CpG-ODN and MPLA Prevent Mortality in a Murine Model of Post-Hemorrhage-Staphyloccocus aureus Pneumonia

Infections are the most frequent cause of complications in trauma patients. Post-traumatic immune suppression (IS) exposes patients to pneumonia (PN). The main pathogen involved in PN is Methicillin Susceptible Staphylococcus aureus (MSSA). Dendritic cells () may be centrally involved in the IS. We assessed the consequences of hemorrhage on pneumonia outcomes and investigated its consequences on DCs functions. A murine model of hemorrhagic shock with a subsequent MSSA pneumonia was used. Hemorrhage decreased the survival rate of infected mice, increased systemic dissemination of sepsis and worsened inflammatory lung lesions. The mRNA expression of Tumor Necrosis Factor-alpha (TNF-α), Interferon-beta (IFN-β) and Interleukin (IL)-12p40 were mitigated for hemorrhaged-mice. The effects of hemorrhage on subsequent PN were apparent on the pDCs phenotype (reduced MHC class II, CD80, and CD86 molecule membrane expression). In addition, hemorrhage dramatically decreased CD8+ cDCs- and CD8- cDCs-induced allogeneic T-cell proliferation during PN compared with mice that did not undergo hemorrhage. In conclusion, hemorrhage increased morbidity and mortality associated with PN; induced severe phenotypic disturbances of the pDCs subset and functional alterations of the cDCs subset. After hemorrhage, a preventive treatment with CpG-ODN or Monophosphoryl Lipid A increased transcriptional activity in DCs (TNF-α, IFN-β and IL-12p40) and decreased mortality of post-hemorrhage MSSA pneumonia

Decreased Numbers of Blood Dendritic Cells and Defective Function of Regulatory T Cells in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

BACKGROUND: Dendritic cells (DC) and regulatory cells (Treg) play pivotal roles in controlling both normal and autoimmune adaptive immune responses. DC are the main antigen-presenting cells to T cells, and they also control Treg functions. In this study, we examined the frequency and phenotype of DC subsets, and the frequency and function of Treg from patients with ANCA-associated vasculitis (AAV). METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from 19 untreated patients with AAV during flares and before any immunosuppressive treatment were analyzed, along with 15 AAV patients in remission and 18 age-matched healthy controls. DC and Treg numbers, and phenotypes were assessed by flow cytometry, and in vitro suppressive function of Treg was determined by co-culture assay. When compared to healthy volunteers, absolute numbers of conventional and plasmacytoid DC were decreased in AAV patients. During the acute phase this decrease was significantly more pronounced and was associated with an increased DC expression of CD62L. Absolute numbers of Treg (CD4(+)CD25(high)CD127(low/-) Tcells) were moderately decreased in patients. FOXP3 and CD39 were expressed at similar levels on Treg from patients as compared to controls. The suppressive function of Treg from AAV patients was dramatically decreased as compared to controls, and this defect was more pronounced during flares than remission. This Treg functional deficiency occurred in the absence of obvious Th17 deviation. CONCLUSION: In conclusion, these data show that AAV flares are associated with both a decrease number and altered phenotype of circulating DC and point to a role for Treg functional deficiency in the pathogenesis of AAV

Etude du rôle des sous-populations de cellules dendritiques dans l'expansion et les fonctions des cellules T régulatrices chez le rat

Nous avons analysé chez le rat l'influence des différentes populations de cellules dendritiques (DC) sur la prolifération et l'activité suppressive des cellules T régulatrices naturelles (Treg) in vitro. Les populations de DC sont purifiées à partir de la rate et séparées en: DC CD 103+ CD45R- CD4+ (DC CD4+), les DC CD103+ CD45R- CD4- (DC CD4-) et les DC CD 103- CD45R+ CD4+ ou DC plasmacytoides (pDC). Nos résultats montrent que les trois sous- populations de DC matures induisent une forte prolifération des cellules TCD4+CD25- allogéniques mais que, contrairement aux DC dites conventionnelles (DC CD4+ et DC CD4-), les pDC matures stimulées via TLR7 ou 9 induisent en absence d'IL-2 exogène une forte prolifération des cellules TCD4+CD25+FOXP-3+ (Treg), réputées hypoprolifératives in vitro. Cette prolifération est indépendante de l'IL-2, nécessite un contact cellulaire et est partiellement dépendante de CD86. Les populations de DC ont également un impact différent sur l'activité suppressive des Treg in vitro. En présence de DC conventionnelles matures, les Treg suppriment fortement la prolifération et la production d'IL-2 et d'IFN-g par les cellules T effectrices alors qu'en présence de pDC, les Treg suppriment la production d'IL-2 mais pas la prolifération ni la production d'IFN-g des cellules T effectrices. Nos résultats indiquent que l'anergie et la fonction suppressive des Treg sont différentiellement contrôlées par les sous- populations de DC et suggèrent un rôle des pDC dans le contrôle des Treg in vivo.We have analysed in the rat the influence of different dendritic cell (DC) subsets on the proliferation and the suppressive activity of naturally occurring T regulatory cells (Treg) in vitro. DC subsets were purified from spleen and separated as: CD103+CD45R-CD4+ (CD4+ DC), CD103+CD45R-CD4- (CD4- DC) and CD103-CD45R+CD4+ or plasmacytoid DC (pDC). Our results show that the three mature DC subsets tested induced a strong proliferation of allogenic CD4+CD25-' Tcell but that, unlike conventional DC subsets (CD4- DC and CD4+ DC), mature pDC stimulated with TLR7 or 9 induce, in the absence of exogenous IL-2, a robust proliferative response of CD4+CD25+FOXP-3+ T cells (Treg), which are reputed hypoproliferative in vitro. Expansion of Treg by pDC is IL-2 independent, requires cell contact and is partially CD86-dependent. DC subsets also differentially control the suppressive activity of Treg in vitro. In the presence of conventional DC, Treg strongly suppress proliferation of, as well as IL-2 and IFN- g production by effector T cells, whereas in the presence of pDC, Treg suppress IL-2 production by effector T cells but not their prolifeation and IFN- g production. Our results indicate that anergy and suppressive activity of Treg are differentially controlled by DC subsets and suggest a role for pDC in the control of Treg in vivo.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Human plasmacytoid dendritic cells induce CD8+LAG-3+Foxp3+CTLA-4+ regulatory T cells that suppress allo-reactive memory T cells

Allo-reactive memory T cells are a major barrier for induction of immunological tolerance to allografts in humans. Here, we report that stimulation of unfractionated human T cells with TLR-stimulated allogeneic plasmacytoid dendritic cells (pDCs) induces CD8(+) regulatory T cells (Tregs) that inhibit T-cell allo-responses, including those of memory T cells. CD3(+) T cells were primed for 7 days with allogeneic pDCs that had been pre-stimulated with TLR-7 or TLR-9 ligands. While the T cells proliferated and produced cytokines during the priming culture, they were profoundly hypo-responsive to re-stimulation with the same allo-antigen in a second culture. Moreover, T cells primed by pDCs exerted donor-specific suppression on allo-responses of both unfractionated and memory CD3(+) T cells. The regulatory capacity of pDC-primed T cells was confined to CD8(+)LAG-3(+)Foxp3(+) CTLA-4(+) T cells, which suppressed allogeneic T-cell responses through a CTLA-4-dependent mechanism. Induction of CD8(+) Tregs by pDCs could be partially prevented by 1-methyl tryptophan, an inhibitor of indoleamine 2,3-dioxygenase. In conclusion, stimulation of human T cells by TLR-stimulated allogeneic pDCs induces CD8(+) Tregs that inhibit allogeneic T-cell responses, including memory T cells. Donor-derived pDCs may be considered as an immunotherapeutic tool to prevent activation of the recipient alloreactive (memory) T-cell repertoire after allogeneic transplantation

Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high) subpopulation. Proliferation of CD4(+) CD25(-) sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co-culture with CD4(+) CD25(high) sorted cells in a dose-dependent manner. The mechanism of suppression by the CD4(+) CD25(high) cell population is mediated by close contact as well as interleukin (IL)-10 and transforming growth factor-beta1 (TGF-beta1) and probably other factors. In addition, we studied the in vitro induction of CD4(+) Treg and their characteristics compared to those of freshly isolated CD4(+) Treg cells. Upon stimulation with a combination of concanavalin A, TGF-beta1 and IL-2, CD4(+) CD25(+) T cells which express FoxP3 and have suppressive capability were induced from CD4(+) CD25(-) cells. The induced CD4(+) CD25(high) express higher levels of IL-10 and TGF-beta1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4(+) CD25(high) subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4(+) CD25(+) cells in horses and offers insights into ex vivo manipulation of Treg cells

IRAK-4-and MyD88-Dependent Pathways Are Essential for the Removal of Developing Autoreactive B Cells in Humans

Most autoreactive B cells are normally counterselected during early B cell development. To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for interleukin-1 receptor-associated kinase 4 (IRAK-4), myeloid differentiation factor 88 (MyD88), and UNC-93B. Indeed, all TLRs except TLR3 require IRAK-4 and MyD88 to signal, and UNC-93B-deficient cells are unresponsive to TLR3, TLR7, TLR8, and TLR9. All patients suffered from defective central and peripheral B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive mature naive B cells in their blood. Hence, TLR7, TLR8, and TLR9 may prevent the recruitment of developing autoreactive B cells in healthy donors. Paradoxically, IRAK-4-, MyD88-, and UNC-93B-deficient patients did not display autoreactive antibodies in their serum or develop autoimmune diseases, suggesting that IRAK-4, MyD88, and UNC-93B pathway blockade may thwart autoimmunity in humans