Search

Gram+ infections are worldwide life-threatening diseases in which the pathological role of type I interferon (IFN) has been highlighted. Plasmacytoid predendritic cells (pDCs) produce high amounts of type I IFN following viral sensing. Despite studies suggesting that pDCs respond to bacteria, the mechanisms underlying bacterial sensing in pDCs are unknown. We show here that human primary pDCs express toll-like receptor 1 (TLR1) and 2 (TLR2) and respond to bacterial lipoproteins. We demonstrated that pDCs differentially respond to gram+ bacteria through the TLR1/2 pathway. Notably, up-regulation of costimulatory molecules and pro-inflammatory cytokines was TLR1 dependent, whereas type I IFN secretion was TLR2 dependent. Mechanistically, we demonstrated that these differences relied on diverse signaling pathways activated by TLR1/2. MAPK and NF-κB pathways were engaged by TLR1, whereas the Phosphoinositide 3-kinase (PI3K) pathway was activated by TLR2. This dichotomy was reflected in a different role of TLR2 and TLR1 in pDC priming of naïve cluster of differentiation 4+ (CD4+) T cells, and T helper (Th) cell differentiation. This work provides the rationale to explore and target pDCs in bacterial infection., It is unclear whether and how plasmacytoid pre-dendritic cells (pDCs) respond to bacteria; this study shows that human pDCs sense Gram-positive bacteria through the Toll-like receptors TLR1 and TLR2, and that these receptors have distinct roles in the activation of pDCs.

T follicular helper cells (Tfh) are implicated in various pathological conditions, but how they differentiate in Th2-skewed environments is unknown. Pattarini et al. delineate a pathway for human Tfh differentiation induced by TSLP through OX40L, relevant to atopic dermatitis., T follicular helper cells (Tfh) are important regulators of humoral responses. Human Tfh polarization pathways have been thus far associated with Th1 and Th17 polarization pathways. How human Tfh cells differentiate in Th2-skewed environments is unknown. We show that thymic stromal lymphopoietin (TSLP)–activated dendritic cells (DCs) promote human Tfh differentiation from naive CD4 T cells. We identified a novel population, distinct from Th2 cells, expressing IL-21 and TNF, suggestive of inflammatory cells. TSLP-induced T cells expressed CXCR5, CXCL13, ICOS, PD1, BCL6, BTLA, and SAP, among other Tfh markers. Functionally, TSLP-DC–polarized T cells induced IgE secretion by memory B cells, and this depended on IL-4Rα. TSLP-activated DCs stimulated circulating memory Tfh cells to produce IL-21 and CXCL13. Mechanistically, TSLP-induced Tfh differentiation depended on OX40-ligand, but not on ICOS-ligand. Our results delineate a pathway of human Tfh differentiation in Th2 environments.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that primes dendritic cells for Th2 induction. It has been implicated in different types of allergic diseases. Recent work suggested that TSLP could play an important role in the tumor microenvironment and influence tumor progression, in particular in breast cancer. In this study we systematically assessed the production of TSLP at the mRNA and protein levels in several human breast cancer cell lines, large-scale public transcriptomics data sets, and primary human breast tumors. We found that TSLP production was marginal, and concerned less than 10% of the tumors, with very low mRNA and protein levels. In most cases TSLP was undetectable and found to be expressed at lower levels in breast cancer as compared to normal breast tissue. Last, we could not detect any functional TSLP receptor (TSLPR) expression neither on hematopoietic cells nor on stromal cells within the primary tumor microenvironment. We conclude that TSLP-TSLPR pathway activity is not significantly detected within human breast cancer. Taken together, these observations do not support TSLP targeting in breast cancer.

In regenerative medicine, new approaches are required for the creation of tissue substitutes, and the interplay between different research areas, such as tissue engineering, microsurgery and gene therapy, is mandatory. In this article, we report a modification of a published model of tissue engineering, based on an arterio-venous loop enveloped in a cross-linked collagen–glycosaminoglycan template, which acts as an isolated chamber for angiogenesis and new tissue formation. In order to foster tissue formation within the chamber, which entails on the development of new vessels, we wondered whether we might combine tissue engineering with a gene therapy approach. Based on the well-described tropism of adeno-associated viral vectors for post-mitotic tissues, a muscular flap was harvested from the pectineus muscle, inserted into the chamber and transduced by either AAV vector encoding human VEGF165 or AAV vector expressing the reporter gene β-galactosidase, as a control. Histological analysis of the specimens showed that muscle transduction by AAV vector encoding human VEGF165 resulted in enhanced tissue formation, with a significant increase in the number of arterioles within the chamber in comparison with the previously published model. Pectineus muscular flap, transduced by adeno-associated viral vectors, acted as a source of the proangiogenic factor vascular endothelial growth factor, thus inducing a consistent enhancement of vessel growth into the newly formed tissue within the chamber. In conclusion, our present findings combine three different research fields such as microsurgery, tissue engineering and gene therapy, suggesting and showing the feasibility of a mixed approach for regenerative medicine.

Background Thymic stromal lymphopoietin (TSLP) is a major proallergic cytokine that promotes T H 2 responses through dendritic cell (DC) activation. Whether it also plays a role in human autoimmune inflammation and associated pathways is not known. Objective In this study we investigated the potential role of several epithelium-derived factors, including TSLP, in inducing IL-23 production by human DCs. We further dissected the role of TSLP in patients with psoriasis, an IL-23–associated skin autoimmune disease. Methods The study was performed in human subjects using primary cells and tissue samples from patients with psoriasis and healthy donors. We analyzed the production of IL-23 in vitro by blood and skin DCs. We studied the function for TSLP and its interaction with other components of the inflammatory microenvironment in situ and ex vivo . Results We found that TSLP synergized with CD40 ligand to promote DC activation and pathogenic IL-23 production by primary blood and skin DCs. In situ TSLP was strongly expressed by keratinocytes of untreated psoriatic lesions but not in normal skin. Moreover, we could demonstrate that IL-4, an important component of the T H 2 inflammation seen in patients with atopic dermatitis, inhibited IL-23 production induced by TSLP and CD40 ligand in a signal transducer and activator of transcription 6–independent manner. Conclusion Our results identify TSLP as a novel player within the complex psoriasis cytokine network. Blocking TSLP in patients with psoriasis might contribute to decreasing DC activation and shutting down the production of pathogenic IL-23.

Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A) promote vessel maturation through the recruitment of a population of circulating monocytes expressing the neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a specific subset of CD11b+ Nrp1+ Gr1− resident monocytes, distinctively recruited by Sema3A. NEMs were found to produce several factors involved in vessel maturation, including PDGFb, TGF-β, thrombospondin-1, and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro. When directly injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness. Tumors treated with NEMs were smaller, better perfused and less hypoxic, and had a reduced level of activation of HIF-1α. We conclude that NEMs represent a novel, unique population of myeloid cells that, once inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth. Cancer Res; 72(24); 6371–81. ©2012 AACR.

Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF-A(165) and VEGF-B(167) on adeno-associated virus-mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor-1 (VEGFR-1) ligand VEGF-B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR-1, VEGFR-2, and neuropilin-1 and that, in particular, VEGFR-1 was specifically up-regulated in hypoxia and on exposure to oxidative stress. VEGF-B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR-1 activation by VEGF-B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of alphaMHC and repression of betaMHC and skeletal alpha-actin, and an increase in SERCA2a, RYR, PGC1alpha, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR-1 activation by VEGF-B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.

Experimental and clinical evidence indicate that bone marrow cells participate in the process of new blood vessel formation. However, the molecular mechanisms underlying their recruitment and their exact role are still elusive. Here, we show that bone marrow cells are recruited to the sites of neoangiogenesis through the neuropilin-1 (NP-1) receptor and that they are essential for the maturation of the activated endothelium and the formation of arteries in mice. By exploiting adeno-associated virus vector-mediated, long-term in vivo gene expression, we show that the 165-aa isoform of VEGF, which both activates the endothelium and recruits NP-1+ myeloid cells, is a powerful arteriogenic agent. In contrast, neither the shortest VEGF121 isoform, which does not bind NP-1 and thus does not recruit bone marrow cells, nor semaphorin 3A, which attracts cells but inhibits endothelial activation, are capable of sustaining arterial formation. Bone marrow myeloid cells are not arteriogenic per se nor are they directly incorporated in the newly formed vasculature, but they contribute to arterial formation through a paracrine effect ensuing in the activation and proliferation of tissue-resident smooth muscle cells.

Skeletal myogenesis is a multistep process starting with progenitor cell proliferation, followed by their exit from the cell cycle, differentiation, alignment, and fusion to form multinucleated myotubes, typical of the differentiated muscle tissue. While the molecular players involved in early myogenesis have been extensively characterized, information about the later steps of the process is scanty. Here, we describe a novel myogenic cell line (MYOP7), composed of highly proliferating Sca-1+ muscle precursor cells, which can be induced to terminally differentiate into spontaneously contracting multinucleated myotubes. By performing high-density microarray analysis on these cells, we identified a series of genes, differentially expressed in proliferating vs differentiating conditions, which are candidates to play a major role in the later phase of myogenesis. In addition, we confirmed that the late stages of muscle differentiation are characterized by a marked upregulation of the cellular receptors for the vascular endothelial growth factor.

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.