S. Giannelli

Human defensins activate monocyte-derived dendritic cells, promote the production of proinflammatory cytokines, and up-regulate the surface expression of CD91

Defensins are endogenous defense peptides with well‐defined antimicrobial activity against a broad spectrum of pathogens including bacteria, fungi, viruses, and parasites. Several lines of evidence suggest that defensins might also contribute to the regulation of host innate and adaptive immunity, but their immunomodulatory functions are still poorly understood. Herein, we studied the impact of human defensins on multiple functions of DCs, which are a central player in all immune responses, bridging innate and adaptive immunity. We challenged DCs differentiated in vitro from human moDCs with HNP‐1 α‐defensin or HBD‐1. HNP‐1 and HBD‐1 were chemotactic for moDCs. Both defensins promoted the activation and maturation of moDCs, as assessed by up‐regulation of surface expression of the costimulatory molecules CD80, CD86, and CD40, the maturation marker CD83, and HLA‐DR. HNP‐1 and HBD‐1 also enhanced the production of the proinflammatory cytokines TNF‐α, IL‐6, and IL‐12p70 but did not affect the production of the regulatory cytokine IL‐10. According to these stimulatory effects, HNP‐1 and HBD‐1 increased the allostimulatory activity of moDCs significantly. Finally, HNP‐1 and HBD‐1 promoted the up‐regulation of CD91 on the DC surface. CD91 is a scavenger receptor involved in the recognition of multiple ligands including defensins, thus suggesting that defensins may amplify their own effects through the activation of an autocrine loop. Taken together, our observations may provide new insight into the immunomodulatory properties of human defensins and may aid the exploration of new therapeutic strategies to potentiate antimicrobial and antitumor immunity.

Application of six-color flow cytometry for the assessment of dendritic cell responses in whole blood assays

Analysis of peripheral blood dendritic cells (PBDCs) is increasingly reaching clinical relevance in a wide range of pathologies, in which investigating the capacity of DC subsets to respond adequately to specific stimuli may aid the comprehension of underlying immunopathologic mechanisms. The evaluation of PBDC responses directly challenged in whole blood (WB) samples offers many advantages over other methods that require DC isolation and culture, but it is limited in multiparametric analysis, currently based on 3- or 4-color assays. Therefore, in this study we developed a 6-color assay dedicated to the analysis of PBDC responses upon WB stimulation. We incubated WB samples with ligands to toll-like receptors (TLRs) with a clear-cut distribution on myeloid DCs (mDCs) or plasmacytoid (pDCs) and analyzed DC responses in terms of upregulation of activation/maturation markers, as well as production of a wide range of regulatory cytokines. Four colors were used to gate on mDCs and pDCs that were identified as lineage-/HLA-DR+/CD11c+ and lineage-/HLA-DR+/CD123+, respectively, and two further colors were used to analyze either the surface expression of CD80, CD86, CD40 or CD83, or the intracellular accumulation of IL-12, tumor-necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IL-6, IL-10 or IL-4. With this method, we could directly compare in the same flow cytometric tube the responses of mDCs and pDCs to TLR stimulation, and investigate the reciprocal coexpression of distinct activation markers or regulatory cytokines. We suggest that the 6-color WB assay presented here may represent a novel tool for investigating the complex biology of DCs.

Incomplete activation of peripheral blood dendritic cells during healthy human pregnancy.

Successful pregnancy relies on the adaptation of immune responses that allow the fetus to grow and develop in the uterus despite being recognized by maternal immune cells. Dendritic cells (DCs) are central to the control of immune tolerance, and their state of activation at the maternal–decidual interface is critical to the feto–maternal immunological equilibrium. So far, the involvement of circulating DCs has been investigated poorly. Therefore, in this study we investigated whether, during healthy human pregnancy, peripheral blood DCs (PBDCs) undergo changes that may be relevant to the adaptation of maternal immune responses that allow fetal tolerance. In a cross‐sectional study, we analysed PBDCs by six‐colour flow cytometry on whole blood samples from 47 women during healthy pregnancy progression and 24 non‐pregnant controls. We demonstrated that both myeloid and plasmacytoid PBDCs undergo a state of incomplete activation, more evident in the third trimester, characterized by increased expression of co‐stimulatory molecules and cytokine production but lacking human leucocyte antigen (HLA)‐DR up‐regulation. To investigate the contribution of soluble circulating factors to this phenomenon, we also performed culture experiments showing that sera from pregnant women added to control DCs conditioned a similar incomplete activation that was associated with reduced DC allostimulatory capacity, supporting the in vivo relevance of our findings. We also obtained evidence that the glycoprotein hormone activin‐A may contribute to DC incomplete activation. We suggest that the changes of PBDCs occurring during late pregnancy may aid the comprehension of the immune mechanisms operated by the maternal immune system to maintain fetal tolerance.

A six‐color flow cytometric assay for the analysis of peripheral blood dendritic cells

Flow cytometric analysis of peripheral blood dendritic cells (PBDCs) and their myeloid (mDCs) and plasmacytoid (pDCs) subsets is a less invasive procedure that is acquiring growing clinical relevance. Because dendritic cells (DCs) lack unique lineage markers, current methods that are based on 3‐ or 4‐color assays do not allow multiparametric analysis of DC subsets. In this study a dedicated 6‐color assay was developed.

Lack of activation of peripheral blood dendritic cells in human pregnancies complicated by intrauterine growth restriction

INTRODUCTIONnThe state of activation of dendritic cells (DCs) at the feto-maternal interface critically contributes to optimal decidual immune responses needed to support fetal-placental development. We recently demonstrated that during healthy pregnancy also peripheral blood DCs (PBDCs), which are easily accessible, are activated as well. In this study, to investigate a possible involvement of DCs in intrauterine growth restriction (IUGR), we evaluated whether PBDCs in pregnancy complicated by IUGR may be altered compared with PBDCs in healthy pregnancy.nnnMETHODSnPBDCs from 12 pregnant women with primary IUGR, 21 healthy pregnant and 19 nonpregnant women were analyzed by flow cytometric analysis of whole-blood samples collected at a single time point.nnnRESULTSnThe number of plasmacytoid PBDCs was significantly reduced in women with IUGR pregnancy. Myeloid and plasmacytoid PBDCs in IUGR lacked the state of activation (assessed as CD80, CD86, CD40 expression) and the shift to a proinflammatory pattern of cytokine production occurring during healthy pregnancy.nnnDISCUSSIONnTo our knowledge, this is the first study investigating the state of PBDC activation in IUGR pregnancy. Our results are in accordance with a previous study reporting a lower expression of activation and maturation markers by decidual DCs in IUGR placentas.nnnCONCLUSIONSnThe reduced activation of PBDCs in IUGR pregnancy may possibly reflect a reduced activation of decidual DCs. If confirmed at the feto-maternal interface, the alterations of DCs described in IUGR pregnancy have the potential to negatively impact on vascular development during gestation. These observations may therefore broaden our understanding of IUGR pathogenesis.