Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix.Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro, and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model.Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA.Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45(+) leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures.Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth

Interactome Networks of FOSL1 and FOSL2 in Human Th17 Cells

Dysregulated function of Th17 cells has implications in immunodeficiencies and autoimmune disorders. Th17 cell differentiation is orchestrated by a complex network of transcription factors, including several members of the activator protein (AP-1) family. Among the latter, FOSL1 and FOSL2 modulate the effector functions of Th17 cells. However, the molecular mechanisms underlying these effects are unclear, owing to the poorly characterized protein interaction networks of FOSL factors. Here, we establish the first interactomes of FOSL1 and FOSL2 in human Th17 cells, using affinity purification–mass spectrometry analysis. In addition to the known JUN proteins, we identified several novel binding partners of FOSL1 and FOSL2. Gene ontology analysis found a significant fraction of these interactors to be associated with RNA-binding activity, which suggests new mechanistic links. Intriguingly, 29 proteins were found to share interactions with FOSL1 and FOSL2, and these included key regulators of Th17 fate. We further validated the binding partners identified in this study by using parallel reaction monitoring targeted mass spectrometry and other methods. Our study provides key insights into the interaction-based signaling mechanisms of FOSL proteins that potentially govern Th17 cell differentiation and associated pathologies. </p

De novo Vessel Formation Through Cross-Talk of Blood-Derived Cells and Mesenchymal Stromal Cells in the Absence of Pre-existing Vascular Structures

Background: The generation of functional blood vessels remains a key challenge for regenerative medicine. Optimized in vitro culture set-ups mimicking the in vivo perivascular niche environment during tissue repair may provide information about the biological function and contribution of progenitor cells to postnatal vasculogenesis, thereby enhancing their therapeutic potential. Aim: We established a fibrin-based xeno-free human 3D in vitro vascular niche model to study the interaction of mesenchymal stromal cells (MSC) with peripheral blood mononuclear cells (PBMC) including circulating progenitor cells in the absence of endothelial cells (EC), and to investigate the contribution of this cross-talk to neo-vessel formation. Materials and Methods: Bone marrow-derived MSC were co-cultured with whole PBMC, enriched monocytes (Mo), enriched T cells, and Mo together with T cells, respectively, obtained from leukocyte reduction chambers generated during the process of single-donor platelet apheresis. Cells were embedded in 3D fibrin matrices, using exclusively human-derived culture components without external growth factors. Cytokine secretion was analyzed in supernatants of 3D cultures by cytokine array, vascular endothelial growth factor (VEGF) secretion was quantified by ELISA. Cellular and structural re-arrangements were characterized by immunofluorescence and confocal laser-scanning microscopy of topographically intact 3D fibrin gels. Results: 3D co-cultures of MSC with PBMC, and enriched Mo together with enriched T cells, respectively, generated, within 2 weeks, complex CD31C /CD34C vascular structures, surrounded by basement membrane collagen type-IVC cells and matrix, in association with increased VEGF secretion. PBMC contained CD31C CD34CCD45dimCD14 progenitor-type cells, and EC of neo-vessels were PBMC-derived. Vascular structures showed intraluminal CD45C cells that underwent apoptosis thereby creating a lumen. Cross-talk of MSC with enriched Mo provided a proangiogenic paracrine environment. MSC co-cultured with enriched T cells formed "cellin-cell" structures generated through internalization of T cells by CD31C CD45dim = cells. No vascular structures were detected in co-cultures of MSC with either Mo or T cells. Conclusion: Our xeno-free 3D in vitro vascular niche model demonstrates that a complex synergistic network of cellular, extracellular and paracrine cross-talk can contribute to de novo vascular development through self-organization via co-operation of immune cells with blood-derived progenitor cells and MSC, and thereby may open a new perspective for advanced vascular tissue engineering in regenerative medicine

Persistent coxsackievirus B1 infection triggers extensive changes in the transcriptome of human pancreatic ductal cells.

Enteroviruses, particularly the group B coxsackieviruses (CVBs), have been associated with the development of type 1 diabetes. Several CVB serotypes establish chronic infections in human cells in vivo and in vitro. However, the mechanisms leading to enterovirus persistency and, possibly, beta cell autoimmunity are not fully understood. We established a carrier-state-type persistent infection model in human pancreatic cell line PANC-1 using two distinct CVB1 strains and profiled the infection-induced changes in cellular transcriptome. In the current study, we observed clear changes in the gene expression of factors associated with the pancreatic microenvironment, the secretory pathway, and lysosomal biogenesis during persistent CVB1 infections. Moreover, we found that the antiviral response pathways were activated differently by the two CVB1 strains. Overall, our study reveals extensive transcriptional responses in persistently CVB1-infected pancreatic cells with strong opposite but also common changes between the two strains. </p

A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

Th17 cells are essential for protection against extracellular pathogens, but their aberrant activity can cause autoimmunity. Molecular mechanisms that dictate Th17 cell-differentiation have been extensively studied using mouse models. However, species-specific differences underscore the need to validate these findings in human. Here, we characterized the human-specific roles of three AP-1 transcription factors, FOSL1, FOSL2 and BATF, during early stages of Th17 differentiation. Our results demonstrate that FOSL1 and FOSL2 co-repress Th17 fate-specification, whereas BATF promotes the Th17 lineage. Strikingly, FOSL1 was found to play different roles in human and mouse. Genome-wide binding analysis indicated that FOSL1, FOSL2 and BATF share occupancy over regulatory regions of genes involved in Th17 lineage commitment. These AP-1 factors also share their protein interacting partners, which suggests mechanisms for their functional interplay. Our study further reveals that the genomic binding sites of FOSL1, FOSL2 and BATF harbour hundreds of autoimmune disease-linked SNPs. We show that many of these SNPs alter the ability of these transcription factors to bind DNA. Our findings thus provide critical insights into AP-1-mediated regulation of human Th17-fate and associated pathologies

Quantitative genome-scale metabolic modeling of human CD4+ T cell differentiation reveals subset-specific regulation of glycosphingolipid pathways

T cell activation, proliferation, and differentiation involve metabolic reprogramming resulting from the interplay of genes, proteins, and metabolites. Here, we aim to understand the metabolic pathways involved in the activation and functional differentiation of human CD4+ T cell subsets (T helper [Th]1, Th2, Th17, and induced regulatory T [iTreg] cells). Here, we combine genome-scale metabolic modeling, gene expression data, and targeted and non-targeted lipidomics experiments, together with in vitro gene knockdown experiments, and show that human CD4+ T cells undergo specific metabolic changes during activation and functional differentiation. In addition, we confirm the importance of ceramide and glycosphingolipid biosynthesis pathways in Th17 differentiation and effector functions. Through in vitro gene knockdown experiments, we substantiate the requirement of serine palmitoyltransferase (SPT), a de novo sphingolipid pathway in the expression of proinflammatory cytokines (interleukin [IL]-17A and IL17F) by Th17 cells. Our findings provide a comprehensive resource for selective manipulation of CD4+ T cells under disease conditions characterized by an imbalance of Th17/natural Treg (nTreg) cells.</p

M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen <i>Chlamydia pneumoniae</i>

<div><p>Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen <i>Chlamydia pneumoniae</i> with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of <i>C</i>. <i>pneumoniae</i>, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 <i>vs</i>. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable <i>C</i>. <i>pneumoniae</i> was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for <i>C</i>. <i>pneumoniae</i>. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence for the ability of <i>C</i>. <i>pneumoniae</i> to evade cellular defense and to persist in human macrophages.</p></div

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix.Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro, and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model.Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA.Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45+ leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures.Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth

Macrophage Polarization.

<p><b>(A)</b> CD14 positive human blood monocytes were treated either with 25 ng/mL GM-CSF or 50 ng/mL M-CSF for 7 days to yield M1-like or M2-like macrophages. M1-like macrophages were activated with GM-CSF, LPS and 50 ng/mL IFN-γ for an additional 48 h to yield M1 macrophages, while M2-like macrophages were treated with M-CSF and IL-4 to yield M2. <b>(B)</b> To examine the impact of macrophage polarization on survival and proliferation of <i>C</i>. <i>pneumoniae</i>, M1 and M2 macrophages were infected and cultured with <i>C</i>. <i>pneumoniae</i> for 24 h. <b>(C)</b> M1-like macrophages were infected with <i>C</i>. <i>pneumoniae</i> and cultured for 48 h in the presence of GM-CSF and IFN-γ, while infected M2-like macrophages were cultured with M-CSF and IL-4. Uninfected macrophages served as control.</p