miR-23a contributes to T cellular redox metabolism in juvenile idiopathic oligoarthritis.

OBJECTIVE: JIA is a chronic inflammatory disease of unknown origin. The regulation of inflammatory processes involves multiple cellular steps including mRNA transcription and translation. Different miRNAs control these processes tightly. We aimed to determine the roles of specific miRNAs within JIA pathogenesis. METHODS: We performed a global miRNA expression analysis in parallel in cells from the arthritic joint and peripheral blood of oligoarticular JIA patients and healthy controls. Quantitative RT-PCR analysis was used to verify expression of miRNA in T cells. Ex vivo experiments and flow cytometric analyses were used to analyse proliferation and redox metabolism. RESULTS: Global miRNA expression analysis demonstrated a different composition of miRNA expression at the site of inflammation compared with peripheral blood. Bioinformatic analysis of predicted miRNA target genes suggest a huge overrepresentation of genes involved in metabolic and oxidative stress pathways in the inflamed joint. Despite enhanced reactive oxygen species (ROS) levels within the local inflammatory milieu, JIA T cells are hyperproliferative and reveal an overexpression of miR-23a, which is an inhibitor of Peptidyl-prolyl isomerase F (PPIF), the regulator of mitochondrial ROS escape. Mitochondrial ROS escape is diminished in JIA T cells, resulting in their prolonged survival. CONCLUSION: Our data suggest that miRNA-dependent mitochondrial ROS shuttling might be a mechanism that contributes to T cell regulation in JIA at the site of inflammation.

Transcriptome analysis defines myocardium gene signatures in children with ToF and ASD and reveals disease-specific molecular reprogramming in response to surgery with cardiopulmonary bypass.

BACKGROUND: Tetralogy of Fallot (ToF) and Atrial Septal Defects (ASD) are the most common types of congenital heart diseases and a major cause of childhood morbidity and mortality. Cardiopulmonary bypass (CPB) is used during corrective cardiac surgery to support circulation and heart stabilization. However, this procedure triggers systemic inflammatory and stress response and consequent increased risk of postoperative complications. The aim of this study was to define the molecular bases of ToF and ASD pathogenesis and response to CPB and identify new potential biomarkers. METHODS: Comparative transcriptome analysis of right atrium specimens collected from 10 ToF and 10 ASD patients was conducted before (Pre-CPB) and after (Post-CPB) corrective surgery. Total RNA isolated from each sample was individually hybridized on Affymetrix HG-U133 Plus Array Strips containing 38,500 unique human genes. Differences in the gene expression profiles and functional enrichment/network analyses were assessed using bioinformatic tools. qRT-PCR analysis was used to validate gene modulation. RESULTS: Pre-CPB samples showed significant differential expression of a total of 72 genes, 28 of which were overexpressed in ToF and 44 in ASD. According to Gene Ontology annotation, the mostly enriched biological processes were represented by matrix organization and cell adhesion in ToF and by muscle development and contractility in ASD specimens. GSEA highlighted the specific enrichment of hypoxia gene sets in ToF samples, pointing to a role for hypoxia in disease pathogenesis. The post-CPB myocardium exhibited significant alterations in the expression profile of genes related to transcription regulation, growth/apoptosis, inflammation, adhesion/matrix organization, and oxidative stress. Among them, only 70 were common to the two disease groups, whereas 110 and 24 were unique in ToF and ASD, respectively. Multiple functional interactions among differentially expressed gene products were predicted by network analysis. Interestingly, gene expression changes in ASD samples followed a consensus hypoxia profile. CONCLUSION: Our results provide a comprehensive view of gene reprogramming in right atrium tissues of ToF and ASD patients before and after CPB, defining specific molecular pathways underlying disease pathophysiology and myocardium response to CPB. These findings have potential translational value because they identify new candidate prognostic markers and targets for tailored cardioprotective post-surgical therapies.

A Proteomic Analysis of GSD-1a in Mouse Livers: Evidence for Metabolic Reprogramming, Inflammation, and Macrophage Polarization.

Glycogen storage disease type 1a (GSD-1a) is a rare genetic disease caused by mutations in the catalytic subunit of the enzyme glucose-6-phosphatase-alpha (G6Pase-α). The majority of patients develop long-term complications including renal failure and hepatocellular adenoma/carcinoma. The purpose of this study was to ascertain the proteomic changes in the liver of LS- G6pc-/- mice, a murine model of GSD-1a, in comparison with wild type mice to identify potential biomarkers of the pathophysiology of the affected liver. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze liver lysates from a total of 20 LS- G6pc-/- and 18 wild type (WT) mice. We compared the proteomic expression profile of LS- G6pc-/- and WT mice. We identified 4138 significantly expressed proteins, 1243 of which were differentially represented. Network and pathway analyses indicate that LS- G6pc-/- livers display an age-dependent modulation of the expression of proteins involved in specific biological processes associated with increased progression of liver disease. Moreover, we found upregulation of proteins involved in the process of tissue inflammation and macrophage polarization toward the M2 phenotype in LS- G6pc-/- mice with adenomas. Our results identify a metabolic reprogramming of glucose-6-P and a pathologic environment in the liver compatible with tumor development and progression.

Development and characterization of an inducible mouse model for glycogen storage disease type Ib.

BACKGROUND AND AIMS: Glycogen storage disease type Ib (GSD1b) is a rare metabolic and immune disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT) and characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenomas. Despite maximal therapy, based on a strict diet and on granulocyte colony-stimulating factor treatment, long-term severe complications still develop. Understanding the pathophysiology of GSD1b is a prerequisite to develop new therapeutic strategies and depends on the availability of animal models. The G6PT-KO mouse mimics the human disease but is very fragile and rarely survives weaning. We generated a conditional G6PT-deficient mouse as an alternative model for studying the long-term pathophysiology of the disease. We utilized this conditional mouse to develop an inducible G6PT-KO model to allow temporally regulated G6PT deletion by the administration of tamoxifen (TM). METHODS: We generated a conditional G6PT-deficient mouse utilizing the CRElox strategy. Histology, histochemistry, and phenotype analyses were performed at different times after TM-induced G6PT inactivation. Neutrophils and monocytes were isolated and analyzed for functional activity with standard techniques. RESULTS: The G6PT-inducible KO mice display the expected disturbances of G6P metabolism and myeloid dysfunctions of the human disorder, even though with a milder intensity. CONCLUSIONS: TM-induced inactivation of G6PT in these mice leads to a phenotype which mimics that of human GSD1b patients. The conditional mice we have generated represent an excellent tool to study the tissue-specific role of the G6PT gene and the mechanism of long-term complications in GSD1b.

Chronic hypoxia reprograms human immature dendritic cells by inducing a proinflammatory phenotype and TREM-1 expression.

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.

Hypoxia downregulates the expression of activating receptors involved in NK-cell-mediated target cell killing without affecting ADCC.

In certain infection sites or tumor tissues, the disruption of homeostasis can give rise to a hypoxic microenvironment, which, in turn, can alter the function of different immune cell types and favor the progression of the disease. Natural killer (NK) cells are directly involved in the elimination of virus-infected or transformed cells, however it is unknown whether their function is affected by hypoxia or not. In this study, we show that NK cells adapt to a hypoxic environment by upregulating the hypoxia-inducible factor 1α. However, NK cells lose their ability to upregulate the surface expression of the major activating NK-cell receptors (NKp46, NKp30, NKp44, and NKG2D) in response to IL-2 (or other activating cytokines, including IL-15, IL-12, and IL-21). These altered phenotypic features correlate with reduced responses to triggering signals resulting in impaired capability of killing infected or tumor target cells. Remarkably, hypoxia does not significantly alter the surface density and the triggering function of the Fc-γ receptor CD16, thus allowing NK cells to maintain their capability of killing target cells via antibody-dependent cellular cytotoxicity. This finding offers an important clue for exploitation of NK cell in antibody-based immunotherapy of cancer.

Hypoxia modulates the gene expression profile of immunoregulatory receptors in human mature dendritic cells: identification of TREM-1 as a novel hypoxic marker in vitro and in vivo.

Dendritic cells (DCs) are a heterogeneous group of professional antigen-presenting cells functioning as sentinels of the immune system and playing a key role in the initiation and amplification of innate and adaptive immune responses. DC development and functions are acquired during a complex differentiation and maturation process influenced by several factors present in the local milieu. A common feature at pathologic sites is represented by hypoxia, a condition of low pO(2), which creates a unique microenvironment affecting cell phenotype and behavior. Little is known about the impact of hypoxia on the generation of mature DCs (mDCs). In this study, we identified by gene expression profiling a significant cluster of genes coding for immune-related cell surface receptors strongly up-regulated by hypoxia in monocyte-derived mDCs and characterized one of such receptors, TREM-1, as a new hypoxia-inducible gene in mDCs. TREM-1 associated with DAP12 in hypoxic mDCs, and its engagement elicited DAP12-linked signaling, resulting in ERK-1, Akt, and IκBα phosphorylation and proinflammatory cytokine and chemokine secretion. Finally, we provided the first evidence that TREM-1 is expressed on mDCs infiltrating the inflamed hypoxic joints of children affected by juvenile idiopathic arthritis, representing a new in vivo marker of hypoxic mDCs endowed with proinflammatory properties.

MicroRNAs in Juvenile Idiopathic Arthritis: State of the Art and Future Perspectives.

Juvenile Idiopathic Arthritis (JIA) represents the most common chronic pediatric arthritis in Western countries and a leading cause of disability in children. Despite recent clinical achievements, patient management is still hindered by a lack of diagnostic/prognostic biomarkers and targeted treatment protocols. MicroRNAs (miRNAs) are short non-coding RNAs playing a key role in gene regulation, and their involvement in many pathologies has been widely reported in the literature. In recent decades, miRNA's contribution to the regulation of the immune system and the pathogenesis of autoimmune diseases has been demonstrated. Furthermore, miRNAs isolated from patients' biological samples are currently under investigation for their potential as novel biomarkers. This review aims to provide an overview of the state of the art on miRNA investigation in JIA. The literature addressing the expression of miRNAs in different types of biological samples isolated from JIA patients was reviewed, focusing in particular on their potential application as diagnostic/prognostic biomarkers. The role of miRNAs in the regulation of immune responses in affected joints will also be discussed along with their potential utility as markers of patients' responses to therapeutic approaches. This information will be of value to investigators in the field of pediatric rheumatology, encouraging further research to increase our knowledge of miRNAs' potential for future clinical applications in JIA.

Plasma-Derived Exosome Proteins as Novel Diagnostic and Prognostic Biomarkers in Neuroblastoma Patients.

Neuroblastoma (NB) is the most common extracranial solid tumor during infancy, causing up to 10% of mortality in children; thus, identifying novel early and accurate diagnostic and prognostic biomarkers is mandatory. NB-derived exosomes carry proteins (Exo-prots) reflecting the status of the tumor cell of origin. The purpose of this study was to characterize, for the first time, the Exo-prots specifically expressed in NB patients associated with tumor phenotype and disease stage. We isolated exosomes from plasma specimens of 24 HR-NB patients and 24 low-risk (LR-NB) patients at diagnosis and of 24 age-matched healthy controls (CTRL). Exo-prot expression was measured by liquid chromatography-mass spectrometry. The data are available via ProteomeXchange (PXD042422). The NB patients had a different Exo-prot expression profile compared to the CTRL. The deregulated Exo-prots in the NB specimens acted mainly in the tumor-associated pathways. The HR-NB patients showed a different Exo-prot expression profile compared to the LR-NB patients, with the modulation of proteins involved in cell migration, proliferation and metastasis. NCAM, NCL, LUM and VASP demonstrated a diagnostic value in discriminating the NB patients from the CTRL; meanwhile, MYH9, FN1, CALR, AKAP12 and LTBP1 were able to differentiate between the HR-NB and LR-NB patients with high accuracy. Therefore, Exo-prots contribute to NB tumor development and to the aggressive metastatic NB phenotype.

Proteomic profiling of extracellular vesicles in synovial fluid and plasma from Oligoarticular Juvenile Idiopathic Arthritis patients reveals novel immunopathogenic biomarkers.

Introduction: New early low-invasive biomarkers are demanded for the management of Oligoarticular Juvenile Idiopathic Arthritis (OJIA), the most common chronic pediatric rheumatic disease in Western countries and a leading cause of disability. A deeper understanding of the molecular basis of OJIA pathophysiology is essential for identifying new biomarkers for earlier disease diagnosis and patient stratification and to guide targeted therapeutic intervention. Proteomic profiling of extracellular vesicles (EVs) released in biological fluids has recently emerged as a minimally invasive approach to elucidate adult arthritis pathogenic mechanisms and identify new biomarkers. However, EV-prot expression and potential as biomarkers in OJIA have not been explored. This study represents the first detailed longitudinal characterization of the EV-proteome in OJIA patients. Methods: Fourty-five OJIA patients were recruited at disease onset and followed up for 24 months, and protein expression profiling was carried out by liquid chromatography-tandem mass spectrometry in EVs isolated from plasma (PL) and synovial fluid (SF) samples. Results: We first compared the EV-proteome of SF vs paired PL and identified a panel of EV-prots whose expression was significantly deregulated in SF. Interaction network and GO enrichment analyses performed on deregulated EV-prots through STRING database and ShinyGO webserver revealed enrichment in processes related to cartilage/bone metabolism and inflammation, suggesting their role in OJIA pathogenesis and potential value as early molecular indicators of OJIA development. Comparative analysis of the EV-proteome in PL and SF from OJIA patients vs PL from age/gender-matched control children was then carried out. We detected altered expression of a panel of EV-prots able to differentiate new-onset OJIA patients from control children, potentially representing a disease-associated signature measurable at both the systemic and local levels with diagnostic potential. Deregulated EV-prots were significantly associated with biological processes related to innate immunity, antigen processing and presentation, and cytoskeleton organization. Finally, we ran WGCNA on the SF- and PL-derived EV-prot datasets and identified a few EV-prot modules associated with different clinical parameters stratifying OJIA patients in distinct subgroups. Discussion: These data provide novel mechanistic insights into OJIA pathophysiology and an important contribution in the search of new candidate molecular biomarkers for the disease.

The SGLT2-inhibitor dapagliflozin improves neutropenia and neutrophil dysfunction in a mouse model of the inherited metabolic disorder GSDIb.

Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells.

Targeting Mononuclear Phagocyte Receptors in Cancer Immunotherapy: New Perspectives of the Triggering Receptor Expressed on Myeloid Cells (TREM-1).

Inflammatory cells are major players in the onset of cancer. The degree of inflammation and type of inflammatory cells in the tumor microenvironment (TME) are responsible for tilting the balance between tumor progression and regression. Cancer-related inflammation has also been shown to influence the efficacy of conventional therapy. Mononuclear phagocytes (MPs) represent a major component of the inflammatory circuit that promotes tumor progression. Despite their potential to activate immunosurveillance and exert anti-tumor responses, MPs are subverted by the tumor to support its growth, immune evasion, and spread. MP responses in the TME are dictated by a network of stimuli integrated through the cross-talk between activatory and inhibitory receptors. Alterations in receptor expression/signaling can create excessive inflammation and, when chronic, promote tumorigenesis. Research advances have led to the development of new therapeutic strategies aimed at receptor targeting to induce a tumor-infiltrating MP switch from a cancer-supportive toward an anti-tumor phenotype, demonstrating efficacy in different human cancers. This review provides an overview of the role of MP receptors in inflammation-mediated carcinogenesis and discusses the most recent updates regarding their targeting for immunotherapeutic purposes. We focus in particular on the TREM-1 receptor, a major amplifier of MP inflammatory responses, highlighting its relevance in the development and progression of several types of inflammation-associated malignancies and the promises of its inhibition for cancer immunotherapy.

The passage from bone marrow niche to bloodstream triggers the metabolic impairment in Fanconi Anemia mononuclear cells.

Fanconi Anemia (FA) is a disease characterized by bone marrow (BM) failure and aplastic anemia. In addition to a defective DNA repair system, other mechanisms are involved in its pathogenesis, such as defective mitochondrial metabolism, accumulation of lipids, and increment of oxidative stress production. To better understand the role of these metabolic alterations in the process of HSC maturation in FA, we evaluated several biochemical and cellular parameters on mononuclear cells isolated from the bone marrow of FA patients or healthy donors. To mimic the cellular residence in the BM niche or their exit from the BM niche to the bloodstream, cells have been grown in hypoxic or normoxic conditions, respectively. The data show that, in normoxic conditions, a switch from anaerobic to aerobic metabolism occurs both in healthy and in pathological samples. However, in FA cells this change is associated with altered oxidative phosphorylation, the increment of oxidative stress production, no activation of the endogenous antioxidant defenses and arrest in the G2M phase of the cell cycle. By contrast, FA cells grown in hypoxic conditions do not show cell cycle and metabolic alterations in comparison to the healthy control, maintaining both an anaerobic flux. The data reported herein suggests that the passage from the BM niche to the bloodstream represents a crucial point in the FA pathogenesis associated with mitochondrial dysfunction.

Exosomal microRNAs from Longitudinal Liquid Biopsies for the Prediction of Response to Induction Chemotherapy in High-Risk Neuroblastoma Patients: A Proof of Concept SIOPEN Study.

Despite intensive treatment, 50% of children with high-risk neuroblastoma (HR-NB) succumb to their disease. Progression through current trials evaluating the efficacy of new treatments for children with HR disease usually depends on an inadequate response to induction chemotherapy, assessed using imaging modalities. In this study, we sought to identify circulating biomarkers that might be detected in a simple blood sample to predict patient response to induction chemotherapy. Since exosomes released by tumor cells can drive tumor growth and chemoresistance, we tested the hypothesis that exosomal microRNA (exo-miRNAs) in blood might predict response to induction chemotherapy. The exo-miRNAs expression profile in plasma samples collected from children treated in HR-NBL-1/SIOPEN before and after induction chemotherapy was compared to identify a three exo-miRs signature that could discriminate between poor and good responders. Exo-miRNAs expression also provided a chemoresistance index predicting the good or poor prognosis of HR-NB patients.

Hypoxia Modifies the Transcriptome of Human NK Cells, Modulates Their Immunoregulatory Profile, and Influences NK Cell Subset Migration.

Hypoxia, which characterizes most tumor tissues, can alter the function of different immune cell types, favoring tumor escape mechanisms. In this study, we show that hypoxia profoundly acts on NK cells by influencing their transcriptome, affecting their immunoregulatory functions, and changing the chemotactic responses of different NK cell subsets. Exposure of human peripheral blood NK cells to hypoxia for 16 or 96 h caused significant changes in the expression of 729 or 1,100 genes, respectively. Gene Set Enrichment Analysis demonstrated that these changes followed a consensus hypoxia transcriptional profile. As assessed by Gene Ontology annotation, hypoxia-targeted genes were implicated in several biological processes: metabolism, cell cycle, differentiation, apoptosis, cell stress, and cytoskeleton organization. The hypoxic transcriptome also showed changes in genes with immunological relevance including those coding for proinflammatory cytokines, chemokines, and chemokine-receptors. Quantitative RT-PCR analysis confirmed the modulation of several immune-related genes, prompting further immunophenotypic and functional studies. Multiplex ELISA demonstrated that hypoxia could variably reduce NK cell ability to release IFNγ, TNFα, GM-CSF, CCL3, and CCL5 following PMA+Ionomycin or IL15+IL18 stimulation, while it poorly affected the response to IL12+IL18. Cytofluorimetric analysis showed that hypoxia could influence NK chemokine receptor pattern by sustaining the expression of CCR7 and CXCR4. Remarkably, this effect occurred selectively (CCR7) or preferentially (CXCR4) on CD56bright NK cells, which indeed showed higher chemotaxis to CCL19, CCL21, or CXCL12. Collectively, our data suggest that the hypoxic environment may profoundly influence the nature of the NK cell infiltrate and its effects on immune-mediated responses within tumor tissues.

Regulation of Human Macrophage M1-M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1.

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.

Therapeutic Potential of Targeting TREM-1 in Inflammatory Diseases and Cancer.

The triggering receptor expressed on myeloid cells (TREM)-1 is a member of the Ig-like immunoregulatory receptor family and a major amplifier of innate immune responses. TREM- 1 has been implicated in the development and perpetuation of a number of inflammatory disorders, and soluble TREM-1 levels are a clinically valuable diagnostic and prognostic biomarker in patients with sepsis and other types of acute and chronic inflammation- associated diseases, easily detectable in biological fluids. High TREM-1 expression in macrophages infiltrating human tumors and increased concentrations of soluble TREM-1 also correlate with aggressive tumor behavior and recurrence and are a relevant independent predictor of poor patient survival. Pharmacological inhibition of TREM-1 has proven effective in preclinical mouse models of infectious and non-infectious inflammatory disorders and malignancies, conferring survival advantages and protecting from organ damage or tumor growth by attenuating inflammatory responses. This review aims at providing a comprehensive overview of the state of the art on TREM-1 research. We review the literature addressing TREM-1 role in the amplification of myeloid cell inflammatory responses at pathologic sites and its relevance in the development, severity, and progression of inflammatory diseases and cancer. Furthermore, we discuss recent advances in the pharmacological use of TREM-1 inhibitors in mouse preclinical models, emphasizing their potential in new strategies for the treatment of acute and chronic inflammatory conditions and for therapeutic intervention in cancer. This information will be of value to investigators in the field of pharmacology, drawing attention to novel therapeutic opportunities to complement current treatment approaches.

Regulation of Langerhans cell functions in a hypoxic environment.

UNLABELLED: Langerhans cells (LCs) are a specialized dendritic cell subset that resides in the epidermis and mucosal epithelia and is critical for the orchestration of skin immunity. Recent evidence suggest that LCs are involved in aberrant wound healing and in the development of hypertrophic scars and chronic wounds, which are characterized by a hypoxic environment. Understanding LCs biology under hypoxia may, thus, lead to the identification of novel pathogenetic mechanisms of wound repair disorders and open new therapeutic opportunities to improve wound healing. In this study, we characterize a previously unrecognized role for hypoxia in significantly affecting the phenotype and functional properties of human monocyte-derived LCs, impairing their ability to stimulate naive T cell responses, and identify the triggering receptor expressed on myeloid (TREM)-1, a member of the Ig immunoregulatory receptor family, as a new hypoxia-inducible gene in LCs and an activator of their proinflammatory and Th1-polarizing functions in a hypoxic environment. Furthermore, we provide the first evidence of TREM-1 expression in vivo in LCs infiltrating hypoxic areas of active hypertrophic scars and decubitous ulcers, pointing to a potential pathogenic role of this molecule in wound repair disorders. KEY MESSAGES: Hypoxia modulates surface molecule expression and cytokine profile in Langerhans cells. Hypoxia impairs human Langerhans cell stimulatory activity on naive T cells. Hypoxia selectively induces TREM-1 expression in human Langerhans cells. TREM-1 engagement stimulates Langerhans cell inflammatory and Th1-polarizing activity. TREM-1 is expressed in vivo in Langerhans cells infiltrating hypoxic skin lesions.

Analysis of the Expression and Single-Nucleotide Variant Frequencies of the Butyrophilin-like 2 Gene in Patients With Uveal Melanoma.

IMPORTANCE: Chromosome 6p amplification is associated with more benign behavior for uveal melanomas (UMs) with an otherwise high risk of metastasis conferred by chromosome 3 monosomy. Chromosome 6p contains several members of the B7 family of immune regulator genes, including butyrophilin-like 2 (BTNL2; OMIM, 606000), which is associated with prostate cancer risk and autoimmune diseases. OBJECTIVE: To investigate the expression and variant allele frequencies of BTNL2, a candidate gene for chromosome 6 amplification, in patients with UM. DESIGN, SETTING, AND PARTICIPANTS: In this case-control study, we analyzed the expression of BTNL2 in UM cell lines and human macrophages in patients with UM. Variants of BTNL2 were analyzed using probes for polymerase chain reaction and high-resolution melting. The association of missense variants rs28362679 and rs41441651 with tumor risk was analyzed in 209 patients with UM and 116 matched control patients as well as 12 UM and 64 other tumor cell lines. Genes that were differentially expressed in M1- and M2-polarized macrophages were identified by microarray analysis of 111 patients with UM, and the association of the expression of these genes with disease-free survival was analyzed by Cox regression analysis. Data were collected from September 2013 to November 2015. MAIN OUTCOMES AND MEASURES: Butyrophilin-like 2 single-nucleotide variants were associated with UM risk; M1 and M2 macrophage-specific gene expression was associated with disease-free survival. RESULTS: We genotyped a total of 325 patients. Of the 209 patients with UM, 124 (59.3%) were male, 114 (54.5%) were Italian, and 95 (45.5%) were German; the mean (range) age was 65 (27-94) years. Of the 116 Italian control patients, 67 (57.8%) were female, and the mean (range) age was 39 (21-88) years. Butyrophilin-like 2 is expressed in patients with UM and macrophages. The frequency of the rs28362679 variant was higher in patients with UM (16 of 209 [7.7%]; 95% CI, 4.7-12.2) than frequencies from European Variation Archive and Exome Aggregation Consortium data (2134 of 118 564 [1.8%]; 95% CI, 1.7-1.9) and Exome Sequencing Project data (100 of 4540 [2.2%]; 95% CI, 1.8-2.7) but were not higher compared with Italian control patients (10 of 116 [8.6%]; 95% CI, 4.6-15.4). The rs41441651 variant was present in 5 patients with UM (2.4%; 95% CI, 0.9-5.7), 2 Italian control patients (1.7%; 95% CI, 0.1-6.5), 2846 patients from European Variation Archive and Exome Aggregation Consortium data (2.4%; 95% CI, 2.3-2.5), and 23 patients from Exome Sequencing Project data (0.5%; 95% CI, 0.3-0.8). Human UM cells express M1 and M2 macrophage-specific genes, whose expression is associated with disease-free survival. CONCLUSIONS AND RELEVANCE: Butyrophilin-like 2, expressed at various levels by UM cells and macrophages, might interfere with the immune control of the tumor. Butyrophilin-like 2 variants showed highly variable frequencies among ethnically related cohorts. There was no enrichment of BTNL2 variants in patients with UM compared with control patients.