Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation

International audienceTocilizumab, a humanized anti-IL-6 receptor α (IL-6Rα) is widely used in the treatment of a panel of pathologies such as adult and juvenile rheumatoid arthritis (RA) and the systemic form of juvenile idiopathic arthritis in children. Its indications are expected to be largely extended to other inflammatory diseases in close future. Dendritic cells (DCs) appear to be deeply involved in the immunopathology of these diseases, yet the effects of tocilizumab on these cells were poorly studied. In this study, we explored the effect of tocilizumab on the regulation of IL-6R subunits [gp130, soluble form of IL-6Rα (sIL-6Rα), and mIL-6Rα] in human monocyte-derived DCs. Human DCs were derived from CD14 + monocytes purified with beads with IL-4 and granulocyte macrophage colony-stimulating factor. Ex vivo cultures of DCs were performed in the presence of tocilizumab. Using lipopolysaccharide (LPS) maturation of DCs, we demonstrated that tocilizumab did not inhibit IL-6 secretion, enhanced mIL-6Rα expression, and largely increased sIL-6Rα secretion. MAPK modulated STAT3 phosphorylation and surface expression of IL-6Rα in LPS-DCs. Tocilizumab had no impact on STAT3 phosphorylation in LPS-DCs while both LPS and IL-6 increased its activation. Tocilizumab modulated the regulation of IL-6R subunits leading to an inflammatory status of DCs and a massive secretion of IL-6Rα. Our results demonstrate that DCs acquire a pro-inflammatory profile following tocilizumab treatment, becoming a major source of IL-6 trans-signaling activation that might explain the poor clinical benefit in some RA patients

IL-2 Phosphorylates STAT5 To Drive IFN-γ Production and Activation of Human Dendritic Cells

International audienceHuman dendritic cells (hDCs) produce IL-2 and express IL-2R α-chain (CD25), but the role of IL-2 in DC functions is not well defined. A recent study suggested that the main function of CD25 on hDCs was to transpresent IL-2 to activate T lymphocytes. Our results demonstrate the expression of the three chains of the IL-2R on hDCs and that IL-2 induces STAT5 phosphorylation. Interestingly, use of inhibitors of p-STAT5 revealed that IL-2 increases LPS-induced IFN-γ through STAT5 phosphorylation. Finally, we report that IL-2 increases the ability of hDCs to activate helpless CD8+ T cells, most likely because of IL-2–triggered IFN-γ synthesis, as we previously described. For the first time, to our knowledge, we disclose that IL-2 induces monocyte-derived hDC's functional maturation and activation through IL-2R binding. Interestingly, our study suggests a direct effect of anti-CD25 mAbs on hDCs that may contribute to their clinical efficacy

Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation

Tocilizumab, a humanized anti-IL-6 receptor α (IL-6Rα) is widely used in the treatment of a panel of pathologies such as adult and juvenile rheumatoid arthritis (RA) and the systemic form of juvenile idiopathic arthritis in children. Its indications are expected to be largely extended to other inflammatory diseases in close future. Dendritic cells (DCs) appear to be deeply involved in the immunopathology of these diseases, yet the effects of tocilizumab on these cells were poorly studied. In this study, we explored the effect of tocilizumab on the regulation of IL-6R subunits [gp130, soluble form of IL-6Rα (sIL-6Rα), and mIL-6Rα] in human monocyte-derived DCs. Human DCs were derived from CD14+ monocytes purified with beads with IL-4 and granulocyte macrophage colony-stimulating factor. Ex vivo cultures of DCs were performed in the presence of tocilizumab. Using lipopolysaccharide (LPS) maturation of DCs, we demonstrated that tocilizumab did not inhibit IL-6 secretion, enhanced mIL-6Rα expression, and largely increased sIL-6Rα secretion. MAPK modulated STAT3 phosphorylation and surface expression of IL-6Rα in LPS-DCs. Tocilizumab had no impact on STAT3 phosphorylation in LPS-DCs while both LPS and IL-6 increased its activation. Tocilizumab modulated the regulation of IL-6R subunits leading to an inflammatory status of DCs and a massive secretion of IL-6Rα. Our results demonstrate that DCs acquire a pro-inflammatory profile following tocilizumab treatment, becoming a major source of IL-6 trans-signaling activation that might explain the poor clinical benefit in some RA patients

Modulation of human dendritic cell functions by bispecific antibody targeting pathogens recognition receptors

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THE VOLTAGE-GATED SODIUM CHANNEL BETA4 SUBUNIT MAINTAINS EPITHELIAL PHENOTYPE IN MAMMARY CELLS

International audienceThe SCN4B gene, encoding for the NaVβ4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells is not characterized. In this study, we demonstrate that reducing NaVβ4 expression in MCF10A non-cancer mammary epithelial cells generates important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably the loss of NaVβ4 induces a complete loss of epithelial organisation in cysts and increases proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of β-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, α-SMA expression. Overall, our results suggest that Navβ4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its down regulation might be a determining step in early carcinogenesis

Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity.

Most circulating strains of Human enterovirus 71 (EV-A71) have been classified primarily into three genogroups (A to C) on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D) and 2 African ones (E and F). Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied

Similarity within the EV-A71 genogroups through comparison of the full-length VP1 gene sequences.

<p>Similarity within the EV-A71 genogroups through comparison of the full-length VP1 gene sequences.</p

Molecular differentiation of the EV-A71 genogroups.

<p>The percentages of nucleotide and amino acid divergences were calculated in pairwise comparisons of 59 sequences selected as representative of the EV-A71 genogroups and subgenogroups. A scatter plot was produced with the divergence values to determine a threshold between intra-and inter-genogroup differences.</p

Bayesian maximum clade credibility phylogram (A) and chronogram (B) inferred with the 1D gene encoding the VP1 capsid protein, depicting the phylogeny of EV-A71.

<p>The trees were estimated with general time reversible nucleotide substitution model, an uncorrelated exponential clock model, and the Bayesian skyline as a tree prior. The posterior probability of the eight nodes used to compare different evolutionary models (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090624#pone-0090624-g004" target="_blank">Figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090624#pone-0090624-g003" target="_blank">3</a>) are indicated. The posterior probability (pp) are indicated for the main nodes.</p

Phylogenetic relationships between EV-A71 full-length VP1 sequences inferred using the Neighbor-Joining method.

<p>The evolutionary distances were computed using the Kimura 2-parameter method. The length of the branches is proportional to the number of nt divergence. The percent of bootstrap replicates is indicated for the main nodes.</p