Macrophages (Mf) are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in vivo in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1) and alternatively activated (anti-inflammatory M2) subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM)-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by oligoarticular juvenile idiopatic arthritis express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment.

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The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia.MethodsChorionic villous PDMSCs were isolated from severe preeclamptic (n = 12) and physiological control term (n = 12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (n = 48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent.ResultsCytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones.ConclusionsHerein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia.

Axl, a prototypic member of the transmembrane tyrosine kinase receptor family, is known to regulate innate immunity. In this study, we show that Axl expression is induced by IFN-α during human dendritic cell (DC) differentiation from monocytes (IFN/DC) and that constitutively Axl-negative, IL-4-differentiated DC (IL-4/DC) can be induced to up-regulate Axl by IFN-α. This effect is inhibited by TLR-dependent maturation stimuli such as LPS, poly(I:C), TLR7/8 ligand, and CD40L. LPS-induced Axl down-regulation on the surface of human IFN-α-treated DC correlates with an increased proteolytic cleavage of Axl and with elevated levels of its soluble form. GM6001 and TAPI-1, general inhibitors of MMP and ADAM family proteases, restored Axl expression on the DC surface and diminished Axl shedding. Furthermore, stimulation of Axl by its ligand, Gas6, induced chemotaxis of human DC and rescued them from growth factor deprivation-induced apoptosis. Our study provides the first evidence that Gas6/Axl-mediated signaling regulates human DC activities, and identifies Gas6/Axl as a new DC chemotaxis pathway. This encourages one to explore whether dysregulation of this novel pathway in human DC biology is involved in autoimmunity characterized by high levels of IFN-α.

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DCs are powerful antigen-presenting cells central in the orchestration of innate and acquired immunity. DC development, migration, and activities are intrinsically linked to the microenvironment. DCs migrate through pathologic tissues before reaching their final destination in the lymph nodes. Hypoxia, a condition of low partial oxygen pressure, is a common feature of many pathologic situations, capable of modifying DC phenotype and functional behavior. We studied human monocyte-derived immature DCs generated under chronic hypoxic conditions (H-iDCs). We demonstrate by gene expression profiling the upregulation of a cluster of genes coding for antigen-presentation, immunoregulatory, and pattern recognition receptors, suggesting a stimulatory role for hypoxia on iDC immunoregulatory functions. In particular, we show that H-iDCs express triggering receptor expressed on myeloid cells (TREM-1), a member of the Ig superfamily of immunoreceptors and an amplifier of inflammation. This effect is reversible because H-iDC reoxygenation results in TREM-1 down-modulation. TREM-1 engagement promotes upregulation of T-cell costimulatory molecules and homing chemokine receptors, typical of mature DCs, and increases the production of proinflammatory, Th1/Th17-priming cytokines/chemokines, resulting in increased T-cell responses. These results suggest that TREM-1 induction by the hypoxic microenvironment represents a mechanism of regulation of Th1-cell trafficking and activation by iDCs differentiated at pathologic sites. Keywords: Dendritic cells r Hypoxia r Immunoregulatory receptors r Innate immunity r Proinflammatory cytokines/chemokinesAdditional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Prof. Mirella Giovarelli e-mail: mirella.giovarelli@unito.it * These authors equally contributed to this work. * * These authors share senior authorship.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 950Daniele Pierobon et al. Eur. J. Immunol. 2013. 43: 949-966 Introduction Myeloid DCs are central in the orchestration of innate and acquired immune responses and in the maintenance of self-tolerance [1]. DC development involves three functionally and phenotypically distinct stages for which the terms "precursors," "immature," and "mature" are commonly used [2][3][4][5]. DCs precursors originate in the bone marrow, circulate via the bloodstream to reach target tissues, and take up residence at sites of potential pathogen entry, where they differentiate into immature DCs (iDCs) specialized for antigen capture [2,4,6]. Peripheral blood monocytes recruited from the circulation to inflammatory sites can also serve as iDC precursors [7,8]. iDC redistribution in the tissues is determined by the local microenvironment through the production of chemotactic mediators, activation of inflammatory chemokine receptors, and regulation of adhesion molecules [7,8]. Tissue injury, inflammation, and transformation cause dramatic changes of the microenvironment,...

Purpose: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 as a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP).Experimental Design: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast (n ¼ 28) and pancreatic (n ¼ 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 fusion proteins and used to activate autologous T cells. Activation was evaluated by IFN-g ELISPOT assay, perforin expression, and ability to halt HER2þ tumor growth in vivo.Results: Specific sustained proliferation and IFN-g production by CD4 and CD8 T cells from HER2-CP was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids. Instead, T cells from healthy donors (n ¼ 22) could be easily stimulated with autologous DCs transfected with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of HER2 þ tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10 and TGF-b1. Conclusions: These results provide the proof of concept that chimeric rat/human HER2 plasmids can be used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin Cancer Res; 20(11); 2910-21. Ó2014 AACR.

Current treatments for allergic asthma primarily ameliorate symptoms rather than inhibit disease progression. Regulating the excessive T helper type 2 (Th2) responses may prevent asthma exacerbation. In this study, we investigated the protective effects of Ad5-gsgAM, an adenovirus vector carrying two mycobacterial antigens Ag85A and Mtb32, against allergic asthma. Using an ovalbumin (OVA)-induced asthmatic mouse model, we found that Ad5-gsgAM elicited much more Th1-biased CD4 + T and CD8 + T cells than bacillus CalmetteGuérin (BCG). After OVA challenge, Ad5-gsgAM-immunized mice showed significantly lowered airway inflammation in comparison with mice immunized with or without BCG. Total serum immunoglobulin E and pulmonary inducible-nitric-oxide-synthase were efficiently reduced. The cytokine profiles in bronchial-alveolar-lavage-fluids (BALFs) were also modulated, as evidenced by the increased level of interferon-γ (IFN-γ) and the decreased level of interleukin (IL)-4, IL-5, and IL-13. Anti-inflammatory cytokine IL-10 was sharply increased, whereas pro-inflammatory cytokine IL-33 was significantly decreased. Importantly, exogenous IL-33 abrogated the protective effects of Ad5-gsgAM, revealing that the suppression of IL-33/ST2 axis substantially contributed to protection against allergic inflammation. Moreover, regulatory T cells were essential for regulating aberrant Th2 responses as well as IL-33/ST2 axis. These results suggested that modulating the IL-33/ST2 axis via adenovirus-vectored mycobacterial antigen vaccination may provide clinical benefits in allergic inflammatory airways disease. Key messages•Ad5-gsgAM elicits Th1 responses and suppresses Th2-mediated allergic asthma in mice.•Ad5-gsgAM inhibits IL-33/ST2 axis by reducing IL-33 secretion but not ILC2 recruiting.•Treg is essential for modulating Th2 responses and IL-33/ST2 axis by Ad5-gsgAM.