ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain.

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1- microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

Genotoxin-producing Salmonella enterica induces tissue-specific types of DNA damage and DNA damage response outcomes.

Introduction: Typhoid toxin-expressing Salmonella enterica causes DNA damage in the intestinal mucosa in vivo, activating the DNA damage response (DDR) in the absence of inflammation. To understand whether the tissue microenvironment constrains the infection outcome, we compared the immune response and DDR patterns in the colon and liver of mice infected with a genotoxigenic strain or its isogenic control strain. Methods: In situ spatial transcriptomic and immunofluorescence have been used to assess DNA damage makers, activation of the DDR, innate immunity markers in a multiparametric analysis. Result: The presence of the typhoid toxin protected from colonic bacteria-induced inflammation, despite nuclear localization of p53, enhanced co-expression of type-I interferons (IfnbI) and the inflammasome sensor Aim2, both classic features of DNA-break-induced DDR activation. These effects were not observed in the livers of either infected group. Instead, in this tissue, the inflammatory response and DDR were associated with high oxidative stress-induced DNA damage. Conclusions: Our work highlights the relevance of the tissue microenvironment in enabling the typhoid toxin to suppress the host inflammatory response in vivo.

Truncated thioredoxin (Trx80) exerts unique mitogenic cytokine effects via a mechanism independent of thiol oxido-reductase activity.

Recently we discovered that a naturally occurring C-terminally truncated thioredoxin (Trx80) is a potent mitogenic cytokine stimulating IL-12 production from CD40(+) monocytes. To further characterise Trx80 we have engineered cysteine to serine mutants of Trx80 corresponding to the active site cysteines of Trx (Trx80SGPS) and to the structural cysteine at position 72 (Trx80C72S). Trx80SGPS and Trx80C72S retained the cell stimulatory activity of Trx80 and increased peripheral blood mononuclear cell (PBMC) proliferation three- to five-fold in vitro (P<0.01, n=18). Both Trx80SGPS and Trx80C72S significantly stimulated IL-12 and IFN-gamma secretion from PBMCs in the same manner as Trx80 (P<0.01, n=9 and 10). The previously described Trx80 dimer is caused by non-covalent interactions, and not by any intermolecular disulphide bonds.

Cytokines and Epstein Barr virus (EBV) genes expression in blood chronic lymphocytic leukaemia (CLL) cells and their immortalised CLL cell lines.

We have encountered two unique chronic lymphocytic leukaemia (CLL) patients, PG and NN. Some blood CLL cells of these patients have been infected and carry Epstein Barr virus (EBV) in vivo. In spite of their early-activated G0/G1 stage of post germinal center (GC) memory cells, ex vivo EBV-carrying blood CLL cells of PG clone expressed LMPs and used specific QUK splice for their EBNA1 expression, similar to the EBV-carrying cells of non-B origin. Interestingly, EBV-carrying CLL cells of NN clone expressed LMP2a and used UK-splice for their EBNA1 expression, similar to the in vivo EBV-carrying high density normal B cells in the blood of healthy individuals. The CLL-derived lines but not normal lymphoblastoid cell line (LCL) used QUK- and YUK-splice for their EBNA1 expression. As expected, LCL and their permanent CLL-derived lines used Cp promoter and up-regulated their EBNA2 expression. Blood CLL cells and the CLL-derived cell lines of these patients spontaneously produced cytokines as shown by microarray assay. The types and quantities of cytokines might relate to their CLL origin and viral strain in the given CLL cells. Neither blood CLL nor their CLL-derived cell lines express any detectable apoptosis-inducer ligands, CD95L or Apo 3L. As a consequence of cell cycle progression, CLL-derived cell lines up-regulated their co-stimulator molecules CD80 and apoptosis-related receptor CD95. Since only the rare EBV-carrying CLL cells grew in vitro, the combination of viral genome and cytokines seems to be critical for the outgrowth of EBV-carrying CLL cells over their EBV-negative counterpart in vitro but not in vivo.

Thioredoxin-80 is a product of alpha-secretase cleavage that inhibits amyloid-beta aggregation and is decreased in Alzheimer's disease brain.

Thioredoxin-1 (Trx1) is an endogenous dithiol reductant and antioxidant that was shown to be decreased in Alzheimer's disease (AD) neurons. A truncated form of Trx1, thioredoxin 80 (Trx80), was reported to be secreted from monocytes having cytokine activity. Here, we show that Trx80 is present in human brain in an aggregated form. Trx80 localizes mainly to neurons and is dramatically decreased in AD brains. Trx80 levels in cerebrospinal fluid (CSF) correlate with those of the classical AD biomarkers amyloid-ß (Aß) 1-42 and total tau. Moreover, Trx80 measurements in CSF discriminate between patients with stable mild cognitive impairment, prodomal AD and mild AD. We report that ADAM10 and 17, two α-secretases processing the Aß precursor protein, are responsible for Trx80 generation. In contrast to the periphery, Trx80 has no pro-inflammatory effects in glia, either by itself or in combination with Aß or apolipoprotein E. Instead, Trx80 inhibits Aß(1-42) aggregation and protects against its toxicity. Thus, a reduction in Trx80 production would result in increased Aß polymerization and enhanced neuronal vulnerability. Our data suggest that a deficit in Trx80 could participate in AD pathogenesis.

Thioredoxin 80-activated-monocytes (TAMs) inhibit the replication of intracellular pathogens.

BACKGROUND: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). PRINCIPAL FINDINGS: In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. SIGNIFICANCE: Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria.

Rescue of the immunotherapeutic potential of a novel T cell epitope in the Epstein-Barr virus latent membrane protein 2.

Epstein-Barr virus (EBV)-associated tumors express a limited number of viral antigens but most of them express the latent membrane protein 2 (LMP2). This article describes a peptide derived from LMP2 (residues 396-404, designated LLL) as a potentially useful vaccine. This peptide could at first be defined as an unlikely T cell target as it could not stabilize MHC surface expression in transporter associated with antigen-processing (TAP)-deficient cells. Nevertheless, T lymphocytes reactive to LLL were detected in the peripheral blood of four EBV-seropositive healthy individuals. We have constructed a chimeric molecule in which LLL was fused to the amino-terminal end of the beta(2) microglobulin (beta(2)m). Autologous dendritic cells constitutively expressing the LLLbeta(2)m molecule were capable of expanding in vitro HLA-A2-restricted anti-LLL T lymphocytes from the peripheral blood of one of the donors. These T lymphocytes exhibited cytolytic activity against target cells expressing the chimeric molecules as well as against EBV-infected lymphoblastoid cells expressing natural LLL-MHC complexes.

Truncated thioredoxin (Trx80) induces differentiation of human CD14+ monocytes into a novel cell type (TAMs) via activation of the MAP kinases p38, ERK, and JNK.

Thioredoxin truncated at its carboxy terminal (Trx80) acts as a cytokine that stimulates monocytes and eosinophils. In the present study, Trx80 was shown to induce differentiation of human CD14(+) monocytes into a cell type not described previously, which we designate as Trx80-activated monocytes (TAMs). TAMs resemble immature dendritic cells (iDCs) generated in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in that both these cell populations exhibit increased proportions of CD1a(+) and mannose receptor (MR)(+) cells. However, in contrast to iDCs, TAMs express high proportion of CD14 and lower proportion of CD83 and HLA-DR. Functional assays revealed that, in comparison to iDCs, TAMs 1) exhibit a higher pinocytic capacity; 2) release significantly higher amounts of the proinflammatory cytokines tumor necrosis factor-alpha (TNF alpha), IL-1 beta, and IL-6 and of the anti-inflammatory cytokine IL-10; and 3) induce a significantly lower proliferative response in allogeneic peripheral blood mononuclear cells (PBMCs). Indeed, Trx80 appears to be the first endogenous substance shown to have the capacity on its own to induce IL-10 production by monocytes. Analysis of the mitogen-activated protein (MAP) kinase signaling pathway revealed that Trx80 induces phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). We propose that Trx80 is an early signal in response to danger, and that TAMs may play a major role in triggering innate immune responses.