Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation.

Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-ß1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-ß1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-ß1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-ß1-ALK5 signaling. Conversely, TGF-ß1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-ß1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-ß1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-ß1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-ß1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.

ß-Catenin promotes the differentiation of epidermal Langerhans dendritic cells.

The epithelial signaling protein and transcriptional regulator ß-catenin has recently been implicated in hematopoietic dendritic cell (DC) differentiation as well as in DC-mediated tolerance. We here observed that epidermal Langerhans cells (LCs) but not interstitial/dermal DCs express detectable ß-catenin. LCs are unique among the DC family members in that LC networks critically depend on epithelial adhesion molecules as well as on the cytokine transforming growth factor-ß1 (TGF-ß1). However, despite the important functions of LCs in the immune system, the molecular mechanisms governing LC differentiation and maintenance remain poorly defined. We found that TGF-ß1 induces ß-catenin in progenitor cells undergoing LC differentiation and that ß-catenin promotes LC differentiation. Vitamin D, another epidermal signal, enhanced TGF-ß1-mediated ß-catenin induction and promoted the expression of multiple epithelial genes by LCs. Moreover, full-length vitamin D receptor (VDR) promoted, whereas a truncated VDR diminished, the positive effects of ectopic ß-catenin on LC differentiation. Therefore, we here identified ß-catenin as a positive regulator of LC differentiation in response to TGF-ß1 and identified a functional interaction between ß-catenin and VDR in these cells.

Expressional and functional studies of CKLF1 during dendritic cell maturation.

Chemokine-like factor 1 (CKLF1) was the first member of the CKLF-like MARVEL transmembrane domain containing member (CMTM) family to be discovered. Its expression level is increased clearly in peripheral blood lymphocytes upon phytohemagglutinin stimulation, but little is known about the expression and function of CKLF1 in dendritic cells (DCs), which are the most potent antigen-presenting cells. In the present study, we showed that CKLF1 was highly expressed in monocytes. During differentiation from monocytes to immature DCs, CKLF1 was increased dramatically on day 2 and then decreased from day 3 to 5. Upon maturation with different stimuli, CKLF1 was down-regulated. Two peptides of CKLF1, C19 and C27, stimulated the effect of immature DCs (imDCs) on T-cell proliferation and IFN-gamma production. Further study on DC maturation showed that C19 and C27 up-regulated HLA-DR expression and IL-12 secretion, with no obvious effects on CD80, CD83 or CD86. Thus, CKLF1-C19 and -C27 can stimulate antigen-presenting capability of imDCs.

Human rhinoviruses induce IL-35-producing Treg via induction of B7-H1 (CD274) and sialoadhesin (CD169) on DC.

IL-35 is a heterodimer of EBV-induced gene 3 and of the p35 subunit of IL-12, and recently identified as an inhibitory cytokine produced by natural Treg in mice, but not in humans. Here we demonstrate that DC activated by human rhinoviruses (R-DC) induce IL-35 production and release, as well as a suppressor function in CD4(+) and CD8(+) T cells derived from human peripheral blood but not in naïve T cells from cord blood. The induction of IL-35-producing T cells by R-DC was FOXP3-independent, but blocking of B7-H1 (CD274) and sialoadhesin (CD169) on R-DC with mAb against both receptors prevented the induction of IL-35. Thus, the combinatorial signal delivered by R-DC to T cells via B7-H1 and sialoadhesin is crucial for the induction of human IL-35(+) Treg. These results demonstrate a novel pathway and its components for the induction of immune-inhibitory T cells.

Epigenetic regulation of dendritic cell differentiation and function by oxidized phospholipids.

Dendritic cells (DCs) are the key cell type in the regulation of an adaptive immune response. Under inflammatory conditions monocytes can give rise to immunostimulatory DCs, depending on microenvironmental stimuli. Here we show that oxidized phospholipids (Ox-Pls), which are generated during inflammatory reactions, dysregulate the differentiation of DCs. DCs generated in the presence of Ox-Pls up-regulated the typical DC marker DC-SIGN but did not express CD1a, CD1b, and CD1c. These DCs generated in the presence of Ox-Pls had a substantially diminished T cell-stimulating capacity after stimulation with Toll-like receptor ligands. Toll-like receptor ligand-induced production of interleukin-12 also was strongly diminished, whereas induction of CD83 was not altered. In addition, we found that Ox-Pls strongly inhibit inflammatory stimuli-induced phosphorylation of histone H3, a key step of interleukin-12 production, yet leaving activation of nuclear factor-kappaB unaltered. Taken together, Ox-Pls present during differentiation yielded DCs with a reduced capacity to become immunostimulatory mature DCs. Furthermore, the presence of Ox-Pls blocked histone modifications required for full activation of DCs. Therefore, inflammation-derived Ox-Pls control DC functions in part by epigenetic mechanisms.

The ssRNA genome of human rhinovirus induces a type I IFN response but fails to induce maturation in human monocyte-derived dendritic cells.

Dendritic cells (DCs) use pattern recognition receptors to sense invading viruses and triggering of these receptors induces a maturation program. Human rhinoviruses (HRVs) belong to the family of Picornaviridae, which have a single-stranded, coding RNA genome. Because HRV does not replicate in DCs, we used genomic RNA from HRV in this study to analyze the impact of natural occurring viral ssRNA on DC function. We found that transfection of human monocyte-derived DCs with viral ssRNA induced type I IFN production but failed to activate the NF-kappaB pathway in DCs. In line with this observation, the up-regulation of typical maturation markers such as CD83 or the production of the proinflammatory cytokines IL-12p40, IL-6, and TNF-alpha was not detectable. Most importantly, the T cell stimulatory capacity of viral ssRNA-treated DCs was not enhanced and remained at the level of immature DCs. Taken together, our results demonstrate that viral ssRNA efficiently activates the innate defense arm of DCs, whereas it is insufficient to activate the stimulatory capacity of DCs for the adaptive defense responses.

The oxidation state of phospholipids controls the oxidative burst in neutrophil granulocytes.

The activation of neutrophil granulocytes has to be carefully controlled to balance desired activity against invading pathogens while avoiding overwhelming activation leading to host tissue damage. We now show that phospholipids are potential key players in this process by either enhancing or dampening the production of reactive oxygen species (ROS) during the oxidative burst. Unoxidized phospholipids induce the production of ROS, and they also work synergistically with FMLP in potentiating the oxidative burst in neutrophil granulocytes. Oxidation of these phospholipids, however, turns them into potent inhibitors of the oxidative burst. OxPls specifically inhibit ROS production by inhibiting the assembly of the phagocyte oxidase complex but do not alter neutrophil viability, nor do they interfere with MAPK activation. Furthermore, up-regulation of the activation marker Mac-1 and phagocytosis of bacteria is not affected. Therefore, phospholipids may act as sensors of oxidative stress in tissues and either positively or negatively regulate neutrophil ROS production according to their oxidation state.

Oxidized phospholipids induce anergy in human peripheral blood T cells.

Lipids are key regulators of immune responses. In this study we investigated the direct impact of oxidized phospholipids (ox-PL) on T cell activation and function. We could demonstrate that ox-PL strongly inhibit proliferation of purified human T cells induced with anti-CD3/CD28 or anti-CD3/CD63 mAb, whereas proliferation of naive T cells from human cord blood was not affected by ox-PL. Unoxidized phospholipids showed no such effect. Inhibition of T cell proliferation by ox-PL was not due to cell death. Moreover, T cell proliferation triggered by PMA/ionomycin activation was not diminished by ox-PL. T cells activated in the presence of ox-PL produced and released low amounts of IFN-gamma and IL-2, whereas IL-4 was only slightly diminished. Ox-PL prevented the expression of de novo synthesized activation markers (CD25, MHC class II) but not expression of CD63 or CD69. We further observed that T cells stimulated in the presence of ox-PL are poorly cytotoxic T cells. Most importantly, T cells activated in the presence of ox-PL failed to proliferate in response to restimulation. This hypo-proliferative state was accompanied with an up-regulation of early growth response gene 3 and Casitas B-lineage lymphoma protein B. Taken together, our results demonstrate that ox-PL are potent and specific regulators of T cell activation and function.