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TGF-β is required for both Foxp3+ regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-β on pDC, and to our knowledge, no study has assessed the capacity of TGF-β-exposed pDC to polarize naive CD4+ T cells. In this paper, we show that TGF-β-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-β/Smad signal, because TGF-β treatment induced Smad2 phosphorylation in pDC and blockade of TGF-β signaling with the SD208 TGF-βRI kinase inhibitor abrogated Th17 commitment induced by TGF-β-treated pDC. Moreover, TGF-β mRNA synthesis and active TGF-β release were induced in TGF-β-treated pDC and anti-TGF-β Ab blocked Th17 commitment. Unexpectedly, TGF-β treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-β-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-β-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-β-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-β-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens. [ABSTRACT FROM AUTHOR]

Background: Reinstating inflammation resolution represents an innovative concept to regain inflammation control in diseases marked by chronic inflammation. While most therapeutics target inflammatory molecules and inflammatory effector cells and mediators, targeting macrophages to initiate inflammation resolution to control neuroinflammation has not yet been attempted. Resolution-phase macrophages are critical in the resolution process to regain tissue homeostasis, and are programmed through the presence and elimination of apoptotic leukocytes. Hence, inducing resolution-phase macrophages might represent an innovative therapeutic approach to control and terminate dysregulated neuroinflammation. Methods: Here, we investigated if the factors released by in vitro induced resolution-phase macrophages (their secretome) are able to therapeutically reprogram macrophages to control neuroinflammation in the model of experimental autoimmune encephalomyelitis (EAE). Results: We found that injection of the pro-resolutive secretome reduced demyelination and decreased inflammatory cell infiltration in the CNS, notably through the in vivo reprogramming of macrophages at the epigenetic level. Adoptive transfer experiments with in vivo or in vitro reprogrammed macrophages using such pro-resolutive secretome confirmed the stability and transferability of this acquired therapeutic activity. Conclusions: Overall, our data confirm the therapeutic activity of a pro-resolution secretome in the treatment of ongoing CNS inflammation, via the epigenetic reprogramming of macrophages and open with that a new therapeutic avenue for diseases marked by neuroinflammation. [ABSTRACT FROM AUTHOR]

Several APCs participate in apoptotic cell-induced immune modulation. Whether plasmacytoid dendritic cells (PDCs) are involved in this process has not yet been characterized. Using a mouse model of allogeneic bone marrow engraftment, we demonstrated that donor bone marrow PDCs are required for both donor apoptotic cell-induced engraftment and regulatory T cell (Treg) increase. We confirmed in naive mice receiving i.v. syngeneic apoptotic cell infusion that PDCs from the spleen induce ex vivo Treg commitment. We showed that PDCs did not interact directly with apoptotic cells. In contrast, in vivo macrophage depletion experiments using clodronate-loaded liposome infusion and coculture experiments with supernatant from macrophages incubated with apoptotic cells showed that PDCs required macrophage-derived soluble factors-including TGF-ß-to exert their immunomodulatory functions. Overall, PDCs may be considered as the major APC involved in Treg stimulation/generation in the setting of an immunosuppressive environment obtained by apoptotic cell infusion. These findings show that like other APCs, PDC functions are influenced, at least indirectly, by exposure to blood-borne apoptotic cells. This might correspond with an additional mechanism preventing unwanted immune responses against self-antigens clustered at the cell surface of apoptotic cells occurring during normal cell turnover. [ABSTRACT FROM AUTHOR]

Background information. Efficient clearance of apoptotic cells, named efferocytosis, is a fundamental physiological process for tissue development and homeostasis. The contribution of non-professional phagocytes like fibroblasts to efferocytosis has been established, although the underlying mechanisms are not well understood. We recently demonstrated that horizontal DNA transfer can occur through the uptake of apoptotic human papillomaviruspositive cancer cells by human primary fibroblasts leading to their transformation. The aim of this present study was to analyse the cellular and molecular mechanisms that drive the phagocytic activity of human primary fibroblasts in the context of apoptotic cervical cancer cell removal. Results. Here we provide evidence that human primary fibroblasts engulf late more efficiently than early apoptotic cells, but their phagocytic ability remains limited compared to professional phagocytes such as human monocyte-derived macrophages. The engulfment occurs in a time-, temperature- and calcium-dependent manner. Remodelling of actin-fibers contributes to the biogenesis of apoptotic cell containing macroendocytic vacuoles. Both morphological analyses and pharmacological approaches confirmed the involvement of actin-driven phagocytosis and likely macropinocytotic mechanisms in apoptotic target internalization. The uptake of apoptotic cells requires phosphatidylserine recognition, which is mainly mediated by phosphatidylserine-receptor brain-specific angiogenesis inhibitor 1. Confocal microscopy analyses with organelle-specific markers revealed that internalised apoptotic material traffics into late phagolysosomes and specific features of microtubule-associated protein 1 light chain 3-associated phagocytosis were observed. Conclusions. Our in vitro data show that fibroblasts contribute to apoptotic tumour cell removal by phagocytosis and likely macropinocytotic mechanisms. Efferocytosis by fibroblasts involves phosphatidylserine receptor brain-specific angiogenesis inhibitor 1, which participates in subsequent uptake orchestration via actin cytoskeleton remodelling. Significance. Our results highlight the cellular and molecular mechanisms of fibroblast-mediated clearance of apoptotic tumour cells. Consequences regarding alternative mechanism of carcinogenesis or tumour progression should be addressed. [ABSTRACT FROM AUTHOR]

Background aims. This study aimed to characterize the immune effectors contained in the grafts from donor mice mobilized by granulocyte colony-stimulating factor (G-CSF) and plerixafor and to evaluate their impact on the development of acute graft-versus-host-disease (aGVHD). Methods. Mobilization was done with G-CSF alone or G-CSF plus plerixafor (G+P). Results. In grafts collected after G+P mobilization, we observed a significantly higher proportion of c-kit+Sca-l+ hematopoietic stem cells compared with G-CSF. A significant increase in the percentage of plasmacytoid dendritic cells was detected in the G+P graft compared with G-CSF graft. We also studied the ability of stem cell grafts mobilized with G+P to induce GVHD in a mouse model. We observed higher mortality (P < 0.001) associated with increased aGVHD clinical score (P < 0.0001) as well as higher pathology score in the intestine of mice receiving G+P as compared with G-CSF grafts (P < 0.001). Moreover, the exacerbated aGVHD severity was associated with upregulation of CCR6 expression on both CD4+ and CD84 T cells from the G+P grafts, as well as on T cells from mice transplanted with G+P grafts. Conclusions. In conclusion, we showed that grafts mobilized with G+P exhibited functional features different from those mobilized with G-CSF alone, which increase the severity of aGVHD in the recipients. [ABSTRACT FROM AUTHOR]

CD3- and CD28-activated T cells expanded for 12 days ex vivo to produce suicide gene-modified T cells are hyporesponsive to alloantigens. To investigate whether this impaired alloreactivity is a result of preferential expansion of regulatory T (Treg) cells, we compared peripheral blood mononuclear cells (PBMC) activated with CD3 and CD28 antibodies co-immobilized on beads and expanded for 12 days with interleukin (IL)-2 (CoCD3/CD28 cells) to the respective unactivated PBMC in terms of proliferation, cytokine production, and expression of Treg markers [cytotoxic T-lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumour necrosis factor receptor (GITR) and forkhead box P3 (FoxP3)] after allostimulation. Alloreactive cells were identified by carboxyfluoresceine succinimidyl ester staining dilution. Alloreactive cells in CoCD3/CD28 cells had a lower proliferative response and a lower potential for IL-2 and interferon-γ secretion than did those in PBMC, demonstrating a functional impairment of alloreactive cells during ex vivo expansion. Expression of Treg markers transiently increased during ex vivo expansion and was unaffected by depletion of CD25+ cells (containing Treg cells) before ex vivo PBMC expansion. Such prior CD25+ depletion did not restore the alloreactivity of CoCD3/CD28 cells. After allostimulation, expression of Treg markers was restricted to proliferative (alloreactive) cells among PBMC or CoCD3/CD28 cells. Lastly, CD4+ CD25+ cells purified from CoCD3/CD28 cells lacked suppressive activity when used as a third party, in contrast to CD4+ CD25+ cells purified from PBMC. In conclusion, the impaired alloreactivity of T cells expanded ex vivo is not a result of preferential Treg cell expansion and/or enhanced suppressive Treg activity. [ABSTRACT FROM AUTHOR]