Toll-like receptor-9 stimulated plasmacytoid dendritic cell precursors suppress autoimmune neuroinflammation in a murine model of multiple sclerosis.

Early innate education of hematopoietic progenitors within the bone marrow (BM) stably primes them for either trained immunity or instead immunoregulatory functions. We herein demonstrate that in vivo or in vitro activation within the BM via Toll-like receptor-9 generates a population of plasmacytoid dendritic cell (pDC) precursors (CpG-pre-pDCs) that, unlike pDC precursors isolated from PBS-incubated BM (PBS-pre-pDCs), are endowed with the capacity to halt progression of ongoing experimental autoimmune encephalomyelitis. CpG activation enhances the selective migration of pDC precursors to the inflamed spinal cord, induces their immediate production of TGF-ß, and after migration, of enhanced levels of IL-27. CpG-pre-pDC derived TGF-ß and IL-27 ensure protection at early and late phases of the disease, respectively. Spinal cords of CpG-pre-pDC-protected recipient mice display enhanced percentages of host-derived pDCs expressing TGF-ß as well as an accumulation of IL-10 producing B cells and of CD11c+ CD11b+ dendritic cells. These results reveal that pDC precursors are conferred stable therapeutic properties by early innate activation within the BM. They further extend to the pDC lineage promising perspectives for cell therapy of autoimmune diseases with innate activated hematopoietic precursor cells.

Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis.

Achieving immunoregulation via in vivo expansion of Foxp3+ regulatory CD4+ T cells (Treg) remains challenging. We have shown that mobilization confers to multipotent hematopoietic progenitors (MPPs) the capacity to enhance Treg proliferation. Transcriptomic analysis of Tregs co-cultured with MPPs revealed enhanced expression of genes stabilizing the suppressive function of Tregs as well as the activation of IL-1ß-driven pathways. Adoptive transfer of only 25,000 MPPs effectively reduced the development of experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for multiple sclerosis (MS). Production of the pathogenic cytokines IL-17 and GM-CSF by spinal cord-derived CD4+ T-cells in MPP-protected recipients was reduced while Treg expansion was enhanced. Treg depletion once protection by MPPs was established, triggered disease relapse to the same level as in EAE mice without MPP injection. The key role of IL-1ß was further confirmed in vivo by the lack of protection against EAE in recipients of IL-1ß-deficient MPPs. Mobilized MPPs may thus be worth considering for cell therapy of MS either per se or for enrichment of HSC grafts in autologous bone marrow transplantation already implemented in patients with severe refractory multiple sclerosis.

Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells.

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases.

Dengue virus activates membrane TRAIL relocalization and IFN-α production by human plasmacytoid dendritic cells in vitro and in vivo.

BACKGROUND: Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro. METHODS FINDINGS: Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production. CONCLUSIONS: This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.

Innate pro-B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells.

Diverse hematopoietic progenitors, including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells, mobilized by hematopoietic growth factors or emerging during parasitic infections, display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-γ released by activated effector T cells (Teffs), by up-regulating their Fas ligand (FasL) expression, which enabled them to kill Teffs through apoptosis. In turn, IFN-γ derived from CpG-proBs enhanced IFN-γ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D, adoptively transferred IFN-γ-deficient CpG-proBs did not prevent T1D development. Additionally, CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation.

TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

Plasmacytoid dendritic cells (pDCs) from HIV controllers produce interferon-α and differentiate into functional killer pDCs under HIV activation.

BACKGROUND: Human immunodeficiency virus (HIV) controllers spontaneously control viremia and CD4 T-cell depletion in contrast to viremic patients. After HIV exposure, plasmacytoid dendritic cells (pDCs) produce high levels of interferon alpha (IFN-α) and express the apoptotic ligand TRAIL (tumor necrosis factor-related apoptosis inducing ligand). Simian models have shown that prolonged high levels of IFN-α production could be responsible for AIDS progression. METHODS: We studied pDC activation in response to human immunodeficiency virus (HIV) using flow cytometry and 3D microscopy. RESULTS: We show here that pDCs from controller patients produced higher levels of IFN-α in response to HIV than pDCs from viremic patients but similar levels to pDCs from healthy donors. Because binding of HIV to CD4 is essential for pDC activation, the low CD4 expression by pDCs from viremic patients may explain the weak IFN-α response to HIV. Three-dimensional microscopy revealed that pDCs from controllers and healthy donors expressed intracellular TRAIL that is relocalized to the membrane after HIV exposure. In contrast, pDCs from viremic patients expressed membrane TRAIL without any stimulation. CONCLUSIONS: We demonstrate that, in response to HIV, pDCs from controller patients produce IFN-α, express membrane TRAIL, and induce apoptosis of T-cell lines.

Free HTLV-1 induces TLR7-dependent innate immune response and TRAIL relocalization in killer plasmacytoid dendritic cells.

A recent report demonstrated that free human T-cell leukemia virus 1 (HTLV-1) could infect plasmacytoid dendritic cells (pDCs). The major role of pDCs is to secrete massive levels of interferon-alpha (IFN-alpha) upon virus exposure; however, the induction of IFN-alpha by HTLV-1 remains unknown. We demonstrate here that cell-free HTLV-1 generated a pDC innate immune response by producing massive levels of IFN-alpha that were inhibited by anti-HTLV-1 antibodies. HTLV-1 induced costimulatory molecules and rapid expression of the apoptotic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Furthermore, HTLV-1 stimulated pDC-induced apoptosis of CD4(+) T cells expressing DR5, transforming pDCs into IFN-producing killer pDCs. We also observed that an endosomal acidification inhibitor and a Toll-like receptor-7 (TLR7)-specific blocker drastically inhibited pDC response to HTLV-1. Three-dimensional microscopy analysis revealed that unstimulated pDCs were "dormant" IFN-producing killer pDCs with high levels of intracellular TRAIL that could be rapidly mobilized to the surface in response to TLR7 activation. Inhibition of viral degradation in endosomes by chloroquine maintained viral integrity, allowing virus detection by 3-dimensional microscopy. We demonstrate that pDCs respond to cell-free HTLV-1 by producing high levels of IFN-alpha and by mobilizing TRAIL on cell surface after TLR7 triggering. This is the first demonstration of an innate immune response induced by free HTLV-1.

Molecular polymorphism of native gonadotropin-releasing hormone (GnRH) is restricted to mammalian GnRH and [hydroxyproline9] GnRH in the developing rat brain.

Although chicken gonadotropin-releasing hormone (GnRH)-II is thought to occur in most animal species, its presence and that of two other variants (lamprey GnRH-III, salmon GnRH) is questionable in rodents. Here we report on the GnRH peptides present in the hypothalamus and the remaining brain of rat of both sexes during development. No immunoreactivity was detected in the elution zone of either native or hydroxylated forms of the above three variants in any of brain extracts chromatographed. The main peptides detected were mammalian GnRH (mGnRH) and m[hydroxyproline9]GnRH (mHypGnRH). In the hypothalamus, these peptides were associated with their free acid and precursor forms. N-terminal fragments from both native decapeptides (GnRH) and mGnRH (GnRH) were observed only in the hypothalamus. C-terminal fragments were detected in both tissues. The relative proportions of mGnRH and mHypGnRH showed no developmental changes in the remaining brain. The hypothalamic proportions of mHypGnRH were high on day 5, and decreased from day 15 onwards. The [Gly11]-precursor to mHypGnRH molar ratio was twofold lower than with the non-hydroxylated peptides. The mGnRH to GnRH molar ratio increased in males but decreased in females during development. No sex-related differences were observed in the native decapeptide to GnRH molar ratio. It was concluded that (1) chicken GnRH-II is not present in all mammals, (2) mGnRH and mHypGnRH are the main GnRH isoforms present in the rat brain, (3) the processing of [Gly11]-precursor into mHypGnRH occurs at a higher rate than that of mGnRH, and (4) the catabolism does not interfere with the developmental changes undergone by the mGnRH and mHypGnRH brain contents.