Extracellular vesicles from type-2 macrophages increase the survival of chronic lymphocytic leukemia cells ex vivo.

The resistance of Chronic Lymphocytic Leukemia (CLL) B-cells to cell death is mainly attributed to interactions within their microenvironment, where they interact with various types of cells. Within this microenvironment, CLL-B-cells produce and bind cytokines, growth factors, and extracellular vesicles (EVs). In the present study, EVs purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL-B-cells cultures. EVs were rapidly internalized by B-cells, leading to a decrease in apoptosis (P = 0.0162 and 0.0469, respectively) and an increased proliferation (P = 0.0335 and 0.0109). Additionally, they induced an increase in the resistance of CLL-B-cells to Ibrutinib, the Bruton kinase inhibitor in vitro (P = 0.0344). A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene BCL-2 (P = 0.0286) but not MCL-1 and an increase in the expression of proliferation-inducing gene APRIL (P = 0.0286) following treatment with EVs. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2 (P = 0.0338), CD40 (P = 0.0338), p53 (P = 0.0219) and BCL-2 (P = 0.0338). Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of CLL-B-cells treated or not with NLCs EVs show various differentially expressed genes.

Targeting the NTSR2/TrkB oncogenic pathway in chronic lymphocytic leukemia.

Current therapies that target the B-cell receptor pathway or the inhibition of anti-apoptotic proteins do not prevent the progressive forms of chronic lymphocytic leukemia (CLL), have low long-term efficacy and are subject to therapeutic resistance. Deciphering the mechanisms of leukemic cell survival and searching for new specific targets therefore remain major challenges to improve the management of this disease. It was evidenced that NTSR2 (neurotensin receptor 2), through the recruitment of TRKB (tropomyosin related kinase B), induces survival pathways in leukemic B cells. We have investigated the therapeutic potential of this protein complex as a new target. The binding domain of NTSR2 and TRKB was identified and a peptide targeting the latter was designed. The peptide binds TRKB and efficiently decreases the interaction of the two proteins. It is also effectively internalized by CLL-B cells in which it notably affects Src family kinase signaling and anti-apoptotic proteins levels. It demonstrated a cytotoxic effect both in vitro on the MEC-1 cell line and ex vivo on a cohort of 30 CLL patients. Altogether, these results underline the therapeutic potential of the NTSR2/TRKB protein complex as a target in CLL and open new perspectives for the development of targeted therapies.

BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells.

Evading apoptosis and sustained survival signaling pathways are two central hallmarks of B-cell chronic lymphocytic leukemia (B-CLL) cells. In this regard, nurse-like cells (NLC), the monocyte-derived type 2 macrophages, deliver stimulatory signals via B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL), and the C-X-C Motif Chemokine Ligand 12 (CXCL12). Previously, we demonstrated that brain-derived neurotrophic factor (BDNF) protects B-CLL cells from spontaneous apoptosis by activating the oncogenic complex NTSR2-TrkB (neurotensin receptor 2-tropomyosin-related kinase receptor B), only overexpressed in B-CLL cells, inducing anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) expression and Src kinase survival signaling pathways. Herein, we demonstrate that BDNF belongs to the NLC secretome and promotes B-CLL survival. This was demonstrated in primary B-CLL co-cultured with their autologous NLC, compared to B-CLL cells cultured alone. Inhibition of BDNF in co-cultures, enhances B-CLL apoptosis, whereas its exogenous recombinant activates pro-survival pathways in B-CLL cultured alone (i.e. Src activation and Bcl-2 expression), at a higher level than those obtained by the exogenous recombinant cytokines BAFF, APRIL and CXCL12, the known pro-survival cytokines secreted by NLC. Together, these results showed that BDNF release from NLC trigger B-CLL survival. Blocking BDNF would support research strategies against pro-survival cytokines to limit sustained B-CLL cell survival.

Regulatory Roles of Sortilin and SorLA in Immune-Related Processes.

Sortilin, also known as Neurotensin Receptor-3, and the sorting-related receptor with type-A repeats (SorLA) are both members of the Vps10p domain receptor family. Initially identified in CNS cells, they are expressed in various other cell types where they exert multiple functions. Although mostly studied for its involvement in Alzheimer's disease, SorLA has recently been shown to be implicated in immune response by regulating IL-6-mediated signaling, as well as driving monocyte migration. Sortilin has been shown to act as a receptor, as a co-receptor and as an intra- and extracellular trafficking regulator. In the last two decades, deregulation of sortilin has been demonstrated to be involved in many human pathophysiologies, including neurodegenerative disorders (Alzheimer and Parkinson diseases), type 2 diabetes and obesity, cancer, and cardiovascular pathologies such as atherosclerosis. Several studies highlighted different functions of sortilin in the immune system, notably in microglia, pro-inflammatory cytokine regulation, phagosome fusion and pathogen clearance. In this review, we will analyze the multiple roles of sortilin and SorLA in the human immune system and how their deregulation may be involved in disease development.

Heparan sulfates and the decrease of N-glycans promote early adipogenic differentiation rather than myogenesis of murine myogenic progenitor cells.

In vitro, extracted muscle satellite cells, called myogenic progenitor cells, can differentiate either in myotubes or preadipocytes, depending on environmental factors and the medium. Transcriptomic analyses on glycosylation genes during satellite cells differentiation into myotubes showed that 31 genes present a significant variation of expression at the early stages of murine myogenic progenitor cells (MPC) differentiation. In the present study, we analyzed the expression of 383 glycosylation related genes during murine MPC differentiation into preadipocytes and compared the data to those previously obtained during their differentiation into myotubes. Fifty-six glycosylation related genes are specifically modified in their expression during early adipogenesis. The variations correspond mainly to: a decrease of N-glycans, and of alpha (2,3) and (2,6) linked sialic acids, and to a high level of heparan sulfates. A high amount of TGF-ß1 in extracellular media during early adipogenesis was also observed. It seems that the increases of heparan sulfates and TGF-ß1 favor pre-adipogenic differentition of MPC and possibly prevent their myogenic differentiation.

Variation in Chst8 gene expression level affects PrPC to PrPSc conversion efficiency in prion-infected Mov cells.

The conversion of the endogenous cellular prion protein to an abnormally folded isoform is a hallmark of transmissible spongiform encephalopathies. It occurs when a misfolded prion protein contacts the cellular PrP. Among the molecular partners suggested to be involved in the misfolding process, the glycosaminoglycans seem to be good candidates. The present study was aimed to examine a possible link between PrP conversion efficiency and transcript level of Chst8 gene that encodes the carbohydrate N-acetylgalactosamine 4-O-sulfotransferase 8. Mov cells expressing ovine PrP were transfected with shRNA directed against Chst8 transcripts. Resulting clones were characterized for their Chst8 and Prnp transcript levels, and for their content in sulfated glycosaminoglycans, more particularly sulfated chondroitins. Unexpectedly, the decreased amount of Chst8 transcript induced an increase of the chondroitin sulfate percentage among total GAGs, with an increased amount of 4-O-sulfation of GalNAc residues. Upon to infection by a sheep prion, a slight amount of PrP(Sc) was observed, which rapidly disappeared upon subpassaging. Together, these findings indicate that the Chst8 transcript level affects the glycosaminoglycan environment of the cellular prion protein, and as a consequence its ability for conversion into PrP(Sc).

Glycosylation-related gene expression profiling in the brain and spleen of scrapie-affected mouse.

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). The molecular requirements for efficient PrP conversion remain unknown. Altered glycosylation has been linked to various pathologies and the N-glycans harbored by two prion protein isoforms are different. In order to search for glycosylation-related genes that could mark prion infection, we used a glycosylation-dedicated microarray that allowed the simultaneous analysis of the expression of 165 glycosylation-related genes encoding proteins of the glycosyltransferase, glycosidase, lectin, and sulfotransferase families to compare the gene expression profiles of normal and scrapie-infected mouse brain and spleen. Eight genes were found upregulated in "scrapie brain" at the final state of the disease. In the spleen, five genes presented a modified expression. Three genes were also upregulated in the spleen of infected mice, and two (Pigq and St3gal5) downregulated. All changes were confirmed by qPCR and biochemical analyses applied to Pigq and St3gal5 proteins.

Polymorphism of the prion protein in mammals: a phylogenetic approach.

PrP, the principal factor modulating resistance/susceptibility to transmissible spongiform encephalopathies, is a well conserved protein bearing strong phylogenetic information, in spite of its relatively short sequence. The construction of the PrP tree allows inferring the probable ancestral sequence for Bovidae where variants were recorded. This ancestral PrP sequence is constituted by a series of 5 octa-repeats, 3 alpha-helices and 2 beta-strands which combines together to form an antiparallel beta-sheet. The appearance of a 6(th) octa-repeat in the Bovinae ancestor during the evolution of Cetartiodactyla is discussed. Additionally, the variation of the substitution rates of amino acids along the sequence revealed that the sites associated to resistance/susceptibility to TSE are mostly located in conservative regions, including alpha-helices and beta-strands. The composition of most variants very sensitive to TSE in sheep and human corresponds to derived sequences compared to the Eutherian ancestor. However, a homozygous resistant variant in sheep differs from the ancestral state.

Glycosylation-related genes are variably expressed depending on the differentiation state of a bioaminergic neuronal cell line: implication for the cellular prion protein.

A striking feature of the cellular prion protein (PrP(C)) is the heterogeneity of its glycoforms, whose contribution to PrP(C) function has yet to be defined. Using the 1C11 neuronal bioaminergic differentiation model and a glycomics approach, we show here a correlation between differential PrP(C) N-glycosylations in 1C11(5-HT) serotonergic and 1C11(NE) noradrenergic cells compared to their 1C11 precursor cells and a variation of the glycogenome expression status in these cells. In particular, expression of genes involved in N-glycan synthesis or in the modeling of chondroitin and heparan sulfate proteoglycans appeared to be modulated. Our results highlight that, the expression of glycosylation-related genes is regulated during bioaminergic neuronal differentiation, consistent with a participation of glycoconjugates in neuronal development and plasticity. A neuronal regulation of glycosylation processes may have direct implications on some neurospecific functions of PrP(C) and may participate in specific brain targeting of prion strains.

Glycosylation-related gene expression in prion diseases: PrPSc accumulation in scrapie infected GT1 cells depends on beta-1,4-linked GalNAc-4-SO4 hyposulfation.

Several lines of evidence indicate that some glycoconjugates are efficient effectors of the cellular prion protein (PrP(C)) conversion into its pathogenic (PrP(Sc)) isoform. To assess how glycoconjugate glycan moieties participate in the biogenesis of PrP(Sc), an exhaustive comparative analysis of the expression of about 200 glycosylation-related genes was performed on prion-infected or not, hypothalamus-derived GT1 cells by hybridization of DNA microarrays, semiquantitative RT-PCR, and biochemical assays. A significant up- (30-fold) and down- (17-fold) regulation of the expression of the ChGn1 and Chst8 genes, respectively, was observed in prion-infected cells. ChGn1 and Chst8 are involved in the initiation of the synthesis of chondroitin sulfate and in the 4-O-sulfation of non-reducing N-acetylgalactosamine residues, respectively. A possible role for a hyposulfated chondroitin in PrP(Sc) accumulation was evidenced at the protein level and by determination of chondroitin and heparan sulfate amounts. Treatment of Sc-GT1 cells with a heparan mimetic (HM2602) induced an important reduction of the amount of PrP(Sc), associated with a total reversion of the transcription pattern of the N-acetylgalactosamine-4-O-sulfotransferase 8. It suggests a link between the genetic control of 4-O-sulfation and PrP(Sc) accumulation.