Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction.Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively.In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity.
Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.
HCMV is a beta-herpes virus, which induces a life long latency in hematopoietic stem cells after an asymptomatic primary infection. In hematopoietic stem cell transplantation, donor hematopoietic stem cells will engraft and differentiate in the recipient. This differentiation induces the reactivation of HCMV in the myeloid compartment and the release of new virions from the donor cells. The HCMV spread leads a primary infection of the immunocompromised HCMV negative recipient and can cause severe end organ diseases. The common treatments of HCMV in immunosuppressed patients are viral DNA replication inhibitors, which cause strong side effects, such as inhibition of hematopoiesis, kidney toxicity, and are likely to promote resistant strain emergence. As a consequence, none of the available drugs can target the HCMV in latent state. To circumvent this, we aim to directly alter the HCMV genome by using the CRIPSR/Cas9 system to knock-out the immediate early gene (IE) encoding essential viral proteins for lytic replication as well as the end of latency. We transduced the low HCMV-permissive U373-MG cells with a lentiviral vector encoding the Cas9 and tree different single gRNA targeting the IE gene. We FACS sorted those cell lines based on their Cas-9-GFP expression and then infected them with a HCMV laboratory strain. We detected mutations at the target site in up to 70% of the viral genomes. We observed a concomitant reduction of 50% less infected cell by FACS staining of the IE protein. Moreover, we set up an assay to analyze the virion release of infected U373-MG cells, which will allow us to address the decrease or inability to release new virions form U373-MG-gRNA-Cas9 HCMV infected cells. We are currently investigating a multiplex strategy with 3 gRNA targeting different parts of the IE gene which are delivered within the same lentiviral vector. The multiplex strategy is auspicious to show higher efficiency than a single gRNA. This strategy will be also tested on HCMV latently infected CD34+ hematopoietic stem cells to prevent viral reactivation. The CRISPR/Cas9 system anti-HCMV is a promising tool to block viral replication. With this anti-HCMV tool we will be able to treat HCMV positive donor cells in order to prevent the primary infection of the immunosuppressed recipient.
Lnk, with APS and SH2-B (Src homology 2-B), belongs to a family of SH2-containing proteins with potential adaptor functions. Lnk regulates growth factor and cytokine receptor-mediated pathways implicated in lymphoid, myeloid, and platelet homeostasis. We have previously shown that Lnk is expressed and up-regulated in vascular endothelial cells (ECs) in response to tumor necrosis factor-α (TNFα). In this study, we have shown that, in ECs, Lnk down-regulates the expression, at both mRNA and protein levels, of the proinflammatory molecules VCAM-1 and E-selectin induced by TNFα. Mechanistically, our data indicated that, in response to TNFα, NFκB/p65 phosphorylation and translocation as well as IκBα phosphorylation and degradation were unchanged, suggesting that Lnk does not modulate NFκB activity. However, Lnk activates phosphatidylinositol 3-kinase (PI3K) as reflected by Akt phosphorylation. Our results identify endothelial nitric-oxide synthase as a downstream target of Lnk-mediated activation of the PI3K/Akt pathway and HO-1 as a new substrate of Akt. We found that sustained Lnk-mediated activation of PI3K in TNFα-activated ECs correlated with the inhibition of ERK1/2 phosphorylation, whereas phosphorylation of p38 and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was unchanged. ERK1/2 inhibition decreases VCAM-1 expression in TNFα-treated ECs. Collectively, our results identify the adaptor Lnk as a negative regulator in the TNFα-signaling pathway mediating ERK inhibition and suggest a role for Lnk in the interplay between PI3K and ERK triggered by TNFα in ECs.
Summary Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. Here we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in RORγt + cells and conventional dendritic cells (cDCs) using germline and conditional double knock-out (DKO) mice. While Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells (ILC3s) in the intestinal mucosa, we found that they were required in cDCs for optimal antigen processing and presentation to CD4 + T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC II compartment (MIIC) of DCs for the fine regulation of antigen presentation and naive CD4 + T cell priming.
CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.
