VSV replication and necessary for NFκB activation, and AKT2, but not AKT1, to be supporting VSV replication. Integrated analysis using the omics data revealed co-regulation of transmembrane transporters including SLC7A11, which was subsequently validated as a host factor in the VSV replication. The data sets are predicted to greatly empower future studies on the functional organization of the response of macrophages to viral challenges.

Acrolein, a highly reactive unsaturated aldehyde, is generated in large amounts during smoking and is best known for its genotoxic capacity. Here, we aimed to assess whether acrolein at concentrations relevant for smokers may also exert immunomodulatory effects that could be relevant in allergy or cancer. In a BALB/c allergy model repeated nasal exposure to acrolein abrogated allergen-specific antibody and cytokine formation, and led to a relative accumulation of regulatory T cells in the lungs. Only the acrolein-treated mice were protected from bronchial hyperreactivity as well as from anaphylactic reactions upon challenge with the specific allergen. Moreover, grafted D2F2 tumor cells grew faster and intratumoral Foxp3+ cell accumulation was observed in these mice compared to sham-treated controls. Results from reporter cell lines suggested that acrolein acts via the aryl-hydrocarbon receptor which could be inhibited by resveratrol and 3′-methoxy-4′-nitroflavone Acrolein- stimulation of human PBMCs increased Foxp3+ expression by T cells which could be antagonized by resveratrol. Our mouse and human data thus revealed that acrolein exerts systemic immunosuppression by promoting Foxp3+ regulatory cells. This provides a novel explanation why smokers have a lower allergy, but higher cancer risk.

Host factor requirements for different classes of viruses have not been fully unraveled. Replication of the viral genome and synthesis of viral proteins within the human host cell are associated with an increased demand for nutrients and specific metabolites. With more than 400 acknowledged members to date in humans, solute carriers (SLCs) represent the largest family of transmembrane proteins dedicated to the transport of ions and small molecules such as amino acids, sugars and nucleotides. Consistent with their impact on cellular metabolism, several SLCs have been implicated as host factors affecting the viral life cycle and the cellular response to infection. In this study, we aimed at characterizing the role of host SLCs in cell survival upon viral infection by performing unbiased genetic screens using a focused CRISPR knockout library. Genetic screens with the cytolytic vesicular stomatitis virus (VSV) showed that the loss of two SLCs genes, encoding the sialic acid transporter SLC35A1/CST and the zinc transporter SLC30A1/ZnT1, affected cell survival upon infection. Further characterization of these genes suggests a role for both of these transporters in the apoptotic response induced by VSV, offering new insights into the cellular response to oncolytic virus infections.

Host factor requirements for different classes of viruses are still to be fully unraveled. Replication of the viral genome and synthesis of viral proteins inside the human host cell are associated with altered, often enhanced, cellular metabolism and increased demand for nutrients as well as specific metabolites. With more than 400 members listed to date in humans, solute carriers (SLCs) represent the largest family of transmembrane proteins dedicated to the transport of ions and small molecules such as amino acids, sugars and nucleotides. Consistent with their impact on cellular metabolism, several SLCs have been implicated as host factors affecting the viral life cycle and the cell response to infection. In this study, we aimed at characterizing the role of host SLCs in cell survival upon viral infection by performing unbiased genetic screens using a focused CRISPR knockout library. Genetic screens with the cytolytic vesicular stomatitis virus (VSV) showed that loss of two SLCs genes, encoding the sialic acid transporter SLC35A1/CST and the zinc transporter SLC30A1/ZnT1, affected cell survival upon infection. Further characterization of these genes pointed to a role of both transporters in the apoptotic response induced by VSV, offering new insights into the cellular response to oncolytic virus infections.

Allergic sensitization has been linked to active and passive smoking in exposed individuals and even their pets. We here investigated the contribution of acrolein, a compound generated in large amounts during smoking, during nasal sensitization andbased on the surprising preliminary resultson tumor growth. As a model antigen we used KLH with or without acrolein.

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