Microenvironmental and cell intrinsic factors governing human cDC2 differentiation and monocyte reprogramming.

cDC2s occur abundantly in peripheral tissues and arise from circulating blood cDC2s. However, the factors governing cDC2 differentiation in tissues, especially under inflammatory conditions, remained poorly defined. We here found that psoriatic cDC2s express the efferocytosis receptor Axl and exhibit a bone morphogenetic protein (BMP) and p38MAPK signaling signature. BMP7, strongly expressed within the lesional psoriatic epidermis, cooperates with canonical TGF-ß1 signaling for inducing Axl+cDC2s from blood cDC2s in vitro. Moreover, downstream induced p38MAPK promotes Axl+cDC2s at the expense of Axl+CD207+ Langerhans cell differentiation from blood cDC2s. BMP7 supplementation allowed to model cDC2 generation and their further differentiation into LCs from CD34+ hematopoietic progenitor cells in defined serum-free medium. Additionally, p38MAPK promoted the generation of another cDC2 subset lacking Axl but expressing the non-classical NFkB transcription factor RelB in vitro. Such RelB+cDC2s occurred predominantly at dermal sites in the inflamed skin. Finally, we found that cDC2s can be induced to acquire high levels of the monocyte lineage identity factor kruppel-like-factor-4 (KLF4) along with monocyte-derived DC and macrophage phenotypic characteristics in vitro. In conclusion, inflammatory and psoriatic epidermal signals instruct blood cDC2s to acquire phenotypic characteristics of several tissue-resident cell subsets.

A novel 3D cell culture model to study the human small intestinal immune landscape.

Several subsets of mononuclear phagocytes and DCs (MDC) populate the small intestine (SI), and these cells reportedly exert specialized functions in anti-microbial immunity and tolerance. Given the specialized phenotype of these cells, differing from other MDC family members, including their putative circulating blood precursors, local intestinal factors play key instructive roles in their differentiation. We designed an SI cell culture model composed of three intestinal epithelial cell (IEC) types, including absorptive enterocytes (E cells), antigen delivering microfold (M) cells, and mucus-producing goblet (G) cells plus T lymphocytes and soluble B cell-derived factors. This model was used to study the differentiation fate of CD34+ hematopoietic progenitor cell-derived monocyte/DC precursors. Progeny cells can be analyzed after a 3-week co-culture period, mimicking the physiologic turn-over time of intestinal MDC. A dominant monocyte differentiation pathway was suppressed, in favor of partial differentiation along DC and macrophage pathways, with low percentages of cells acquired DC or macrophage markers. Moreover, E and G cells play opposing roles in CX3CR1+ vs CD103dim cell differentiation, indicating that both together might counter-balance M/DC differentiation. Thus, SI epithelial cells suppress M/DC differentiation, supporting a key role for exogenous factors in M/DC differentiation.

BMPR1a Is Required for the Optimal TGFß1-Dependent CD207+ Langerhans Cell Differentiation and Limits Skin Inflammation through CD11c+ Cells.

The cytokine TGFß1 induces epidermal Langerhans cell (LC) differentiation from human precursors, an effect mediated through BMPR1a/ALK3 signaling, as revealed from ectopic expression and receptor inhibition studies. Whether TGFß1‒BMPR1a signaling is required for LC differentiation in vivo remained incompletely understood. We found that TGFß1-deficient mice show defective perinatal expansion and differentiation of LCs. LCs can be identified within the normal healthy human epidermis by anti-BMPR1a immunohistology staining. Deletion of BMPR1a in all (vav+) hematopoietic cells revealed that BMPR1a is required for the efficient TGFß1-dependent generation of CD207+ LC-like cells from CD11c+ intermediates in vitro. Similarly, BMPR1a was required for the optimal induction of CD207 by preformed major histocompatibility complex II‒positive epidermal resident LC precursors in the steady state. BMPR1a expression is strongly upregulated in epidermal cells in psoriatic lesions, and BMPR1aΔCD11c mice showed a defect in the resolution phase of allergic and psoriatic skin inflammation. Moreover, whereas LCs from these mice expressed CD207, BMPR1a counteracted LC activation and migration from skin explant cultures. Therefore, TGFß1‒BMPR1a signaling seems to be required for the efficient induction of CD207 during LC differentiation in the steady state, and bone marrow‒derived lesional CD11c+ cells may limit established skin inflammation through enhanced BMPR1a signaling.

Matrix metalloproteinase 15 plays a pivotal role in human first trimester cytotrophoblast invasion and is not altered by maternal obesity.

Adequate anchoring of the placenta in the uterus through invasion of first trimester cytotrophoblasts (CTB) is required for a successful pregnancy. This process is mediated by matrix metalloproteinases (MMPs) and regulated by the maternal environment. Obesity is known to alter the intrauterine milieu and has been related to impaired invasion. We hypothesized that placental MMP15, a novel membrane-type MMP, is involved in CTB invasion and regulated by maternal obesity in early pregnancy. Thus, in this study MMP15 was immunolocalized to invasive extravillous and interstitial CTB. MMP15 silencing in chorionic villous explants using two different siRNAs reduced trophoblast outgrowth length (-35%, P ≤ .001 and -26%, P < .05) and area (-43%, P ≤ .001 and -36%, P ≤ .01) without altering trophoblast proliferation or apoptosis. Short-term treatment of primary first trimester trophoblasts with IL-6 (10 ng/mL), interleukin 10 (IL-10) (50 ng/mL), and tumor necrosis factor α (TNF-α) (10 ng/mL) did not affect MMP15 protein levels. Likewise, MMP15 mRNA and protein levels were unaltered between human first trimester placentas from control pregnancies vs those complicated with maternal obesity. Overall, our results suggest that the role of MMP15 in placental development and function in early pregnancy is limited to CTB invasion without being affected by short- and long-term inflammation.

Increased Aggregation Tendency of Alpha-Synuclein in a Fully Disordered Protein Complex.

The recent discovery of biologically active fully disordered, so called random fuzzy protein-protein interactions leads to the question of how the high flexibility of these protein complexes correlates to aggregation and pathologic misfolding. We identify the structural mechanism by which a random fuzzy protein complex composed of the intrinsically disordered proteins alpha-Synuclein and SERF1a is able to potentiate cytotoxic aggregation. A structural model derived from an integrated NMR/SAXS analysis of the reconstituted aSyn:SERF1a complex enabled us to observe the partial deprotection of one precise aSyn amyloid nucleation element in the fully unstructured ensemble. This minimal exposure was sufficient to increase the amyloidogenic tendency of SERF1a-bound aSyn. Our findings provide a structural explanation of the previously observed pro-amyloid activity of SERF1a. They further demonstrate that random fuzziness can trigger a structurally organized disease-associated reaction such as amyloid polymerization.

MicroRNA-451a overexpression induces accelerated neuronal differentiation of Ntera2/D1 cells and ablation affects neurogenesis in microRNA-451a-/- mice.

MiR-451a is best known for its role in erythropoiesis and for its tumour suppressor features. Here we show a role for miR-451a in neuronal differentiation through analysis of endogenous and ectopically expressed or silenced miR-451a in Ntera2/D1 cells during neuronal differentiation. Furthermore, we compared neuronal differentiation in the dentate gyrus of hippocampus of miR-451a-/- and wild type mice. MiR-451a overexpression in lentiviral transduced Ntera2/D1 cells was associated with a significant shifting of mRNA expression of the developmental markers Nestin, ßIII Tubulin, NF200, DCX and MAP2 to earlier developmental time points, compared to control vector transduced cells. In line with this, accelerated neuronal network formation in AB.G.miR-451a transduced cells, as well as an increase in neurite outgrowth both in number and length was observed. MiR-451a targets genes MIF, AKT1, CAB39, YWHAZ, RAB14, TSC1, OSR1, POU3F2, TNS4, PSMB8, CXCL16, CDKN2D and IL6R were, moreover, either constantly downregulated or exhibited shifted expression profiles in AB.G.miR-451a transduced cells. Lentiviral knockdown of endogenous miR-451a expression in Ntera2/D1 cells resulted in decelerated differentiation. Endogenous miR-451a expression was upregulated during development in the hippocampus of wildtype mice. In situ hybridization revealed intensively stained single cells in the subgranular zone and the hilus of the dentate gyrus of wild type mice, while genetic ablation of miR-451a was observed to promote an imbalance between proliferation and neuronal differentiation in neurogenic brain regions, suggested by Ki67 and DCX staining. Taken together, these results provide strong support for a role of miR-451a in neuronal maturation processes in vitro and in vivo.

Different Preference of Degradome in Invasion versus Angiogenesis.

Proteases are required for a multitude of cellular processes including homeostatic tissue remodelling, invasion and angiogenesis. The physiological function of a cell or tissue is reflected by the set of proteases expressed, also termed degradome. The role of proteases in invasion and angiogenesis has been studied intensively, mostly in cancer. We aimed to compare the set of proteases required for physiological invasion versus physiological angiogenesis from cells deriving from the same organ, and to identify the proteases specific for each process. The human placenta comprises trophoblasts that invade the maternal uterus in a regulated, physiological manner, and it is the source of primary endothelial cells. We isolated the trophoblasts and endothelial cells and verified their invasive phenotype and angiogenic properties, respectively. We then performed gene expression analysis of the degradome, e.g. cysteine, metallo, serine, threonine and aspartic proteases, identified the differentially expressed proteases among the trophoblasts and endothelial cells, and clustered them hierarchically. The results revealed that the set of proteases in trophoblasts versus in endothelial cells overlaps, with a total of 69% in common. Nevertheless, 42% of the studied degradomes differed, with a fold change ≥2. For instance, metalloproteinases were predominantly expressed in trophoblasts, and 31% of the proteases were exclusively expressed in either trophoblasts or endothelial cells; this suggests particular roles for these proteases in either invasion or angiogenesis. Our data identify common and distinct proteases in cells capable of performing invasion and angiogenesis, and may provide basic information for the design of techniques to specifically investigate invasion or angiogenesis.