Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors.

Precise molecular characterization of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is hampered by their mixed composition of mature and immature cells and lack of specific markers. Here, we focus on mature CD66b+CD10+CD16+CD11b+ PMN-MDSCs (mPMN-MDSCs) from either cancer patients or healthy donors receiving G-CSF for stem cell mobilization (GDs). By RNA sequencing (RNA-seq) experiments, we report the identification of a distinct gene signature shared by the different mPMN-MDSC populations under investigation, also validated in mPMN-MDSCs from GDs and tumor-associated neutrophils (TANs) by single-cell RNA-seq (scRNA-seq) experiments. Analysis of such a gene signature uncovers a specific transcriptional program associated with mPMN-MDSC differentiation and allows us to identify that, in patients with either solid or hematologic tumors and in GDs, CD52, CD84, and prostaglandin E receptor 2 (PTGER2) represent potential mPMN-MDSC-associated markers. Altogether, our findings indicate that mature PMN-MDSCs distinctively undergo specific reprogramming during differentiation and lay the groundwork for selective immunomonitoring, and eventually targeting, of mature PMN-MDSCs.

Implementation of a combined bioinformatics and experimental approach to address lncRNA mechanism of action: The example of NRIR.

In this study, we demonstrate the benefit of applying combined strategies to analyze lncRNA action based on bioinformatics and experimental information. This strategy was developed to identify the molecular function of negative regulator of interferon response (NRIR), a type I interferon-stimulated gene (ISG), that we have previously demonstrated to be involved in the upregulation of a subset of ISGs in LPS-stimulated human monocytes. In this study, we provide experimental evidence that NRIR is localized in cellular nuclei, enriched on the chromatin fraction, and upregulates ISGs acting at the transcriptional level. In silico analysis of secondary structures identified distinct NRIR structural domains, comprising putative DNA- and protein-binding regions. In parallel, the presence of a putative DNA-binding domain in NRIR and the five putative NRIR-binding sites in the promoter of NRIR-target genes support the function of NRIR as a transcriptional regulator of its target genes. By use of integrated experimental/bioinformatics approaches, comprising database and literature mining together with in silico analysis of putative NRIR-binding proteins, we identified a list of eight transcription factors (TFs) shared by the majority of NRIR-target genes and simultaneously able to bind TF binding sites enriched in the NRIR-target gene promoters. Among these TFs, the predicted NRIR:STAT interactions were experimentally validated by RIP assay.

Characterization of Long Non-Coding RNAs in Systemic Sclerosis Monocytes: A Potential Role for PSMB8-AS1 in Altered Cytokine Secretion.

Systemic sclerosis (SSc) is a chronic autoimmune disease mainly affecting the connective tissue. In SSc patients, monocytes are increased in circulation, infiltrate affected tissues, and show a pro-inflammatory activation status, including the so-called interferon (IFN) signature. We previously demonstrated that the dysregulation of the IFN response in SSc monocytes is sustained by altered epigenetic factors as well as by upregulation of the long non-coding RNA (lncRNA) NRIR. Considering the enormously diverse molecular functions of lncRNAs in immune regulation, the present study investigated the genome-wide profile of lncRNAs in SSc monocytes, with the aim to further unravel their possible role in monocyte dysregulation and disease pathogenesis. Transcriptomic data from two independent cohorts of SSc patients identified 886 lncRNAs with an altered expression in SSc monocytes. Differentially expressed lncRNAs were correlated with neighboring protein coding genes implicated in the regulation of IFN responses and apoptotic signaling in SSc monocytes. In parallel, gene co-expression network analysis identified the lncRNA PSMB8-AS1 as a top-ranking hub gene in co-expression modules implicated in cell activation and response to viral and external stimuli. Functional characterization of PSMB8-AS1 in monocytes demonstrated that this lncRNA is involved in the secretion of IL-6 and TNFα, two pivotal pro-inflammatory cytokines altered in the circulation of SSc patients and associated with fibrosis and disease severity. Collectively, our data showed that lncRNAs are linked to monocyte dysregulation in SSc, and highlight their potential contribution to disease pathogenesis.

Impact of Sex on Circulating Leukocytes Composition in COPD Patients.

PURPOSE: Chronic obstructive pulmonary disease is characterized by chronic inflammatory response both at the lung site and at the systemic level. Abnormalities in circulating leukocytes have been reported to occur in COPD patients and have been often shown to correlate with the decline in lung function. COPD affects men and women at a virtually comparable rate, even though distinct sex specific symptoms, progression and therapeutic implications have been described. Nonetheless, these sex-associated differences have not been analyzed in terms of circulating leukocytes. To assess the impact of sex on the changes of circulating immune cells in COPD patients. PATIENTS AND METHODS: Blood samples were collected from 50 COPD patients (31 males, 19 females) and 63 age and sex-matched controls (35 males, 28 females) enrolled in this pilot study. Complete blood cell count and multi-parametric flow cytometry analysis were performed to characterize the leukocyte populations and subsets. RESULTS: Male COPD patients are distinguished from controls by a significant increase in white blood cell counts, neutrophil total and differential counts, and neutrophil-to-lymphocyte ratio. Conversely, a generalized leukocyte decrease discriminated female COPD patients from the related controls. The impact of sex is further remarked by a decrease in adaptive immune cell subpopulations in males as opposed to a consistent increase of innate immune cell types in females correlating with disease severity. CONCLUSION: These data indicate that the definition of specific changes of circulating leukocytes to be used as reliable biomarkers of the disease severity cannot be accomplished irrespectively of sex.

Identification of a miR-146b-Fas ligand axis in the development of neutropenia in T large granular lymphocyte leukemia.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4- phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

Fast and Accurate Quantitative Analysis of Cytokine Gene Expression in Human Neutrophils by Reverse Transcription Real-Time PCR.

Polymorphonuclear neutrophils, traditionally viewed as short-lived effector cells, are nowadays regarded as important components of effector and regulatory circuits in the innate and adaptive immune systems. Most of the physiological functions of neutrophils as crucial players in the host immune response, able not only to act in the early phases of acute inflammation but also to condition the progression of the inflammatory reaction and the subsequent initiation of the specific immune response, relies on their capacity to produce and release a number of proinflammatory and immunoregulatory cytokines. This fact has reevaluated the importance, the role, and the physiological and pathological significance of neutrophils in the pathogenesis of inflammatory, infectious, autoimmune, and neoplastic diseases and has identified neutrophils as an important potential target for selective pharmacological intervention to both promote and restrain inflammation. In this context, understanding the mechanisms of modulation of neutrophil-derived cytokines and chemokines represents a critical step toward a better understanding of how neutrophils may influence pathophysiological processes in vivo. Herein, we describe and discuss an updated version of the methods that we have developed to rapidly and precisely characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the reverse transcription quantitative real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool, in the neutrophil research area, to test many experimental conditions for gene expression analysis.

Neutrophil-derived miR-223 as local biomarker of bacterial peritonitis.

Infection remains a major cause of morbidity, mortality and technique failure in patients with end stage kidney failure who receive peritoneal dialysis (PD). Recent research suggests that the early inflammatory response at the site of infection carries diagnostically relevant information, suggesting that organ and pathogen-specific "immune fingerprints" may guide targeted treatment decisions and allow patient stratification and risk prediction at the point of care. Here, we recorded microRNA profiles in the PD effluent of patients presenting with symptoms of acute peritonitis and show that elevated peritoneal miR-223 and reduced miR-31 levels were useful predictors of bacterial infection. Cell culture experiments indicated that miR-223 was predominantly produced by infiltrating immune cells (neutrophils, monocytes), while miR-31 was mainly derived from the local tissue (mesothelial cells, fibroblasts). miR-223 was found to be functionally stabilised in PD effluent from peritonitis patients, with a proportion likely to be incorporated into neutrophil-derived exosomes. Our study demonstrates that microRNAs are useful biomarkers of bacterial infection in PD-related peritonitis and have the potential to contribute to disease-specific immune fingerprints. Exosome-encapsulated microRNAs may have a functional role in intercellular communication between immune cells responding to the infection and the local tissue, to help clear the infection, resolve the inflammation and restore homeostasis.

The Long Non-coding RNA NRIR Drives IFN-Response in Monocytes: Implication for Systemic Sclerosis.

TLR4 activation initiates a signaling cascade leading to the production of type I IFNs and of the downstream IFN-stimulated genes (ISGs). Recently, a number of IFN-induced long non-coding RNAs (lncRNAs) that feed-back regulate the IFN response have been identified. Dysregulation of this process, collectively known as the "Interferon (IFN) Response," represents a common molecular basis in the development of autoimmune and autoinflammatory disorders. Concurrently, alteration of lncRNA profile has been described in several type I IFN-driven autoimmune diseases. In particular, both TLR activation and the upregulation of ISGs in peripheral blood mononuclear cells have been identified as possible contributors to the pathogenesis of systemic sclerosis (SSc), a connective tissue disease characterized by vascular abnormalities, immune activation, and fibrosis. However, hitherto, a potential link between specific lncRNA and the presence of a type I IFN signature remains unclear in SSc. In this study, we identified, by RNA sequencing, a group of lncRNAs related to the IFN and anti-viral response consistently modulated in a type I IFN-dependent manner in human monocytes in response to TLR4 activation by LPS. Remarkably, these lncRNAs were concurrently upregulated in a total of 46 SSc patients in different stages of their disease as compared to 18 healthy controls enrolled in this study. Among these lncRNAs, Negative Regulator of the IFN Response (NRIR) was found significantly upregulated in vivo in SSc monocytes, strongly correlating with the IFN score of SSc patients. Weighted Gene Co-expression Network Analysis showed that NRIR-specific modules, identified in the two datasets, were enriched in "type I IFN" and "viral response" biological processes. Protein coding genes common to the two distinct NRIR modules were selected as putative NRIR target genes. Fifteen in silico-predicted NRIR target genes were experimentally validated in NRIR-silenced monocytes. Remarkably, induction of CXCL10 and CXCL11, two IFN-related chemokines associated with SSc pathogenesis, was reduced in NRIR-knockdown monocytes, while their plasmatic level was increased in SSc patients. Collectively, our data show that NRIR affects the expression of ISGs and that dysregulation of NRIR in SSc monocytes may account, at least in part, for the type I IFN signature present in SSc patients.

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

BACKGROUND AND OBJECTIVE: Systemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. METHODS: Chromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression. RESULTS: 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. CONCLUSION: SSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster.

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance.

Cancer-related CD15/FUT4 overexpression decreases benefit to agents targeting EGFR or VEGF acting as a novel RAF-MEK-ERK kinase downstream regulator in metastatic colorectal cancer.

BACKGROUND: Cancer-related immune antigens in the tumor microenvironment could represent an obstacle to agents targeting EGFR "cetuximab" or VEGF "bevacizumab" in metastatic colorectal cancer (mCRC) patients. METHODS: Infiltrating immune cells into tumor tissues, cancer-related expression of immune antigens (CD3, CD8, CD68, CD73, MPO, CD15/FUT4) from 102 mCRC patients receiving first-line Cetuximab or Bevacizumab plus chemotherapy were assessed by immunohistochemistry and validated in an independent tissue microarrays of 140 patients. Genome-wide expression profiles from 436 patients and 60 colon cancer cell lines were investigated using bioinformatics analysis. In vitro kinase assays of target genes activated by chemokines or growth factors were performed. RESULTS: Here, we report that cancer-related CD15/FUT4 is overexpressed in most of mCRCs patients (43 %) and associates with lower intratumoral CD3+ and CD8+ T cells, higher systemic inflammation (NLR at diagnosis >5) and poorer outcomes, in terms of response and progression-free survival than those CD15/FUT4-low or negative ones (adjusted hazard ratio (HR) = 2.92; 95 % CI = 1.86-4.41; P < 0.001). Overexpression of CD15/FUT4 is induced through RAF-MEK-ERK kinase cascade, suppressed by MEK inhibitors and exhibits a close connection with constitutive oncogenic signalling pathways that respond to ERBB3 or FGFR4 activation (P < 0.001). CD15/FUT4-high expressing colon cancer cells with primary resistance to cetuximab or bevacizumab are significantly more sensitive to MEK inhibitors than CD15/FUT4-low counterparts. CONCLUSION: Cancer-related CD15/FUT4 overexpression participates in cetuximab or bevacizumab mechanisms of resistance in mCRC patients. CD15/FUT4 as a potential target of the antitumor immune response requires further evaluation in clinical studies.

IL-10 disrupts the Brd4-docking sites to inhibit LPS-induced CXCL8 and TNF-α expression in monocytes: Implications for chronic obstructive pulmonary disease.

BACKGROUND: IL-10 is well known for its ability to block the expression and production of numerous proinflammatory cytokines, in this manner preventing the development of excessive or chronic immune activation. IL-10-induced transcriptional repression of CXCL8 and TNFA genes consists of 2 distinct phases: an early phase, occurring rapidly and in a protein synthesis-independent manner, followed by a second phase that is more delayed and dependent on protein synthesis. OBJECTIVE: We sought to identify the mechanisms through which IL-10 rapidly and directly suppresses LPS-induced CXCL8 and TNF-α transcription, which might be defective under pathologic conditions. METHODS: The molecular events triggered by IL-10 in LPS-activated monocytes at the CXCL8 and TNFA loci were investigated by using the chromatin immunoprecipitation assay. RESULTS: Inhibition of LPS-induced CXCL8 and TNF-α expression by IL-10 proceeds through a common mechanism targeting LPS-induced phosphorylation of the nuclear factor κB p65 serine 276 residue (pS276p65). As a result, all the pS276p65-dependent events occurring at the CXCL8 and TNFA loci are consistently reduced, ultimately leading to a reduction in transcript elongation. Additionally, IL-10 selectively controls CXCL8 transcript elongation through histone deacetylase (HDAC) 2-dependent covalent chromatin modifications, disrupting the assembly of the transcriptional machinery. Remarkably, PBMCs from patients with acute-phase chronic obstructive pulmonary disease, which express negligible HDAC2 levels, are scarcely affected by IL-10 in terms of inhibition of CXCL8 expression. CONCLUSIONS: This study provides mechanistic evidence that IL-10 creates a chromatin environment that decreases the transcriptional rate of CXCL8 and TNF-α to Toll-like receptor 4-activating signals. Data identify novel molecular targets for therapeutic strategies aimed at dampening inflammation in pathologies such as chronic obstructive pulmonary disease, in which reduced intracellular HDAC2 levels have been described.

Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils.

Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPß recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells.

Fast and accurate quantitative analysis of cytokine gene expression in human neutrophils.

Polymorphonuclear neutrophils, traditionally viewed as short-lived effector cells, are nowadays regarded as important components of effector and regulatory circuits in the innate and adaptive immune systems. Most of the physiological functions of neutrophils as crucial players in the host immune response, able not only to act in the early phases of acute inflammation but also to condition the progression of the inflammatory reaction and the subsequent initiation of the specific immune response, rely on their capacity to produce and release a number of proinflammatory and immunoregulatory cytokines. This fact has reevaluated the importance, role, and physiological and pathological significance of neutrophils in the pathogenesis of inflammatory, infectious, autoimmune, and neoplastic diseases and has identified neutrophils as an important potential target for selective pharmacological intervention to both promote and restrain inflammation. In this context, understanding the mechanisms of modulation of neutrophil-derived cytokines and chemokines represents a critical step toward a better understanding of how neutrophils may influence pathophysiological processes in vivo. Herein, we describe and discuss an updated version of the methods that we have developed to rapidly and precisely characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the reverse transcription quantitative real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool, in the neutrophil research area, to test many experimental conditions for gene expression analysis.

Optimizing the purification and analysis of miRNAs from urinary exosomes.

BACKGROUND: Exosomes are cytoplasm containing vesicles released by many cells that can be found in several biological fluids including urine. Urinary exosomes are released from every segment of the nephron, are detectable in urine, constitutively contain RNA (small RNAs and mRNAs) and harbor unique subset of proteins, reflecting their cellular source. METHODS: With the aim of establishing the optimal protocol for high throughput analysis of exosomal miRNAs, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dye, capillary electrophoresis and analysis of spectrophotometric parameters. MiRNAs detection and abundance was determined by RT-qPCR. RESULTS: Among the exosomes isolation methods, Ultrafiltration resulted to be the most suited. The highest exosomal RNA yield quantified by RiboGreen® staining was obtained with the combination of TRI Reagent™ with miRNeasy®, followed by TRI Reagent™, SeraMir™, miRCURY™, mirVana™ and miRNeasy®; but after a multivariate analysis, SeraMir™ scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analyzed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. CONCLUSIONS: The selection of appropriate urinary exosomal miRNA isolation method was dependent on various validation results. Ultrafiltration in combination with SeraMir™ exoRNA columns represents the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes, perfectly suited for further applicative research in the field of miRNAs in kidney physiology and pathology.

Negative regulation of Toll-like receptor 4 signaling by IL-10-dependent microRNA-146b.

Toll-like receptors (TLRs) play key roles in detecting pathogens and initiating inflammatory responses that, subsequently, prime specific adaptive responses. Several mechanisms control TLR activity to avoid excessive inflammation and consequent immunopathology, including the anti-inflammatory cytokine IL-10. Recently, several TLR-responsive microRNAs (miRs) have also been proposed as potential regulators of this signaling pathway, but their functional role during the inflammatory response still is incompletely understood. In this study, we report that, after LPS engagement, monocytes up-regulate miR-146b via an IL-10-mediated STAT3-dependent loop. We show evidence that miR-146b modulates the TLR4 signaling pathway by direct targeting of multiple elements, including the LPS receptor TLR4 and the key adaptor/signaling proteins myeloid differentiation primary response (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6). Furthermore, we demonstrate that the enforced expression of miR-146b in human monocytes led to a significant reduction in the LPS-dependent production of several proinflammatory cytokines and chemokines, including IL-6, TNF-α, IL-8, CCL3, CCL2, CCL7, and CXCL10. Our results thus identify miR-146b as an IL-10-responsive miR with an anti-inflammatory activity based on multiple targeting of components of the TLR4 pathway in monocytes and candidate miR-146b as a molecular effector of the IL-10 anti-inflammatory activity.

Cutting edge: An inactive chromatin configuration at the IL-10 locus in human neutrophils.

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. We analyzed posttranslational modifications of histones associated with genes that are active, repressed, or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac, and H3K4me1 marks. Differently from autologous IL-10-producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils from healthy subjects or melanoma patients. By contrast, increased H3K4me3, H4Ac, H3K4me1, and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10.

IL-10-induced microRNA-187 negatively regulates TNF-α, IL-6, and IL-12p40 production in TLR4-stimulated monocytes.

IL-10 is a potent anti-inflammatory molecule that, in phagocytes, negatively targets cytokine expression at transcriptional and posttranscriptional levels. Posttranscriptional checkpoints also represent the specific target of a recently discovered, evolutionary conserved class of small silencing RNAs known as "microRNAs" (miRNAs), which display the peculiar function of negatively regulating mRNA processing, stability, and translation. In this study, we report that activation of primary human monocytes up-regulates the expression of miR-187 both in vitro and in vivo. Accordingly, we identify miR-187 as an IL-10-dependent miRNA playing a role in IL-10-mediated suppression of TNF-α, IL-6, and the p40 subunit of IL-12 (IL-12p40) produced by primary human monocytes following activation of Toll-like receptor 4 (TLR4). Ectopic expression of miR-187 consistently and selectively reduces TNFα, IL-6, and IL-12p40 produced by LPS-activated monocytes. Conversely, the production of LPS-induced TNF-α, IL-6, and IL-12p40 is increased significantly when miR-187 expression is silenced. Our data demonstrate that miR-187 directly targets TNF-α mRNA stability and translation and indirectly decreases IL-6 and IL-12p40 expression via down-modulation of IκBζ, a master regulator of the transcription of these latter two cytokines. These results uncover an miRNA-mediated pathway controlling cytokine expression and demonstrate a central role of miR-187 in the physiological regulation of IL-10-driven anti-inflammatory responses.

IFN-ß expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling.

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-ß. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-ß in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-ß mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-ß transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-ß promoter, revealed that IFN-ß mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-ß mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.