MMP7 cleavage of amino-terminal CD95 death receptor switches signaling toward non-apoptotic pathways.

CD95 is a death receptor that can promote oncogenesis through molecular mechanisms that are not fully elucidated. Although the mature CD95 membrane receptor is considered to start with the arginine at position 17 after elimination of the signal peptide, this receptor can also be cleaved by MMP7 upstream of its leucine at position 37. This post-translational modification occurs in cancer cells but also in normal cells such as peripheral blood leukocytes. The non-cleaved CD95 amino-terminal region consists in a disordered domain and its in silico reconstitution suggests that it might contribute to receptor aggregation and thereby, regulate the downstream death signaling pathways. In agreement with this molecular modeling analysis, the comparison of CD95-deficient cells reconstituted with full-length or N-terminally truncated CD95 reveals that the loss of the amino-terminal region of CD95 impairs the initial steps of the apoptotic signal while favoring the induction of pro-survival signals, including the PI3K and MAPK pathways.

Immunotherapy perspectives in the new era of B-cell editing.

Since the early days of vaccination, targeted immunotherapy has gone through multiple conceptual changes and challenges. It now provides the most efficient and up-to-date strategies for either preventing or treating infections and cancer. Its most recent and successful weapons are autologous T cells carrying chimeric antigen receptors, engineered purposely for binding cancer-specific antigens and therefore used for so-called adoptive immunotherapy. We now face the merger of such achievements in cell therapy: using lymphocytes redirected on purpose to bind specific antigens and the  clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) revolution, which conferred genome-editing methodologies with both safety and efficacy. This unique affiliation will soon and considerably expand the scope of diseases susceptible to adoptive immunotherapy and of immune cells available for being reshaped as therapeutic tools, including B cells. Following the monumental success story of passive immunotherapy with monoclonal antibodies (mAbs), we are thus entering into a new era, where a combination of gene therapy/cell therapy will enable reprogramming of the patient's immune system and notably endow his B cells with the ability to produce therapeutic mAbs on their own.

Serine 165 phosphorylation of SHARPIN regulates the activation of NF-κB.

The adaptor SHARPIN composes, together with the E3 ligases HOIP and HOIL1, the linear ubiquitin chain assembly complex (LUBAC). This enzymatic complex catalyzes and stamps atypical linear ubiquitin chains onto substrates to modify their fate and has been linked to the regulation of the NF-κB pathway downstream of most immunoreceptors, inflammation, and cell death. However, how this signaling complex is regulated is not fully understood. Here, we report that a portion of SHARPIN is constitutively phosphorylated on the serine at position 165 in lymphoblastoid cells and can be further induced following T cell receptor stimulation. Analysis of a phosphorylation-resistant mutant of SHARPIN revealed that this mark controls the linear ubiquitination of the NF-κB regulator NEMO and allows the optimal activation of NF-κB in response to TNFα. These results identify an additional layer of regulation of the LUBAC and unveil potential strategies to modulate its action.

Oncostatin M induces IL-33 expression in liver endothelial cells in mice and expands ST2+CD4+ lymphocytes.

Interleukin (IL)-33 is crucially involved in liver pathology and drives hepatoprotective functions. However, the regulation of IL-33 by cytokines of the IL-6 family, including oncostatin M (OSM) and IL-6, is not well studied. The aim of the present study was to determine whether OSM mediates regulation of IL-33 expression in liver cells. Intramuscular administration in mice of an adenovirus encoding OSM (AdOSM) leads to increase in expression of OSM in muscles, liver, and serum of AdOSM-infected mice compared with control mice. The increase of circulating OSM markedly regulated mRNA of genes associated with blood vessel biology, chemotaxis, cellular death, induction of cell adhesion molecules, and the alarmin cytokine IL-33 in liver. Steady-state IL-33 mRNA was upregulated by OSM at an early phase (8 h) following AdOSM infection. At the protein level, the expression of IL-33 was significantly induced in liver endothelial cells [liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells] with a peak at 8 days post-AdOSM infection in mice. In addition, we found OSM-stimulated human microvascular endothelial HMEC-1 cells and human LSEC/TRP3 cells showed a significant increase in expression of IL-33 mRNA in a dose-dependent manner in cell culture. The OSM-mediated overexpression of IL-33 was associated with the activation/enrichment of CD4(+)ST2(+) cells in liver of AdOSM-infected mice compared with adenovirus encoding green fluorescent protein-treated control mice. In summary, these data suggest that the cytokine OSM regulates the IL-33 expression in liver endothelial cells in vivo and in HMEC-1/TRP3 cells in vitro and may specifically expand the target CD4(+)ST2(+) cells in liver.

Interleukin-27 and IFNγ regulate the expression of CXCL9, CXCL10, and CXCL11 in hepatitis.

UNLABELLED: Interleukin-27 (IL-27) belongs to the IL-6/IL-12 family of cytokines, associated with different inflammatory diseases and orchestrates its biological activity via common heterodimeric receptor composed of WSX-1 (IL-27Rα) and gp130. The present study was aimed to investigate the regulation of CXCL9, CXCL10, and CXCL11 chemokines in hepatic cells (human LX-2 cell line derived from normal human stellate cells (HSC), primary human hepatocytes, HSC, and HepG2 cells) and concanavalin A (ConA)-induced liver inflammation. We demonstrated that IL-27, but not IL-6, induced/up-regulated CXCR3 ligand genes (CXCL9, CXCL10, and CXCL11; out of 26 selected genes) in a STAT1-dependent manner in hepatic cells in vitro both at transcript and protein levels. In ConA-induced T cell-mediated hepatic model, we showed that soluble IL-27/IFNγ was elevated following ConA hepatitis in association with increased CXCL9, CXCL10, and CXCL11 expression in the liver. The exogenous IL-27 administration induced CXCR3 ligands in mouse liver at 4 h with any significant effect on recruitment of CXCR3(+) immune cells in the liver. The neutralization of IL-27 during ConA hepatitis differentially modulated (transcript vs protein expression) CXCR3 ligands and IFNγ during ConA-induced hepatitis with down-regulated expression of CXCL9 and CXCL10 at transcript level. The IFNγ, complementary regulated the expression of CXCR3 ligands as their up-regulation during ConA hepatitis, was abolished in IFNγ KO mice. In summary, IL-27 up-regulated the CXCL9, CXCL10, and CXCL11 chemokine expression in hepatic cells. IL-27 regulated CXCR3 ligand expression in IFNγ-dependent manner during acute hepatitis suggesting a complementary role of IL-27 and IFNγ to moderate liver inflammation via regulation of CXCR3 ligands. KEY MESSAGE: IL-27 up-regulated CXCR3 ligand expression in human hepatic cells in vitro. IL-27 up-regulated CXCR3 ligand expression and secretion in ConA hepatitis in vivo. CXCR3 ligand expression was down-regulated by blocking IL-27 or IFNγ deficiency. IL-27 modulated liver injury by regulation of CXCR3 ligands in IFNγ-dependent manner.

Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling.

Bone resorption by osteoclasts and bone formation by osteoblasts are tightly coupled processes implicating factors in TNF, bone morphogenetic protein, and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating bone formation by osteoblasts but the coupling factors were not identified. Using a high-throughput approach, we identified here Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages that induce osteoblast differentiation and matrix mineralization from human mesenchymal stem cells while inhibiting adipogenesis. Upon activation of toll-like receptors (TLRs) by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA to OSM, OSM receptor subunits gp130 and OSMR, or to the downstream transcription factor STAT3. The induced osteoblast differentiation program culminated with enhanced expression of CCAAT-enhancer-binding protein δ, Cbfa1, and alkaline phosphatase. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines have also a potent role in bone formation induced by monocytes/macrophages and activation of TLRs: IL-6 and leukemia inhibitory factor. We propose that during bone inflammation, infection, or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies.

Definition and characterization of an inhibitor for interleukin-31.

Interleukin-31 (IL-31) is a recently described T cell-derived cytokine, mainly produced by T helper type 2 cells and related to the IL-6 cytokine family according to its structure and receptor. IL-31 is the ligand for a heterodimeric receptor composed of a gp130-like receptor (GPL) associated with the oncostatin M receptor (OSMR). A link between IL-31 and atopic dermatitis was shown by studying the phenotype of IL-31 transgenic mice and IL-31 gene haplotypes in patients suffering from dermatitis. In this study, we generated a potent IL-31 antagonist formed by external portions of OSMR and GPL fused with a linker. This fusion protein, OSMR-L-GPL, consisting of 720 amino acids, counteracted the binding of IL-31 to its membrane receptor complex and the subsequent signaling events involving the STATs and MAPK pathways. Neutralizing effects were found in IL-31-sensitive cell lines, including brain-derived cells and primary cultures of keratinocytes.

Molecular dissection of human interleukin-31-mediated signal transduction through site-directed mutagenesis.

Interleukin (IL)-31 is a recently described cytokine, preferentially produced by T helper 2 lymphocytes and associated with skin diseases, such as atopic dermatitis. IL-31 is a member of the four alpha-helix bundle cytokine family and is related to the IL-6 subgroup. Its heterodimeric membrane receptor is composed of the gp130-like receptor (GPL) subunit associated to the oncostatin M receptor subunit. We identified critical amino acids implicated in the ligand receptor interaction by computational analysis combined with site-directed mutagenesis. Six IL-31 residues selected for their putative involvement in cytokine receptor contact sites were alanine-substituted, and the corresponding proteins were expressed in mammalian and bacterial systems. Biochemical, membrane binding, cell signaling, and cell proliferation analyses showed that mutation E44A, E106A, or H110A abolished IL-31 binding to GPL and the subsequent signaling events. A second ligand receptor-binding site involved Lys(134), with alanine substitution leading to a protein that still binds GPL, but is unable to recruit the second receptor subunit and the subsequent signaling pathways. The results indicate that IL-31 recognizes its receptor complex through two different binding sites, and we propose a three-dimensional model for IL-31.

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes.

Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.

Development of murine monoclonal antibodies to methamphetamine and methamphetamine analogues.

Methamphetamine and ecstasy are addictive drugs that cause major health problems in young people. Here we report on the development of high-affinity monoclonal antibodies to methamphetamine and its analogues, which may constitute powerful tools for antibody-based therapy. Six haptens, methamphetamine and ecstasy analogues, were synthesized, linked to a carrier protein and injected into mice. Several specific monoclonal antibodies were subsequently obtained following fusion of splenocytes from the immunized animals, with Sp2/O cells. Antibody specificity was fully investigated by competition ELISA, using a series of analogues, to identify specific amphetamine and/or ecstasy-specific antibodies. Antibody affinity was estimated to be in the range of 10(8) M(-1) with an enantiomeric hapten. Finally, two characteristic hybridoma clones (DAS-M243-6H5 and DAS-M278-4B12), secreting specific and potent mAbs were isolated. The development of drug-specific antibodies as in this study may provide promising therapeutic insight into how to neutralize methamphetamine in vivo during acute intoxication.

Design of cocaethylene and cocaine conjugates to produce highly selective polyclonal antibodies.

With the aim to obtain specific anti-cocaine antibodies directed against cocaine and active metabolites for use in immunotherapy, a series of six haptens were prepared, based on the structure of cocaine. The haptens differed by 3 positions of linkers: nitrogen, carboxyl group, and aromatic nucleus. The haptens were grafted onto 3 carrier proteins: bovine serum albumin, tetanus toxoid or keyhole limpet hemocyanin according to different methods of coupling: carbodiimide or mixed anhydride techniques. The immuno-conjugates were administered to rabbits and the antisera elicited were analyzed in term of titer, affinity and specificity. Variation in antisera properties were observed and attributed to the site of coupling the hapten, to the carrier proteins, and to the method of coupling. Antisera titers were in the range of 1/1 (no significant response) to 1/12,832, with antisera affinity up to 5.9 × 10(11) M-1. This strategy allowed the selection of a new hapten, which after coupling on carrier proteins, led to the production of antisera with a high specificity toward cocaine and cocaethylene, but exclude the inactive metabolites of cocaine.

Development of monoclonal antibodies directed against cocaine and cocaethylene: potential new tools for immunotherapy.

Cocaine abuse is a major health problem, with the number of overdose-related incidents on a constant increase. Monoclonal antibodies against cocaine and its major toxic metabolite cocaethylene, have been developed for immunotherapeutical neutralization in vivo. A series of monoclonal antibodies with high affinity for cocaethylene and cocaine were obtained. Clones DASm244-4D8A4A4 (4D8) and DASm244-5B3C3C6 (5B3) were selected and fully characterized. The antibodies secreted exhibited 1.40 x 10(8) and 3.69 x 10(7) M(-1) affinity constants for [3H]-cocaine and cocaethylene, respectively. In addition to cocaine, they bound to cocaethylene and did not recognize non-toxic cocaine metabolites. They did not bind to blood cells, indicating that they may be potential tools for cocaine neutralization in vivo in cases of overdose.