ESAT-6 and HspX improve the effectiveness of BCG to induce human dendritic cells-dependent Th1 and NK cells activation

The limited efficacy of the BCG vaccine against tuberculosis is partly due to the missing expression of immunogenic proteins. We analyzed whether the addition to BCG of ESAT-6 and HspX, two Mycobacterium tuberculosis (Mtb) antigens, could enhance its capacity to activate human dendritic cells (DCs). BCG showed a weak ability to induce DC maturation, cytokine release, and CD4+ lymphocytes and NK cells activation. The addition of ESAT-6 or HspX alone to BCG-stimulated DC did not improve these processes, whereas their simultaneous addition enhanced BCG-dependent DC maturation and cytokine release, as well as the ability of BCG-treated DCs to stimulate IFN-\u3b3 release and CD69 expression by CD4+ lymphocytes and NK cells. Addition of TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DCs incubated with ESAT-6 and HspX, as well as IFN-\u3b3 secretion by CD4+ lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DCs to induce the expression of memory phenotype marker CD45RO in na\uefve CD4+ T cells. Our results indicate that ESAT-6 and HspX cooperation enables BCG-treated human DCs to induce T lymphocyte and NK cell-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of BCG vaccination

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes ex-press five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane micro -domains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphor-ylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 con-trols resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking

Fam65b is a new transcriptional target of FOXO1 that regulates RhoA signaling for T lymphocyte migration

Forkhead box Os (FOXOs) transcription factors favor both T cell quiescence and trafficking through their control of the expression of genes involved in cell cycle progression, adhesion and homing. Here, we report that the product of the fam65b gene is a new transcriptional target of FOXO1 that regulates RhoA activity. We show that Fam65b binds the small GTPase RhoA via a non canonical domain and represses its activity by decreasing its GTP loading. As a consequence, Fam65b negatively regulates chemokine-induced responses such as adhesion, morphological polarisation and migration. Therefore, these results show the existence of a new functional link between FOXO1 and RhoA pathways, through which the FOXO1 target Fam65b tonically dampens chemokine-induced migration by repressing RhoA activity

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage

This work was supported by the European Project Innovative Medicines Initiative Joint Undertaking project BeTheCure (Contract 115142-2), Associazione Italiana Ricerca sul Cancro, Fondazione Berlucchi, Interuniversity Attraction Poles 7-40 program, and by grants from the Spanish Ministry of Economy and Competitiveness (SAF-2014-53416-R) and the RETICS Program (RD12/0009/009 RIER, RD16/0012/0006) (M.M. and L.M.-M)

Towards a scientific interpretation of the terroir concept: plasticity of the grape berry metabolome

BACKGROUND: The definition of the terroir concept is one of the most debated issues in oenology and viticulture. The dynamic interaction among diverse factors including the environment, the grapevine plant and the imposed viticultural techniques means that the wine produced in a given terroir is unique. However, there is an increasing interest to define and quantify the contribution of individual factors to a specific terroir objectively. Here, we characterized the metabolome and transcriptome of berries from a single clone of the Corvina variety cultivated in seven different vineyards, located in three macrozones, over a 3-year trial period. RESULTS: To overcome the anticipated strong vintage effect, we developed statistical tools that allowed us to identify distinct terroir signatures in the metabolic composition of berries from each macrozone, and from different vineyards within each macrozone. We also identified non-volatile and volatile components of the metabolome which are more plastic and therefore respond differently to terroir diversity. We observed some relationships between the plasticity of the metabolome and transcriptome, allowing a multifaceted scientific interpretation of the terroir concept. CONCLUSIONS: Our experiments with a single Corvina clone in different vineyards have revealed the existence of a clear terroir-specific effect on the transcriptome and metabolome which persists over several vintages and allows each vineyard to be characterized by the unique profile of specific metabolites.Andrea Anesi, Matteo Stocchero, Silvia Dal Santo, Mauro Commisso, Sara Zenoni, Stefania Ceoldo, Giovanni Battista Tornielli, Tracey E. Siebert, Markus Herderich, Mario Pezzotti and Flavia Guzz

ESAT-6 and HspX improve the effectiveness of BCG to induce human Dendritic Cells-dependent T cells and NK cells activation

The limited efficacy of Bacillus Calmette-Guerin (BCG) vaccination against some forms of tuberculosis is partly due to a missing expression of critical immunogenic proteins. We hypothesized that addition of ESAT-6 and HspX Mycobacterium Tuberculosis (Mtb) antigens could ameliorate the BCG ability to activate human dendritic cells (DC), that play an essential role in immune response. Here we report that BCG showed a weak ability to induce DC maturation, cytokine release and the subsequent CD4+ lymphocytes and NK cells activation. Addition of single ESAT-6 or HspX to BCG-stimulated DC did not significantly improve these processes. However, simultaneous addition of ESAT-6 and HspX enhanced BCG-dependent DC maturation and IL-12, IL-1\u3b2, IL-23, IL-6 and TNF\u3b1 release. Moreover, DC incubated with BCG in presence of both ESAT-6 and HspX elicited IFN-\u3b3 release by CD4+ lymphocytes, and increased IFN-\u3b3 secretion and CD69 cytolysis marker expression in NK cells. These effects were inhibited by IL-12-blocking antibodies. A specific TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DC incubated with ESAT-6 and HspX, as well as IFN-\u3b3 secretion by CD4+ lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DC to induce the expression of the memory phenotype marker CD45RO in na\uefve CD4+ T cells. Our results indicate that ESAT-6 and HspX cooperation enhances the ability of BCG to stimulate human DC, that become able to induce T lymphocytes and NK cells-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of vaccination with BCG

ESAT-6 and HspX improve the effectiveness of BCG to induce human Dendritic Cells-dependent T cells and NK cells activation

The limited efficacy of Bacillus Calmette-Guerin (BCG) vaccination against some forms of tuberculosis is partly due to a missing expression of critical immunogenic proteins. We hypothesized that addition of ESAT-6 and HspX Mycobacterium Tuberculosis (Mtb) antigens could ameliorate the BCG ability to activate human dendritic cells (DC), that play an essential role in immune response. Here we report that BCG showed a weak ability to induce DC maturation, cytokine release and the subsequent CD4+ lymphocytes and NK cells activation. Addition of single ESAT-6 or HspX to BCG-stimulated DC did not significantly improve these processes. However, simultaneous addition of ESAT-6 and HspX enhanced BCG-dependent DC maturation and IL-12, IL-1\u3b2, IL-23, IL-6 and TNF\u3b1 release. Moreover, DC incubated with BCG in presence of both ESAT-6 and HspX elicited IFN-\u3b3 release by CD4+ lymphocytes, and increased IFN-\u3b3 secretion and CD69 cytolysis marker expression in NK cells. These effects were inhibited by IL-12-blocking antibodies. A specific TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DC incubated with ESAT-6 and HspX, as well as IFN-\u3b3 secretion by CD4+ lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DC to induce the expression of the memory phenotype marker CD45RO in na\uefve CD4+ T cells. Our results indicate that ESAT-6 and HspX cooperation enhances the ability of BCG to stimulate human DC, that become able to induce T lymphocytes and NK cells-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of vaccination with BCG

SOS1, ARHGEF1, and DOCK2 rho-GEFs mediate JAK-Dependent LFA-1 activation by chemokines

JAK-dependent activation of the rho module of integrin affinity triggering mediates chemokine-induced leukocyte adhesion. However, the signaling events linking JAKs to rho small GTPase activation by chemokines is still incompletely described. In this study, we show that son of sevenless 1 (SOS1), rho guanine nucleotide exchange factor (GEF)1 (ARHGEF1), and dedicator of cytokinesis (DOCK)2 GEFs mediate CXCL12-induced LFA-1 activation in human primary T lymphocytes. Downregulated expression of SOS1, ARHGEF1, and DOCK2 impairs LFA-1-mediated rapid T lymphocyte adhesion as well as underflow arrest on ICAM-1 induced by CXCL12. Moreover, LFA-1 affinity triggering by CXCL12 is impaired by SOS1, ARHGEF1, and DOCK2 downregulation. Notably, the three GEFs are all critically involved in chemokine-induced RhoA and Rac1 activation, thus suggesting the occurrence of a SOS1 specificity shift in the context of chemokine signaling. Accordingly, SOS1, ARHGEF1, and DOCK2 are tyrosine phosphorylated upon chemokine signaling with timing coherent with rapid LFA-1 affinity activation. Importantly, chemokine-induced tyrosine phosphorylation of these GEFs is fully mediated by JAK protein tyrosine kinases. Unexpectedly, and differently from VAV1, tyrosine phosphorylation of SOS1, ARHGEF1, and DOCK2 is completely inhibited by pertussis toxin pretreatment, thus suggesting different routes of rho-GEF triggering upon CXCR4 engagement. Taken together, these findings reveal a deeper level of complexity in the rho-signaling module, with at least four different rho-GEFs cooperating in the regulation of chemokine-induced integrin activation, possibly suggesting the emergence of stochastic concurrency in signaling mechanisms controlling leukocyte trafficking

Protein Tyrosine Phosphatase Receptor Type \u3b3 Is a JAK Phosphatase and Negatively Regulates Leukocyte Integrin Activation

Regulation of signal transduction networks depends on protein kinase and phosphatase activities. Protein tyrosine kinases of the JAK family have been shown to regulate integrin affinity modulation by chemokines and mediated homing to secondary lymphoid organs of human T lymphocytes. However, the role of s in leukocyte recruitment is still elusive. In this study, we address this issue by focusing on protein tyrosine phosphatase receptor type \u3b3 (PTPRG), a tyrosine phosphatase highly expressed in human primary monocytes. We developed a novel methodology to study the signaling role of receptor type tyrosine phosphatases and found that activated PTPRG blocks chemoattractant-induced \u3b22 integrin activation. Specifically, triggering of LFA-1 to high-affinity state is prevented by PTPRG activation. High-throughput phosphoproteomics and computational analyses show that PTPRG activation affects the phosphorylation state of at least 31 signaling proteins. Deeper examination shows that JAKs are critically involved in integrin-mediated monocyte adhesion and that PTPRG activation leads to JAK2 dephosphorylation on the critical 1007-1008 phosphotyrosine residues, implying JAK2 inhibition and thus explaining the antiadhesive role of PTPRG. Overall, the data validate a new approach to study receptor tyrosine phosphatases and show that, by targeting JAKs, PTPRG downmodulates the rapid activation of integrin affinity in human monocytes, thus emerging as a potential novel critical regulator of leukocyte trafficking