Valerie Garceau

HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA.

The mammalian innate immune system is activated by foreign nucleic acids. Detection of double-stranded DNA (dsDNA) in the cytoplasm triggers characteristic antiviral responses and macrophage cell death. Cytoplasmic dsDNA rapidly activated caspase 3 and caspase 1 in bone marrow–derived macrophages. We identified the HIN-200 family member and candidate lupus susceptibility factor, p202, as a dsDNA binding protein that bound stably and rapidly to transfected DNA. Knockdown studies showed p202 to be an inhibitor of DNA-induced caspase activation. Conversely, the related pyrin domain–containing HIN-200 factor, AIM2 (p210), was required for caspase activation by cytoplasmic dsDNA. This work indicates that HIN-200 proteins can act as pattern recognition receptors mediating responses to cytoplasmic dsDNA.

Pivotal Advance: Avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products

Macrophages are involved in many aspects of development, host defense, pathology, and homeostasis. Their normal differentiation, proliferation, and survival are controlled by CSF‐1 via the activation of the CSF1R. A recently discovered cytokine, IL‐34, was shown to bind the same receptor in humans. Chicken is a widely used model organism in developmental biology, but the factors that control avian myelopoiesis have not been identified previously. The CSF‐1, IL‐34, and CSF1R genes in chicken and zebra finch were identified from respective genomic/cDNA sequence resources. Comparative analysis of the avian CSF1R loci revealed likely orthologs of mammalian macrophage‐specific promoters and enhancers, and the CSF1R gene is expressed in the developing chick embryo in a pattern consistent with macrophage‐specific expression. Chicken CSF‐1 and IL‐34 were expressed in HEK293 cells and shown to elicit macrophage growth from chicken BM cells in culture. Comparative sequence and co‐evolution analysis across all vertebrates suggests that the two ligands interact with distinct regions of the CSF1R. These studies demonstrate that there are two separate ligands for a functional CSF1R across all vertebrates.

Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Background The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. Methodology/Principal Findings Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. Conclusions Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.

Histone Deacetylase 7 Promotes Toll-like Receptor 4-dependent Proinflammatory Gene Expression in Macrophages

Background: Histone deacetylase (HDAC) inhibitors reduce LPS-induced inflammatory mediator production from macrophages, but the relevant HDAC targets are unknown. Results: A specific isoform of Hdac7 amplifies expression of LPS-inducible genes via a HIF-1α-dependent mechanism in macrophages. Conclusion: The class IIa HDAC Hdac7 promotes inflammatory responses in macrophages. Significance: Hdac7 may be a viable target for developing new anti-inflammatory drugs. Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target.

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.

Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34.

Highlights ► The active region of porcine CSF-1 and full length CSF-1R have been cloned. ► Biological activity of porcine CSF-1 has been demonstrated. ► The cross species reactivity of IL-34 has been investigated. ► Both mouse and human IL-34 are biologically active on the pig CSF-1R. ► CSF-1 and IL-34 structure analysis of species-specific activity has been performed.

The development and maintenance of the mononuclear phagocyte system of the chick is controlled by signals from the macrophage colony-stimulating factor receptor.

BackgroundMacrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model.ResultsBased upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP+ donors. In both cases, the transferred cells gave rise to large numbers of EGFP+ tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP+ cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings.ConclusionsThe data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling.

Cloning and expression of feline colony stimulating factor receptor (CSF-1R) and analysis of the species specificity of stimulation by colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34)

Highlights ► Full-length feline CSF-1R has been cloned and expressed in a stable cell line. ► The biological activity of human, mouse and porcine CSF-1 has been evaluated. ► Human and mouse IL-34 are also biologically active on the feline CSF-1R. ► The potential therapeutic applications of CSF-1 is discussed.

Production and characterisation of a monoclonal antibody that recognises the chicken CSF1 receptor and confirms that expression is restricted to macrophage-lineage cells

Macrophages contribute to innate and acquired immunity as well as many aspects of homeostasis and development. Studies of macrophage biology and function in birds have been hampered by a lack of definitive cell surface markers. As in mammals, avian macrophages proliferate and differentiate in response to CSF1 and IL34, acting through the shared receptor, CSF1R. CSF1R mRNA expression in the chicken is restricted to macrophages and their progenitors. To expedite studies of avian macrophage biology, we produced an avian CSF1R-Fc chimeric protein and generated a monoclonal antibody (designated ROS-AV170) against the chicken CSF1R using the chimeric protein as immunogen. Specific binding of ROS-AV170 to CSF1R was confirmed by FACS, ELISA and immunohistochemistry on tissue sections. CSF1 down-regulated cell surface expression of the CSF1R detected with ROS-AV170, but the antibody did not block CSF1 signalling. Expression of CSF1R was detected on the surface of bone marrow progenitors only after culture in the absence of CSF1, and was induced during macrophage differentiation. Constitutive surface expression of CSF1R distinguished monocytes from other myeloid cells, including heterophils and thrombocytes. This antibody will therefore be of considerable utility for the study of chicken macrophage biology.

Cross species reactivity of Interleukin 34 (IL-34)

Background: Polyvalent vaccination represents a recent attempt to improve the effectiveness of lung cancer immunotherapy. This study aimed to investigate whether a gene expression pattern of tumor-associated antigens (TAA) would exist indicating that their use will be most appropriate for the polyvalent vaccination of Caucasian non-small cell lung carcinoma (NSCLC) patients. We examined the concomitant expression of genes belonging to different TAA families for which expression frequencies either have never been detected in NSCLC or vary widely in the literature. Methods: Tumor material from 23 patients with NSCLC (12 adenocarcinomas, 8 squamous cell carcinomas, 3 bronchoalveolar carcinomas) was examined. mRNA transcripts were detected for 5 genes of the survivin family, 5 MAGE-A genes as well as the genes of human telomerase reverse transcriptase (hTERT) and p53, by the use of quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) or semiquantitative RT-PCR. Results: 15/23 (65%) and 8/23 (35%) tumor samples were found expressing 6-11 and 2-5 out of the 12 examined TAAs, respectively, at levels >1% of the testis reference sample. The most prevalent TAA patterns observed were those of survivin standard (survivinstd)/survivin-2B expressed by 22/23 (95.5%) tumor samples and of survivin-std/survivin-2B/hTERT expressed by 19/23 (82.5%) tumor samples. The expression levels of the survivin-std gene strongly positively correlated to those of the survivin-2B (p=0.001) and the hTERT genes (p=0.031). The number of concomitantly expressed genes was found to be positively correlated to the age of the patients (p=0.001) and the tumor size (p=0.048). Conclusion: This study provides evidence that, in Caucasian patients with NSCLC, highly prevalent expression patterns of TAA genes, predominantly of overexpressed TAAs, do exist. This result implies that the combined use of these TAA could help in designing more effective NSCLC immunotherapeutic protocols.Purpose/Objective: Rheumatoid arthritis (RA) is a chronic autoimmune disease that leads to joint destruction. The recruitment of effectors cells, including monocytes to the joint space is an important step in RA progression and is mediated by chemokines (Ch) and their receptors (ChR). MicroRNAs are a recently discovered class of posttranscriptional regulators. Many members of the miR family are implicated in the regulation of cell movement and migration. Our previous study showed miR-155 is upregulated in RA synovial fluid (SF) monocytes suggesting that this miR may be involved in activation of these cells, including their migration into joint space. We hypothesized that miR-155 could regulates migration of monocytes in RA by modulating the expression of the chemokine and chemokine receptor system. Materials and methods: Peripheral blood (PB) CD14+ cells from healthy controls (HC) and RA patients were transfected with miR-155 mimic or scramble mimic using N-TER nanoparticles and cultured for 48 h. TaQman Low Density Array and multiplex assay was used to evaluate ChR expression and Ch production, respectively. Similar analysis was carried out on bone marrow monocytes (BMM) from miR-155-/- and WT mice. In addition, absolute copy numbers of miR- 155 transcripts in PB and SF CD14+ of RA and HC were assessed by QPCR. Results: PB and SF monocytes in RA patients showed higher copy number of miR-155 compared to HC. Overexpression of miR-155 in HC and RA monocytes did not affect the production of CCL2, CCL7, CCL21, CXCL5, CXCL8, CXCL7, CXCL10 and CX3CL1. In contrast, overexpression of miR-155 induced the production of chemokines such as CCL4, CCL5 and CCL22 in RA monocytes and CCL3 in both RA and HC. Analysis of chemokine receptors in BMM of miR-155-/- and WT mice revealed significantly higher levels of CCR1, CCR2, CCR5 and CXCR4 in miR-155 deficient cells suggesting that miR-155 can act as a negative regulator of these receptors in homeostatic state. As expected, TLR-4 ligand significantly suppressed expression of these receptors in both WT and miR-155-/- cells. Analysis of 3’UTRs of Ch/ ChR (TargetScan) suggests that miR-155 is likely interfering with signaling pathways implicated in Ch/ChR system expression. Conclusions: Deregulation of miR-155 in RA monocytes can contribute to the production of pro-inflammatory chemokines by these cells and to their accumulation at sites of inflammation.Purpose/Objective: Sphingosine kinase (SPHKs), SphK1 and SphK2, have been identified to phosphorylate sphingosine into sphingosine-1- phosphate (S1P). They are involved in a wide variety of cellular responses. S1P acts via S1P Receptors, S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5, all of which can be bound and activated specifically by S1P. A defect either in S1P signalling or S1PRs has been associated with many pathologies. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by high levels of proinflammatory cytokine production. Elevated SPHK1, S1P, and S1P1 have been reported in RA synovium. S1P signalling via S1P1 promotes synoviocyte proliferation, increases COX-2 expression and prostaglandin E2 production. This study comprehensively evaluated expression of SPHK1/2 and S1PRs in RA patients compare to healthy controls (HC) and osteoarthritis (OA) in peripheral blood (PB) and synovial tissues, respectively. Materials and methods: mRNA and protein expression of SPHK1/2 and SIPRs were examined in neutrophils, monocytes and T lymphocytes of peripheral blood of 10 HC and RA patients, who met the diagnostic criteria of 2010 ARC / EULAR by QPCR and FACS, respectively. Competitive ELISA assessed SIP in serum of RA patients with remission and relapse and HC. We also performed SPHK 1/2 and SIPRs immunohistochemistry in synovial tissue from 4 RA/ OA patients. Results: S1P was three times high in RA than those observed in HC, also was statistically higher in RA patient with relapse than remission. Intracellular expression of hSPHK1 in RA patients, with opposed to HC, was up regulated 1.4-folds in monocytes and T- lymphocytes with significance expression in CD4T cells. hS1P1 and hS1P3 exhibited a similar expression were up-regulated in neutrophils, while, hS1P5 was statistical high in T cells. In contrast, hS1P4 was down regulated in all sorted cells particularly in CD4T cells. As opposed to OA synovial tissue, RA synovial tissues were strongly positive for hSPHK1 and hS1P1, 3 expressions. Quantitative analysis showed, SPHK1 and hS1P3 are expressed in lining, sub lining and vascular endothelial layer, while hS1P1 expressed mainly in lining and sub lining layers of the RA synovial tissue compared with OA. Conclusions: These results suggest that SPHKs/S1P and its S1PRs might play a role in RA pathogenesis. The clinical significance of S1P as a biomarker for disease activity deserves further attention.