Jagged-1 signaling has recently been reported to be involved in the Th17 cell differentiation. However, little is known about its mechanisms. Soluble Jagged-1 was used to activate the Jagged-1-Notch signaling to interfere with the IL-6 and TGF-β-induced Th17 cell skewing. Genes relevant to the autoimmunity or inflammation were screened for the first time in this system by qPCR array for the differential expressions. The 18 genes out of 84, including Clec7a, Il12b, Il12rb1, Il12rb2, Csf3, Il15, Il17a, Il17f, Il17rc, Il17rd, Il17re, Il23a, Myd88, Socs1, Stat4, Stat5a, Sykb and Tbx21, were downregulated, but only Cxcl2, Cxcl12 and Mmp3 were upregulated. The expressions of the genes, Rorγt, Il17a, Il17f, Il12rb1 and Il23a, induced by simultaneous IL-6 and TGF-β treatment were significantly suppressed by Jagged-1, followed by the reduction of RORγt, IL-17A, and IL-17F. Consistent with the attenuation of RORγt, and the reduced production and secretion of IL-17A and IL-17F in the cell supernatant and the in situ stained cells, the number of CD4(+)IL-17(+) cells was also diminished. It is concluded that the Jagged-1-Notch signaling can suppress the IL-6 and TGF-β treatment-induced Th17 cell skewing through the attenuation of RORγt and, hence by, the down-regulation of IL-17A, IL-17F, IL-23a, and IL-12rb1.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

BACKGROUND

Heterotopic ossification (HO) is a frequent complication of modern wartime extremity injuries. The biological mechanisms responsible for the development of HO in traumatic wounds remain elusive.

OBJECTIVE

The aims of our study were to (1) characterize the expression profile of osteogenesis-related gene transcripts in traumatic war wounds in which HO developed; and (2) determine whether expression at the mRNA level correlated with functional protein expression and HO formation.

METHODS

Biopsy specimens from 54 high-energy penetrating extremity wounds obtained at the initial and final surgical débridements were evaluated. The levels of selected osteogenic-related gene transcripts from RNA extracts were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. As a result of its key role in osteogenesis, the concentration of BMP-2 in the effluent of 29 wounds also was determined.

RESULTS

The transcripts of 13 genes (ALPL [p = 0.006], BMP-2 [p < 0.001], BMP-3 [p = 0.06], COL2A1 [p < 0.001], COLL10A1 [p < 0.001], COL11A1 [p = 0.006], COMP [p = 0.02], CSF2 [p = 0.003], CSF3 [p = 0.012], MMP8 [p < 0.001], MMP9 [p = 0.014], SMAD1 [p = 0.024], and VEGFA [p = 0.017]) were upregulated greater than twofold in wounds in which HO developed compared with wounds in which it did not develop. Gene transcript expression of BMP-2 also correlated directly with functional protein expression in the wounds that formed HO (p = 0.029).

CONCLUSIONS

Important differences exist in the osteogenic gene expression profile of wounds in which HO developed compared with wounds in which it did not develop. The upregulation of multiple osteogenesis-related gene transcripts indicates the presence of a proosteogenic environment necessary for ectopic bone formation in traumatic wounds.

CONCLUSIONS

Understanding the osteogenic environment associated with war wounds may allow for the development of novel therapeutic strategies for HO.

Lower socioeconomic position (SEP) has consistently been associated with poorer health. To explore potential biological embedding and the consequences of SEP experiences from early life to adulthood, we investigate how SEP indicators at different points across the life course may be related to a combination of 28 inflammation markers. Using blood-derived inflammation profiles measured by a multiplex array in 268 participants from the Italian component of the European Prospective Investigation into Cancer and Nutrition cohort, we evaluate the association between early life, young adulthood and later adulthood SEP with each inflammatory markers separately, or by combining them into an inflammatory score. We identified an increased inflammatory burden in participants whose father had a manual occupation, through increased plasma levels of CSF3 (G-CSF; β = 0.29; P = 0.002), and an increased inflammatory score (β = 1.96; P = 0.029). Social mobility was subsequently modelled by the interaction between father's occupation and the highest household occupation, revealing a significant difference between "stable Non-manual" profiles over the life course versus "Manual to Non-manual" profiles (β = 2.38, P = 0.023). Low SEP in childhood is associated with modest increase in adult inflammatory burden; however, the analysis of social mobility suggests a stronger effect of an upward social mobility over the life course.

OBJECTIVE

To determine the effects of primary antiphospholipid syndrome (PAPS)-derived anti-beta(2)GPI antibodies on gene expression in human umbilical vein endothelial cells (HUVEC) by gene profiling using microarrays.

METHODS

Anti-beta(2)GPI antibodies purified from sera of patients with PAPS or control IgG isolated from normal subjects were incubated with HUVEC for 4 h before isolation of RNA and processing for hybridisation to Affymetrix Human Genome U133A-2.0 arrays. Data were analysed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. For selected genes microarray data were confirmed by real-time PCR analysis or at the protein level by ELISA.

RESULTS

A total of 101 genes were found to be upregulated and 14 genes were downregulated twofold or more in response to anti-beta(2)GPI antibodies. A number of novel genes not previously associated with APS were induced, including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1, and growth factors CSF2, CSF3 IL-6, IL1beta and FGF18. The majority of downregulated genes were transcription factors/signalling molecules including ID2. Quantitative real-time RT-PCR analysis confirmed the microarray results for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C).

CONCLUSIONS

This study reveals a complex gene expression response in HUVEC to anti-beta(2)GPI antibodies with multiple chemokines, pro-inflammatory cytokines, pro-thrombotic and pro-adhesive genes regulated by these antibodies in vitro. Some of these newly identified anti-beta(2)GPI antibody-regulated genes could contribute to the vasculopathy associated with this disease.

Inflammatory markers have consistently been associated with vascular disease. Evidence of genetic polymorphisms in inflammatory loci that predict severe carotid artery disease (CAAD) would suggest that this relationship is not secondary to other correlated factors, but related to inflammation itself. We examined the full common genetic variation in 42 inflammatory loci for prediction of severe CAAD versus ultrasound proven controls using a tagSNP approach. For selected loci, monocyte RNA levels were contrasted in subjects with and without CAAD. We confirm the association of IL6(-174), FGB (-455), and ALOX5 with CAAD and show that multiple ALOX5 SNPs independently predict CAAD. We provide evidence for previously unreported associations of SNPs in IL4R, NFKBIA, and PLG with CAAD, and weaker evidence for associations with CSF3, IL10RA, and VCAM1. The NFKBIA and IL10RA expression levels significantly differed between subjects with CAAD and controls. These results support a role for genetic variation related to inflammation in CAAD and a causal role for specific gene products.

Gene expression profiling (GEP) of 8 stage 0/I untreated Chronic Lymphocytic Leukemia (CLL) patients showed over-expression of Frizzled 3 (FZD3)/ROR-1 receptor tyrosine kinase (RTK), FLT-3 RTK and CXCR3 G-protein coupled receptor (GPCR). RT-PCR of 24 genes in 21 patients of the WNT pathway corroborated the GEP. Transforming growth factorβ, fibromodulin, TGFβRIII and SMAD2 are also over-expressed by GEP. Serum cytokine profiling of 26 low stage patients showed elevation of IFNγ, CSF3, Flt-3L and insulin-like growth factor binding protein 4. In order to ascertain why CLL cells grow poorly in culture, a GEP of 4 CLL patients cells at 0 hr and 24 hr in culture demonstrated over expression of CXCL5, CCL2 and CXCL3, that may recruit immune cells for survival. Treatment with thalidomide, an immunomodulatory agent, showed elevation of CCL5 by GEP but was not cytotoxic to CLL cells. Our data suggest an interplay of several oncogenic pathways, cytokines and immune cells that promote a survival program in CLL.

The ratio of dihydroxylated to trihydroxylated catechins (RDTC) is an important indicator of tea quality and biochemical marker for the study of genetic diversity. It is reported to be under genetic control but the underlying mechanism is not well understood. Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key enzymes involved in the formation of dihydroxylated and trihydroxylated catechins. The transcriptome and HPLC analysis of tea samples from Longjing43 and Zhonghuang2 under control and shading treatment were performed to assess the F3'H and F3'5'H genes that might affect RDTC. A total of 74.7 million reads of mRNA seq (2×101bp) data were generated. After de novo assembly, 109,909 unigenes were obtained, and 39,982 of them were annotated using 7 public databases. Four key F3'H and F3'5'H genes (including CsF3'5'H1, CsF3'H1, CsF3'H2 and CsF3'H3) were identified to be closely correlated with RDTC. Shading treatment had little effect on RDTC, which was attributed to the stable expression of these key F3'H and F3'5'H genes. The correlation of the coexpression of four key genes and RDTC was further confirmed among 13 tea varieties by real time PCR and HPLC analysis. The coexpression of three F3'H genes and a F3'5'H gene may play a key role in affecting RDTC in Camellia sinensis. The current results may establish valuable foundation for further research about the mechanism controlling catechin composition in tea.

Granulocyte-macrophage colony-stimulating factor (CSF), also known as CSF2, and granulocyte CSF, also known as CSF3, are important survival and proliferation factors for neutrophils and macrophages. The objective of the present study was to determine whether single nucleotide polymorphisms (SNPs) of CSF2 and CSF3 are associated with lung function in smoking-induced chronic obstructive pulmonary disease. In total, five SNPs of CSF2 and CSF3 were studied in 587 non-Hispanic white subjects with the fastest (n = 281) or the slowest (n = 306) decline of lung function selected from among continuous smokers in the National Heart, Lung, and Blood Institute Lung Health Study (LHS). These SNPs were also studied in 1,074 non-Hispanic white subjects with the lowest (n = 536) or the highest (n = 538) baseline lung function at the beginning of the LHS. An increase in the number of CSF3 -1719T alleles was significantly associated with protection against low lung function (odds ratio 0.73, 95% confidence interval 0.56-0.95), and was still significant after adjustment for multiple comparisons. There was also a significant association of a CSF3 haplotype with baseline levels of forced expiratory volume in one second. No association was found for CSF2 SNPs and lung function, nor was there evidence of epistasis. In conclusion, genetic variation in colony-stimulating factor 3 is associated with cross-sectionally measured lung function in smokers.

Exposure to silver nanoparticles (AgNP) used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters) and surface chemistry (citrate or polyvinlypyrrolidone capping) on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb) by human monocyte-derived macrophages (MDM). Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications.

In bone marrow (BM), hematopoietic elements are mingled with adipocytes (BM-A), which are the most abundant stromal component in the niche. BM-A progressively increase with aging, eventually occupying up to 50% of BM cavities. In this work, the role played by BM-A was explored by studying primary human BM-A isolated from hip surgery patients at the molecular level, through microarray analysis, and at the functional level, by assessing their relationship with primary human hematopoietic stem cells (HSC) by the long-term culture initiating cell (LTC-IC) assay. Findings demonstrated that BM-A are capable of supporting HSC survival in the LTC-IC assay, since after 5 weeks of co-culture, HSC were still able to proliferate and differentiate. Furthermore, critical molecules such as C-X-C motif chemokine 12 (CXCL12), interleukin (IL)-8, colony-stimulating factor 3 (CSF3), and leukaemia inhibitory factor (LIF), were expressed at similar levels in BM-A and in primary human BM mesenchymal stromal cells (BM-MSC), whereas IL-3 was higher in BM-A. Interestingly, BM-A displayed a different gene expression profile compared with subcutaneous adipose tissue adipocytes (AT-A) collected from abdominal surgery patients, especially in terms of regulation of lipid metabolism, stemness genes, and white-to-brown differentiation pathways. Accordingly, analysis of the gene pathways involved in hematopoiesis regulation showed that BM-A are more closely related to BM-MSC than to AT-A. The present data suggest that BM-A play a supporting role in the hematopoietic niche and directly sustain HSC survival.

Asthma arises from the complex interplay of inflammatory pathways in diverse cell types and tissues. We sought to undertake a comprehensive transcriptomic assessment of the epithelium and airway T cells that remain understudied in asthma and investigate interactions between multiple cells and tissues. Epithelial brushings and flow-sorted CD3+ T cells from sputum and BAL were obtained from healthy subjects (n = 19) and patients with asthma (mild, moderate, and severe asthma; n = 46). Gene expression was assessed using Affymetrix HT HG-U133+ PM GeneChips, and results were validated by real-time quantitative PCR. In the epithelium, IL-13 response genes (POSTN, SERPINB2, and CLCA1), mast cell mediators (CPA3 and TPSAB1), inducible nitric oxide synthase, and cystatins (CST1, CST2, and CST4) were upregulated in mild asthma, but, except for cystatins, were suppressed by corticosteroids in moderate asthma. In severe asthma-with predominantly neutrophilic phenotype-several distinct processes were upregulated, including neutrophilia (TCN1 and MMP9), mucins, and oxidative stress responses. The majority of the disease signature was evident in sputum T cells in severe asthma, where 267 genes were differentially regulated compared with health, highlighting compartmentalization of inflammation. This signature included IL-17-inducible chemokines (CXCL1, CXCL2, CXCL3, IL8, and CSF3) and chemoattractants for neutrophils (IL8, CCL3, and LGALS3), T cells, and monocytes. A protein interaction network in severe asthma highlighted signatures of responses to bacterial infections across tissues (CEACAM5, CD14, and TLR2), including Toll-like receptor signaling. In conclusion, the activation of innate immune pathways in the airways suggests that activated T cells may be driving neutrophilic inflammation and steroid-insensitive IL-17 response in severe asthma.

The scavenger receptor CD36 is a critical factor initiating ischemic brain injury, but the cell type(s) expressing CD36 and responsible for its harmful effects remain unknown. Using bone marrow (BM) chimeras subjected to transient middle cerebral artery occlusion, we found that CD36(-/-) mice transplanted with wild-type (WT) BM (WT→CD36(-/-)) have smaller infarcts (-67%), comparable with those of mice lacking CD36 both in brain and hematogenous cells (CD36(-/-) →CD36(-/-); - 72%). Conversely, WT mice receiving CD36(-/-) BM (CD36(-/-) →WT) have infarcts similar to WT→WT mice, suggesting that CD36 in the host brain (i.e., in microglia and endothelial cells), and not in hematogenous cells is involved in the damage. As anticipated, postischemic neutrophil infiltration in CD36(-/-) →CD36(-/-) mice was attenuated. Surprisingly, however, in WT→CD36(-/-) mice, in which infarcts were small, neutrophil infiltration was large and similar to that of CD36(-/-) →WT mice, in which infarcts were not reduced. Postischemic neutrophil free radical production was attenuated in WT→CD36(-/-) mice compared with CD36(-/-) →WT mice, whereas expression of the neutrophil activator colony-stimulating factor 3 (CSF3) was suppressed in CD36(-/-) cerebral endothelial cells, but not microglia. In CD36(-/-) cerebral endothelial cultures exposed to extracts from stroke brains, the upregulation of CSF3, but not neutrophil attractant chemokines, was suppressed. Intracerebroventricular administration of CSF3, 24 h after stroke, reconstituted neutrophil radical production and increased infarct volume in WT→CD36(-/-) mice. The findings identify endothelial cells as a key player in the deleterious effects of CD36 in stroke, and unveil a novel role of endothelial CD36 in enabling neutrophil neurotoxicity through CSF3.

UNASSIGNED

Ischemic stroke is a leading cause of death and disability worldwide with limited therapeutic options. The inflammatory response initiated by cerebral ischemia-reperfusion contributes to ischemic brain injury and is a potential therapeutic target. Here we report that CD36, an innate immunity receptor involved in the initiation of postischemic inflammation, is a previously unrecognized regulator of neutrophil cytotoxicity. The effect is mediated by endothelial CD36 via upregulation of the neutrophil activator CSF3 in cerebral endothelial cells. Therefore, approaches to modulate cerebral endothelial CD36 signaling or to neutralize CSF3 may provide novel therapeutic opportunities to ameliorate postischemic inflammatory injury.

BACKGROUND

Experimental autoimmune encephalomyelitis (EAE) is a model of inflammatory demyelinating diseases mediated by different types of leukocytes. How these cells communicate with each other to orchestrate autoimmune attacks is not fully understood, especially in the case of neutrophils, whose importance in EAE is newly established. The present study aimed to determine the expression pattern and role of different components of the IL-36 signaling pathway (IL-36α, IL-36β, IL-36γ, IL-36R) in EAE.

METHODS

EAE was induced by either active immunization with myelin peptide, passive transfer of myelin-reactive T cells or injection of pertussis toxin to transgenic 2D2 mice. The molecules of interest were analyzed using a combination of techniques, including quantitative real-time PCR (qRT-PCR), flow cytometry, Western blotting, in situ hybridization, and immunohistochemistry. Microglial cultures were treated with recombinant IL-36γ and analyzed using DNA microarrays. Different mouse strains were subjected to clinical evaluation and flow cytometric analysis in order to compare their susceptibility to EAE.

RESULTS

Our observations indicate that both IL-36γ and IL-36R are strongly upregulated in nervous and hematopoietic tissues in different forms of EAE. IL-36γ is specifically expressed by neutrophils, while IL-36R is expressed by different immune cells, including microglia and other myeloid cells. In culture, microglia respond to recombinant IL-36γ by expressing molecules involved in neutrophil recruitment, such as Csf3, IL-1β, and Cxcl2. However, mice deficient in either IL-36γ or IL-36R develop similar clinical and histopathological signs of EAE compared to wild-type controls.

CONCLUSIONS

This study identifies IL-36γ as a neutrophil-related cytokine that can potentially activate microglia, but that is only correlative and not contributory in EAE.

Caper (Capparis spinosa L.) fruits have been used as food as well as folk medicine in the treatment of inflammatory disorders, such as rheumatism. The present study was carried out to study the anti-inflammatory activities of C. spinosa L. fruit (CSF) aqueous extract and to isolate main phytochemicals from its bioactive fractions. The CSF aqueous extract were separated into three fractions (CSF1-CSF3) by macroporous adsorption resins. The fractions CSF2 and CSF3 effectively inhibited the carrageenan-induced paw edema in mice. Systematic fractionation and isolation from CSF2+3 led to the identification of 13 compounds (1-13). Their chemical structures were elucidated by spectroscopic analyses including nuclear magnetic resonance (NMR) and mass spectrometry (MS) and literature comparisons. Major compounds found in the bioactive fraction CSF2+3 are flavonoids, indoles, and phenolic acids. To our knowledge, 8 of these 13 compounds (1-4, 6-7, 10, and 13) were identified from caper fruits for the first time. The anti-inflammatory effects of these purified compounds are currently under investigation.

BACKGROUND

Late-term pregnancy may lead to maternal and neonatal morbidity and mortality. Mice null for the progesterone receptor co-regulator Krüppel-like Factor 9 (KLF9) exhibit delayed parturition and increased incidence of neonatal deaths.

OBJECTIVE

Our aim is to evaluate the contribution of myometrial KLF9 to human parturition.

METHODS

Myometrial biopsies were obtained from women with term (>37 to ≤41 wk) and late-term (>41 wk) pregnancies during cesarean delivery and assessed for gene and protein expression. Human myometrial cells transfected with nontargeting or KLF9 small interfering RNAs (siRNA) were treated with the progesterone antagonist RU486 and analyzed for pro-inflammatory chemokine/cytokine gene expression.

METHODS

The study took place in a University-affiliated tertiary care hospital and University research laboratory.

METHODS

Term patients (n = 8) were in spontaneous active labor whereas late-term patients (n = 5) were either in or were induced to active labor, prior to elective cesarean delivery.

METHODS

Steroid hormone receptor, contractility, and inflammation-associated gene expression in myometrial biopsies and in siKLF9-transfected, RU486-treated human myometrial cells was associated with KLF9 expression levels.

RESULTS

Myometrium from women with late-term pregnancy showed lower KLF9, total PGR, and PGR-A/PGR-B isoform expression. Transcript levels of select chemokines/cytokines were up- (CSF3, IL1, IL12A, TGFB2) and down- (CCL3, CCL5, CXCL1, CXCL5, IL15) regulated in late-term relative to term myometrium. Knock-down of KLF9 expression in RU486-treated human myometrial cells modified the expression of PGR and labor-associated cytokines, relative to control siRNA-treated cells.

CONCLUSIONS

Myometrial KLF9 may contribute to the onset of human parturition through its regulation of PGR expression and inflammatory signaling networks.

Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3'H, designated as CsF3'H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3'H was highly homologous with the characterized F3'Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3'H-specific conserved motifs were discovered in the protein sequence of CsF3'H. Enzymatic analysis of the heterologously expressed CsF3'H in yeast demonstrated that tea F3'H catalyzed the 3'-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min(-1), respectively. Transcription level of CsF3'H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3',4'-flavan-3-ols, 3',4',5'-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3'H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3'H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3'H in the biosynthesis of 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols in tea leaves.

Erythropoiesis is regulated such that a sufficient number of mature erythrocytes is produced. Down-regulation of erythropoiesis causes various types of anemia. Although some anemia-related genes have been identified, there are several types of anemic disease for which the molecular mechanisms are yet unclear, suggesting that unidentified genes in addition to the classical cytokine pathways play important roles in anemia. To address this issue, a new animal model for anemia is required. We established a reversible anemic model in zebrafish by keeping fish at 17 degrees C, a low water temperature. In zebrafish kidney marrow, expression of several genes encoding hematopoietic transcription factors (Runx1, scl, c-myb and GATA-2) and particularly erythropoiesis-related factors (klfd, hbaa1, ba1, GATA-1, EPO, and EPOr) was down-regulated, whereas myelopoiesis-related factors (csf1a and csf3) was up-regulated in low temperature conditions. We propose that this zebrafish model is useful to identify novel genes for hematopoiesis, particularly erythropoiesis.

Hemocyanins induce a potent Th1-dominant immune response with beneficial clinical outcomes when used as a carrier/adjuvant in vaccines and nonspecific immunostimulant in cancer. However, the mechanisms by which hemocyanins trigger innate immune responses, leading to beneficial adaptive immune responses, are unknown. This response is triggered by a proinflammatory signal from various components, of which macrophages are an essential part. To understand how these proteins influence macrophage response, we investigated the effects of mollusks hemocyanins with varying structural and immunological properties, including hemocyanins from Concholepas concholepas, Fissurella latimarginata, and Megathura crenulata (keyhole limpet hemocyanin), on cultures of peritoneal macrophages. Hemocyanins were phagocytosed and slowly processed. Analysis of this process showed differential gene expression along with protein levels of proinflammatory markers, including IL-1β, IL-6, IL-12p40, and TNF-α. An extended expression analysis of 84 cytokines during a 24-h period showed a robust proinflammatory response for F. latimarginata hemocyanin in comparison with keyhole limpet hemocyanin and C. concholepas hemocyanin, which was characterized by an increase in the transcript levels of M1 cytokines involved in leukocyte recruitment. These cytokine genes included chemokines (Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3), ILs (Il1b and Ifng), growth factors (Csf2 and Csf3), and TNF family members (Cd40lg). The protein levels of certain cytokines were increased. However, every hemocyanin maintains downregulated key M2 cytokine genes, including Il4 and Il5 Collectively, our data demonstrate that hemocyanins are able to trigger the release of proinflammatory factors with different patterns of cytokine expression, suggesting differential signaling pathways and transcriptional network mechanisms that lead to the activation of M1-polarized macrophages.

Bacterial vaginosis is associated with a 1.4-fold increased risk of preterm birth. We have shown previously that Lactobacillus rhamnosus GR-1 supernatant up-regulates interleukin 10 and down-regulates tumor necrosis factor-alpha output in lipopolysaccharide (LPS)-treated human primary placenta cultures in a fetal sex-dependent manner. We hypothesize that lactobacilli also exert their anti-inflammatory effect by up-regulation of colony-stimulating factor 3 (granulocyte) (CSF3), which is secreted from both immune and placental trophoblast cells, and that this activity is dependent on the sex of the fetus. Placental trophoblast cells were isolated from term elective cesarean section placentae using a Percoll gradient and separated from CD45(+) cells using magnetic purification. Cells were treated with LPS in the presence or absence of pretreatments with L. rhamnosus GR-1 supernatant or chemical inhibitors of the intracellular signaling pathways. Phosphorylations of mitogen-activated protein kinase 14 (MAPK14, previously known as p38) and signal transducer and activator of transcription (STAT) 3 were measured by Western blot analysis, and levels of CSF3 were determined by ELISA. CSF3 output was increased only in the placental trophoblast cells of female fetuses treated with LPS, GR-1 supernatant, and a combination of both treatments. The GR-1 supernatant up-regulated the phosphorylation of STAT3 and MAPK14. CSF3 output was inhibited by both Janus kinases (JAK) and MAPK14 inhibitors. None of the treatments was able to increase CSF3 output in either the pure trophoblast or the CD45(+) cell preparations alone. These results suggest an underlying mechanism for the sex difference in incidence of preterm birth and provide potential evidence for a therapeutic benefit of lactobacilli in reducing the risk of preterm labor.

Mycobacterium avium subspecies paratuberculosis (MAP) can cause a chronic inflammatory bowel disease, Johne's disease (JD), in ruminant animals. This study has explored the molecular basis of resistance and susceptibility to this disease in red deer breeds previously confirmed to express polarised phenotypes by experimental infection trials and following natural infection. Monocyte-derived macrophage cultures were obtained from uninfected red deer selected for either a resistant or susceptible phenotype. Cells were infected with MAP in vitro and gene expression analysed by RNA-Seq. Transcriptome analysis revealed a more disrupted gene expression profile in macrophages from susceptible animals compared with cells from resistant animals in terms of the number of genes up- or downregulated. Highly upregulated genes were related to chemotaxis (CXCL10, CSF3, and CCL8) and type 1 interferon signalling (RSAD2, IFIT1, IFIT2, ISG12, ISG15, USP18, and HERC6). Upregulation of these genes was observed to be greater in macrophages from susceptible animals compared to cells from resistant animals in response to in vitro MAP infection. These data support the use of transcriptomic approaches to enable the identification of markers associated particularly with susceptibility to MAP infection.

Tea is an important economic crop with a 3.02 Gb genome. It accumulates various bioactive compounds, especially catechins, which are closely associated with tea flavor and quality. Catechins are biosynthesized through the phenylpropanoid and flavonoid pathways, with 12 structural genes being involved in their synthesis. However, we found that in Camellia sinensis the understanding of the basic profile of catechins biosynthesis is still unclear. The gene structure, locus, transcript number, transcriptional variation, and function of multigene families have not yet been clarified. Our previous studies demonstrated that the accumulation of flavonoids in tea is species, tissue, and induction specific, which indicates that gene coexpression patterns may be involved in tea catechins and flavonoids biosynthesis. In this paper, we screened candidate genes of multigene families involved in the phenylpropanoid and flavonoid pathways based on an analysis of genome and transcriptome sequence data. The authenticity of candidate genes was verified by PCR cloning, and their function was validated by reverse genetic methods. In the present study, 36 genes from 12 gene families were identified and were accessed in the NCBI database. During this process, some intron retention events of the CsCHI and CsDFR genes were found. Furthermore, the transcriptome sequencing of various tea tissues and subcellular location assays revealed coexpression and colocalization patterns. The correlation analysis showed that CsCHIc, CsF3'H, and CsANRb expression levels are associated significantly with the concentration of soluble PA as well as the expression levels of CsPALc and CsPALf with the concentration of insoluble PA. This work provides insights into catechins metabolism in tea and provides a foundation for future studies.

OBJECTIVE

To evaluate the epicardial adipose tissue (EAT) transcriptome in comparison to subcutaneous fat (SAT) in coronary artery disease (CAD) and type 2 diabetes (T2DM).

RESULTS

SAT and EAT samples were obtained from subjects with T2DM and CAD (n = 5) and those without CAD with or without T2DM (=3) undergoing elective cardiac surgery. RNA-sequencing analysis was performed in both EAT and SAT. Gene enrichment analysis was conducted to identify pathways affected by the differentially expressed genes. Changes of top genes were verified by quantitative RT-PCR (qRT-PCR), western blot, and immunofluorescence. A total of 592 genes were differentially expressed in diabetic EAT, whereas there was no obvious changes in SAT transcriptome between diabetics and non-diabetics. Diabetic EAT was mainly enriched in inflammatory genes, such as Colony Stimulating Factor 3 (CSF3), Interleukin-1b (IL-1b), IL-6. KEGG pathway analysis confirmed that upregulated genes were involved in inflammatory pathways, such as Tumor Necrosis Factor (TNF), Nuclear Factor-κB (NF-κB) and advanced glycation end-products-receptor advanced glycation end products (AGE-RAGE). The overexpression of inflammatory genes in diabetic EAT was largely correlated with upregulated transcription factors such as NF-κB and FOS.

CONCLUSIONS

Diabetic EAT transcriptome is significantly different when compared to diabetic SAT and highly enriched with genes involved in innate immune response and endothelium, like Pentraxin3 (PTX3) and Endothelial lipase G (LIPG). EAT inflammatory genes expression could be induced by upregulated transcription factors, mainly NF-kB and FOSL, primarily activated by the overexpressed AGE-RAGE signaling. This suggests a unique and novel atherogenic pathway in diabetes.

In psoriasis lesions, a diverse mixture of cytokines is up-regulated that influence each other generating a complex inflammatory situation. Although this is the case, the inhibition of IL-17A alone showed unprecedented clinical results in patients, indicating that IL-17A is a critical inducer of psoriasis pathogenesis. To elucidate IL-17A-driven keratinocyte-intrinsic signaling pathways, we treated monolayers of normal human epidermal keratinocytes in vitro with a mixture of six cytokines (IL-17A, TNF-α, IL-17C, IL-22, IL-36γ and IFN-γ) involved in psoriasis to mimic the inflammatory milieu in psoriasis lesions. Microarray and gene set enrichment analysis revealed that this cytokine mixture induced similar gene expression changes with the previous transcriptome studies using psoriasis lesions. Importantly, we identified a set of IL-17A-regulated genes in keratinocytes, which recapitulate typical psoriasis genes exemplified by DEFB4A, S100A7, IL19 and CSF3, based on the differences in the expression profiles of cells stimulated with six cytokines versus cells stimulated with only five cytokines lacking IL-17A. Furthermore, a specific IL-17A-induced gene, NFKBIZ, which encodes IκB-ζ, a transcriptional regulator for NF-κB, was demonstrated to have a significant role for IL-17A-induced gene expression. Thus, we present novel in vitro data from normal human keratinocytes that would help elucidating the IL-17A-driven keratinocyte activation in psoriasis.