Tumor-associated macrophages (TAMs) are enriched in glioblastoma (GBM) that contains glioma stem cells (GSCs) at the apex of its cellular hierarchy. The correlation between TAM density and glioma grade suggests a supportive role of TAMs in tumor progression. Here we interrogated the molecular link between GSCs and TAM recruitment in GBMs and demonstrated that GSCs secrete Periostin (POSTN) to recruit TAMs. TAM density correlates with POSTN levels in human GBMs. Silencing POSTN in GSCs markedly reduced TAM density, inhibited tumor growth, and increased survival of mice bearing GSC-derived xenografts. We found that TAMs in GBMs are not brain-resident microglia, but mainly monocyte-derived macrophages from peripheral blood. Disrupting POSTN specifically attenuated the tumor supportive M2 type of TAMs in xenografts. POSTN recruits TAMs through integrin αvβ3 as blocking this signaling by an RGD peptide inhibited TAM recruitment. Our findings highlight the possibility of improving GBM treatment by targeting POSTN-mediated TAM recruitment.
POSTN; Tumor-associated macrophage (TAM); Glioblastoma (GBM); Cancer stem cell; Microglia
The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IKKα is a critical negative regulator of ASC-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKKi facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.
Background: GLP-1 and its analogs have a variety of anti-diabetic effects. However, short half-life and rapid degraded by DPP-IV limits the therapeutic potential of the native GLP-1. So, many DPP-IV-resistant and long-acting GLP-1 analogs were developed. In this study, an antibody-like extendin-4-IgG4 fusion protein was developed. Methods: The γ4 constant region contains two amino acid substitutions relative to native γ4 (S228P and L235E) lead to affinity for FcγRI to be low and stability of the IgG4 molecular. The fusion protein was expressed in CHO cells and assembled into an immunoglobulin-like structure with molecular weight of approximately 130 kDa. Results: The Exendin-4-IgG4 fusion protein was found to affinity bind GLP-1R in vitro. In vivo when compared the potency and duration of glucose-lowering effects in diabetic (db/db) mice at the same dose, exendin-4 resulted in a glucose-lowering effect that persisted only for 6 hours, but the extendin-4-IgG4 fusion protein for more than 168 hours. Injecting subcutaneously with a high dose of the fusion protein led normal BALB/c mice to the lower blood glucose level but did not cause serious hypoglycemia. Especially, the half-life time of the fusion protein in cynomolgus monkeys was about 180 hours, almost the longest half-life time among the developed GPL-1 analogues, which suggested a longer half-life time in human. Conclusions: The intact antibody-like fusion protein has more advantages than the Fc fusion protein including the intent of prolonging the half-life. These results also suggested the fusion protein was a safe and long-acting potential anti-diabetic agent.
Extendin-4-IgG4 fusion protein; anti-diabetic agent; long-acting; GLP-1 analog; antibody-like
The safety and efficacy of the JXA1-R vaccine, an attenuated strain of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), were examined using an intramuscular challenge model in piglets. The JXA1-R vaccine was obtained by passing HP-PRRSV JXA1 through Marc-145 cells (82nd passage). Genomic sequence comparisons showed that strain JXA1-R and its parental strain, JXA1, differ by 47 amino acids, and most of these differences are scattered throughout the PRRSV genome. Four-week-old PRRSV-free piglets were inoculated intramuscularly with JXA1-R vaccine (103.0, 104.0, 105.0, 106.0, and 107.0 50% tissue culture infective doses [TCID50]/ml for groups 1 to 5, respectively) and then challenged intramuscularly with the 5th passage virus of JXA1 virus (JXA1-F5, 3 ml × 104.5 TCID50/ml) 28 days after inoculation. The humoral immune response, swine growth, clinical signs, and differential organ lesions were monitored. The results showed that all vaccinated piglets had a perceptible humoral immune response to vaccination after day 7, which then promptly increased, almost reaching the maximum sample/positive (S/P) ratio value at 28 days postimmunization. Viremia detection indicated that the viral replication levels of the challenge virus in the immunized groups (immunization doses ≥104.0/ml) were significantly lower than that of the virus-challenged unvaccinated control group. Piglets in groups 2 to 5 were effectively protected against lethal HP-PRRSV infection and did not show any obvious changes in body temperature or clinical signs of disease at any point during the experiment. However, two of five piglets in group 1 showed mild pathological lesions and transitory high fever. These results suggest that JXA1-R (TCID50/ml ≥104.0) is sufficiently attenuated and can provide effective protection against the lethal wild-type HP-PRRSV.
Staphylococcus capitis is an emerging opportunistic pathogen of humans, and found as a colonizer of the human gut. Here, we report a case of S. capitis subsp. urealyticus infection. The strain LNZR-1 was isolated from the blood culture of a patient with sigmoid colon cancer. It was found to be resistant to some important antibiotics, such as linezolid, a highly effective antimicrobial against clinically important Staphylococci pathogens. However, data on the genetic resistance mechanisms in S. capitis subsp. urealyticus are only sparsely available.
The draft genome of S. capitis subsp. urealyticus strain LNZR-1 was sequenced by using next-generation sequencing technologies. Sequence data assembly revealed a genome size of 2,595,865 bp with a G + C content of 32.67%. Genome annotation revealed the presence of antibiotic resistance genes conferring resistance against some of the tested antibiotics as well as non-tested antibiotics. The genome also possesses a lot of genes that may be related to multidrug resistance. Whole genome comparison of the LNZR-1 with five other S. capitis strains showed that some functional regions are highly homologous between the six assemblies made herein. The LNZR-1 genome has high similarity with the genomes of the strains VCU116 and CR01, although some short stretches present in the genomes of strains VCU116 and CR01 were absent in the strain LNZR-1.
The presence of a plethora of genes responsible for antibiotic resistance suggests that strain LNZR-1 could present a potential threat to human health. The comparative genomic analysis of S. capitis strains presented in this study is important for better understanding of multidrug resistance in S. capitis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13099-014-0045-x) contains supplementary material, which is available to authorized users.
Staphylococcus capitis subsp. urealyticus; Multidrug-resistant; Genome sequencing; Comparative genomic analysis
MyD88-dependent GM-CSF production by endothelial cells plays a role in the initiation of obesity-associated inflammation by promoting adipose macrophage recruitment and M1-like polarization.
Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor–MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet–induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.
The interleukin 17 (IL-17) family, a subset of cytokines consisting of IL-17A-F, plays crucial roles in host defense against microbial organisms and in the development of inflammatory diseases. Although IL-17A is the signature cytokine produced by T helper 17 (Th17) cells, IL-17A and other IL-17 family cytokines have multiple sources ranging from immune cells to non-immune cells. The IL-17 family signals via their correspondent receptors and activates downstream pathways that include NFκB, MAPKs and C/EBPs to induce the expression of anti-microbial peptides, cytokines and chemokines. The proximal adaptor Act1 is a common mediator during the signaling of all IL-17 cytokines so far and is thus involved in IL-17 mediated host defense and IL-17-driven autoimmune conditions. This review will give an overview and recent updates on the IL-17family, the activation and regulation of IL-17 signaling as well as diseases associated with this cytokine family
interleukin 17; IL-17R; signaling transduction; Act1
Th17 cells have been implicated in autoimmunity and inflammatory bowel disease (IBD). Antigen presenting cell (APC) derived cytokines such as IL-1β and IL-6 are key mediators supporting Th17 differentiation, yet how these factors are induced in vivo remains unclear. Here we show that IL-27 acting on APCs enhances IL-6 and IL-1β production and Th17 differentiation. IL-27Rα−/− TCRβ−/− recipients fail to develop gut inflammation following naïve CD4 T cell transfer, while IL-27Rα+/+ TCRβ−/− recipients develop severe colitis. Investigation of T cell responses exhibits that IL-27Rα−/− TCRβ−/− mice do not support Th17 differentiation with significantly decreased levels of IL-6 and IL-1β by APCs. Our study has identified a novel proinflammatory role for IL-27 in vivo that promotes Th17 differentiation by inducing Th17-supporting cytokines in APCs.
CD4; APC; IBD
Patients with inborn errors of IL-17F or IL-17RA display chronic mucocutaneous candidiasis (CMC). We report a biallelic missense mutation (T536I) in the adaptor molecule ACT1 in two siblings with CMC. The mutation, located in the SEFIR domain, abolished the homotypic interaction of ACT1 with IL-17 receptors, with no effect on homodimerization. The patients’ fibroblasts failed to respond to IL-17A and IL-17F, and their T cells to IL-17E. By contrast, healthy individuals homozygous for the common variant D10N, located in the ACT1 TNF receptor-associated factor (TRAF)-interacting domain and previously associated with psoriasis, had impaired, but not abolished, responses to IL-17 cytokines. SEFIR-independent interactions of ACT1 with other proteins, such as CD40, heat shock protein (HSP)70 and HSP90, were not affected by the T536I mutation. Overall, human IL-17A and IL-17F depend on ACT1 to mediate protective mucocutaneous immunity. Moreover, other ACT1-dependent IL-17 cytokines seem to be largely redundant in host defense.
Self-reactive B cells in BALB/c AM14 transgenic (AM14 Tg) rheumatoid factor (RF) mice are not subject to central or peripheral toleralization. Instead, they remain at a stage of “clonal ignorance”, i.e. they do not proliferate and differentiate into Ab-producing cells. However, the immunoregulatory mechanisms that prevent autoantibody production in these mice remain unclear. In this study, we show that crossing AM14 Tg mice to a mouse strain deficient in Act1, a molecule involved in the regulation of BAFF-R and CD40-signaling in B cells, results in spontaneous activation of AM14 Tg B cells and production of AM14-specific antibodies. Three to five-month old AM14 Tg Act1−/− mice showed significant expansion of AM14 Tg B cells, including a 2–3 fold increase in the spleen and cLNs compared to AM14 Tg Act1+/+ mice. Furthermore, in the presence of endogenous self-Ag (IgHa congenic background), AM14 Tg Act1−/− B cells were spontaneously activated and differentiated into antibody forming cells (AFC). In contrast with previous studies using AM14 Tg MLR.Faslpr mice, we found that a significant number of AM14 Tg cells AM14 Tg Act1−/− mice displayed phenotypic characteristics of GC B cells. Anti-CD40L treatment significantly limited the expansion and activation of AM14 Tg Act1−/− B cells, suggesting that CD40L-mediated signals are required for the retention of these cells. Our results support the important role of Act1 in the regulation of self-reactive B cells and reveal how Act1 functions to prevent the production of autoantibodies.
Rapid local drug clearance of antimicrobials is a major drawback for the treatment of chronic periodontitis. In the study reported here, minocycline-loaded poly(ethylene glycol)-poly(lactic acid) nanoparticles were prepared and administered locally for long drug retention and enhanced treatment of periodontitis in dogs.
Biodegradable poly(ethylene glycol)-poly(lactic acid) was synthesized to prepare nanoparticles using an emulsion/solvent evaporation technique. The particle size and zeta potential of the minocycline-loaded nanoparticles (MIN-NPs) were determined by dynamic light scattering and the morphology of the nanoparticles was observed by transmission electron microscopy. The in vitro release of minocycline from MIN-NPs and in vivo pharmacokinetics of minocycline in gingival crevice fluid, after local administration of MIN-NPs in the periodontal pockets of beagle dogs with periodontitis, were investigated. The anti-periodontitis effects of MIN-NPs on periodontitis-bearing dogs were finally evaluated.
Transmission electron microscopy examination and dynamic light scattering results revealed that the MIN-NPs had a round shape, with a mean diameter around 100 nm. The in vitro release of minocycline from MIN-NPs showed a remarkably sustained releasing characteristic. After local administration of the MIN-NPs, minocycline concentration in gingival crevice fluid decreased slowly and retained an effective drug concentration for a longer time (12 days) than Periocline®. Anti-periodontitis effects demonstrated that MIN-NPs could significantly decrease symptoms of periodontitis compared with Periocline and minocycline solution. These findings suggest that MIN-NPs might have great potential in the treatment of periodontitis.
minocycline; nanoparticles; periodontitis; local delivery
Endothelial colony forming cells (ECFCs) participate in post-natal vasculogenesis. We previously reported that vascular endothelial growth factor (VEGF) promotes human ECFC differentiation through AMP-activated protein kinase (AMPK) activation. However, the mechanisms underlying transcriptional regulation of ECFC differentiation still remain largely elusive. Here, we investigated the role of transcription factor Krüppel-like factor 2 (KLF2) in the regulation of ECFC function.
Methods and results
Human ECFCs were isolated from cord blood and cultured. Treatment with VEGF significantly increased endothelial markers in ECFCs and their capacity for migration and tube formation. The mRNA and protein levels of KLF2 were also significantly up-regulated. This up-regulation was abrogated by AMPK inhibition or by knockdown of KLF2 with siRNA. Furthermore, adenovirus-mediated overexpression of KLF2 promoted ECFC differentiation by enhancing expression of endothelial cell markers, reducing expression of progenitor cell markers, and increasing the capacity for tube formation in vitro, indicating the important role of KLF2 in ECFC-mediated angiogenesis. Histone deacetylase 5 (HDAC5) was phosphorylated by AMPK activity induced by VEGF and the AMPK agonist AICAR (5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide). In vivo angiogenesis assay revealed that overexpression of KLF2 in bone-marrow-derived pro-angiogenic progenitor cells promoted vessel formation when the cells were implanted in C57BL/6 mice.
Up-regulation of KLF2 by AMPK activation constitutes a novel mechanism of ECFC differentiation, and may have therapeutic value in the treatment of ischaemic heart disease.
Endothelial progenitor cells; VEGF; Differentiation; AMPK; Krüppel-like factor 2
Here we identified a population of bone marrow neutrophils that constitutively express RORγt and which can produce and respond to IL-17A (IL-17). IL-6, IL-23 and RORγt, but not T cells or NK cells, are required for IL-17 production in neutrophils. IL-6 and IL-23 induced IL-17RC and Dectin-2 expression in neutrophils, and expression of IL-17RC was augmented by Aspergillus and Dectin-2 activation. Autocrine IL-17A–IL-17 receptor activity induced production of reactive oxygen species (ROS), and increased fungal killing in vitro and in a model of Aspergillus keratitis. Human neutrophils also expressed RORγt, and induced IL-17A, IL-17RC and Dectin-2 expression following IL-6 and IL-23 stimulation. These findings identify a population of human and murine neutrophils that exhibit autocrine IL-17 activity, and which likely contribute to the etiology of microbial and inflammatory diseases.
Interleukin 17(IL-17) is a signature cytokine of Th17 cells. We previously reported that deletion of NF-κ B activator 1(Act1), the key transducer of IL-17R signaling, from the neuroectodermal lineage in mice (neurons, oligodendrocytes, astrocytes)results in attenuated severity of experimental autoimmune encephalomyelitis (EAE). Here we examined the cellular basis of this observation. EAE disease course was unaffected by deleting Act1 from neurons or mature oligodendrocytes and Act1 deletion from astrocytes only modestly affected disease course. Deletion of Act1 from NG2+ glia resulted in markedly reduced EAE severity. Furthermore, IL-17 induced characteristic inflammatory mediator expression in NG2+ glial cells. Additionally, IL-17 also exhibited strong inhibitory effects on the maturation of oligodendrocyte lineage cells in vitro and reduced their survival. These data identify NG2+ glia as the major CNS cellular target of IL-17 in EAE. The sensitivity of oligodendrocyte lineage cells to IL-17-mediated toxicity further suggests a direct link between inflammation and neurodegeneration in multiple sclerosis (MS).
Campylobacter jejuni is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how C. jejuni colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, C. jejuni typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal C. jejuni colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (Sigirr−/−), a negative regulator of MyD88-dependent signaling led to heavy and widespread C. jejuni colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. C. jejuni heavily colonized the cecal and colonic crypts of Sigirr−/− mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established C. jejuni pathogenicity factors, capsular polysaccharides (kpsM) and motility/flagella (flaA). We also explored the basis for the inflammatory response elicited by C. jejuni in Sigirr−/− mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by C. jejuni. Despite heavy colonization, Tlr4−/−/Sigirr−/− mice were largely unresponsive to infection by C. jejuni, whereas Tlr2−/−/Sigirr−/− mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the C. jejuni capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of Sigirr−/− mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to C. jejuni.
Research into the key virulence strategies of the bacterial pathogen Campylobacter jejuni, as well as the host immune responses that develop against this microbe have, in many ways, been limited by the lack of relevant animal models. Here we describe the use of Sigirr deficient (−/−) mice as a model for C. jejuni pathogenesis. Not only do Sigirr−/− mice develop significant intestinal inflammation in response to colonization by C. jejuni, but the ability of this pathogen to trigger gastroenteritis was dependent on key virulence factors. We also found that the induction of the inflammatory and Th1/Th17 immune responses to infection in these mice depended on specific Toll-like receptors, principally TLR4, which we identified as the main driver of inflammation. In contrast, TLR2 signaling was found to protect mucosal integrity, with Tlr2−/−/Sigirr−/− mice suffering exaggerated mucosal damage and inflammation. Notably, we found that C. jejuni's capsule helped conceal it from the host's immune system as its loss led to significantly increased activation of host TLRs and exaggerated gastroenteritis. Our research shows that the increased sensitivity of Sigirr−/− mice can be used to generate a unique and exciting model that facilitates the study of C. jejuni pathogenesis as well as host immunity to this enteric pathogen.
IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA binding protein HuR in IL-17-induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17-induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. While IL-17 induced the co-shift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translational-active versus translational-inactive IL-17-induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17-induced neutrophilia. In summary, HuR functions to couple receptor proximal signaling to posttranscriptional machinery, contributing to IL-17-induced inflammation.
The purpose of this research was to evaluate the therapeutic effects and prognostic factors of transanal local excision (TAE) for rectal cancer.
We retrospectively analyzed 116 cases that underwent TAE for rectal cancer from 1995 to 2008. A Cox regression analysis was used to analyze prognostic factors.
The survival times for the patients were from 14 to 160.5 months (median time, 58.5 months). The 5-year and 10-year overall survival rates were 72% and 53%, respectively. In all 16 cases experienced local recurrence (13.8%). Pathological type, recurrence or metastasis, and depth of infiltration (T stage) were the prognostic factors according to the univariate analysis, and the latter two were independent factors affecting patient prognosis. For patients with T1 stage who underwent adjuvant radiotherapy, there was no local recurrence; for those in T2 stage, the local recurrence rate was 14.6%. In addition, there was no difference between the patients who received radiotherapy and those who did not (T1: P = 0.260, T2: P = 0.262 for survival rate and T1: P = 0.480, T2: P = 0.560 for recurrence).
The result of TAE for rectal cancer is satisfactory for T1 stage tumors, but it is not suitable for T2 stage tumors.
rectal cancer; transanal local excision; survival; recurrence; radiotherapy
Carex diaoluoshanica, a new species of Carex sect. Lageniformes from Hainan, China, is described and illustrated. The new species is similar to C. breviscapa but differs in having wider leaves with the leaf base gradually narrowed, 5–10 cm long and petiolelike, culms subfiliform, with only two spikes, the lateral female spikes from near the culm base.
Objective. To study the effects and underlying mechanisms of voltage-gated K+ channels on the proliferation of multiple myeloma cells. Methods. RPMI-8226 MM cell line was used for the experiments. Voltage-gated K+ currents and the resting potential were recorded by whole-cell patch-clamp technique. RT-PCR detected Kv channel mRNA expression. Cell viability was analyzed with MTT assay. Cell counting system was employed to monitor cell proliferation. DNA contents and cell volume were analyzed by flow cytometry. Results. Currents recorded in RPMI-8226 cells were confirmed to be voltage-gated K+ channels. A high level of Kv1.3 mRNA was detected but no Kv3.1 mRNA was detected in RPMI-8226 cells. Voltage-gated K+ channel blocker 4-aminopyridine (4-AP) (2 mM) depolarized the resting potential from −42 ± 1.7 mV to −31.8 ± 2.8 mV (P < 0.01). The results of MTT assay showed that there was no significant cytotoxicity to RPMI-8226 cells when the 4-AP concentration was lower than 4 mM. 4-AP arrested cell cycle in G0/G1 phase. Cells were synchronized at the G1/S boundary by treatment of aphidicolin and released from the blockage by replacing the medium with normal culture medium or with culture medium containing 2 mM 4-AP. 4-AP produced no significant inhibitory effect on cell cycle compared with control cells (P > 0.05). Conclusions. In RPMI-8226, voltage-gated K+ channels are involved in proliferation and cell cycle progression its influence on the resting potential and cell volume may be responsible for this process; the inhibitory effect of the voltage-gated K+ channel blocker on RPMI-8226 cell proliferation is a phase-specific event.
Many Poaceae species show a gametophytic self-incompatibility (GSI) system, which is controlled by at least two independent and multiallelic loci, S and Z. Until currently, the gene products for S and Z were unknown. Grass SI plant stigmas discriminate between pollen grains that land on its surface and support compatible pollen tube growth and penetration into the stigma, whereas recognizing incompatible pollen and thus inhibiting pollination behaviors. Leymus chinensis (Trin.) Tzvel. (sheepgrass) is a Poaceae SI species. A comprehensive analysis of sheepgrass stigma transcriptome may provide valuable information for understanding the mechanism of pollen-stigma interactions and grass SI.
The transcript abundance profiles of mature stigmas, mature ovaries and leaves were examined using high-throughput next generation sequencing technology. A comparative transcriptomic analysis of these tissues identified 1,025 specifically or preferentially expressed genes in sheepgrass stigmas. These genes contained a significant proportion of genes predicted to function in cell-cell communication and signal transduction. We identified 111 putative transcription factors (TFs) genes and the most abundant groups were MYB, C2H2, C3H, FAR1, MADS. Comparative analysis of the sheepgrass, rice and Arabidopsis stigma-specific or preferential datasets showed broad similarities and some differences in the proportion of genes in the Gene Ontology (GO) functional categories. Potential SI candidate genes identified in other grasses were also detected in the sheepgrass stigma-specific or preferential dataset. Quantitative real-time PCR experiments validated the expression pattern of stigma preferential genes including homologous grass SI candidate genes.
This study represents the first large-scale investigation of gene expression in the stigmas of an SI grass species. We uncovered many notable genes that are potentially involved in pollen-stigma interactions and SI mechanisms, including genes encoding receptor-like protein kinases (RLK), CBL (calcineurin B-like proteins) interacting protein kinases, calcium-dependent protein kinase, expansins, pectinesterase, peroxidases and various transcription factors. The availability of a pool of stigma-specific or preferential genes for L. chinensis offers an opportunity to elucidate the mechanisms of SI in Poaceae.
Electronic supplementary material
The online version of this article (doi: 10.1186/1471-2164-15-399) contains supplementary material, which is available to authorized users.
Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling.
Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′ins) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′ins helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.
SEFIR domain; interleukin 17 receptor A; Act1 binding; IL-17 signaling
Asthma airway remodeling is linked to T helper-2 (TH2) inflammation. Angiogenesis is a consistent feature of airway remodeling, but its contribution to pathophysiology remains unclear. We hypothesized that nascent endothelial cells in newly forming vessels are sufficient to initiate TH2-inflammation. VE-cadherin is a constitutively expressed endothelial cell adhesion molecule, which is exposed in its monomer form on endothelial tip cells prior to adherens junction formation. Antibody targeted to VE-cadherin monomers inhibits angiogenesis by blocking this adherens junction formation. Here, VE-cadherin monomer antibody reduced angiogenesis in the lungs of the allergen-induced murine asthma model. Strikingly, TH2 responses including, IgE production, eosinophil infiltration of the airway, subepithelial fibrosis, mucus metaplasia and airway-hyperreactivity were also attenuated by VE-cadherin blockade, via mechanisms that blunted endothelial IL-25 and proangiogenic progenitor cell TSLP production. The results identify angiogenic responses in the origins of atopic inflammation.
VE-Cadherin; angiogenesis; asthma; Th2; endothelium
Interleukin 17 (IL-17) cytokines play a crucial role in a variety of inflammatory and autoimmune diseases. They signal through heterodimeric receptor complexes consisting of members of IL-17 receptor (IL-17R) family. A unique intracellular signaling domain was identified within all IL-17Rs, termed SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R]. SEFIR is also found in nuclear factor κB (NF-κB) activator 1 (Act1), an E3 ubiquitin ligase, and mediates its recruitment to IL-17Rs. Here we report the structure of the first SEFIR domain from IL-17RB at 1.8Å resolution. SEFIR displays a five-stranded parallel β-sheet that is wrapped by six helices. Site-directed mutagenesis on IL-17RB identified helix αC as being critical for its interaction with Act1 and IL-25 (IL-17E) signaling. Using the current SEFIR structure as a template, the key functional residues in Act1 are also mapped as part of helix αC, which is conserved in IL-17RA and RC, suggesting this helix as a common structural signature for heterotypic SEFIR-SERIR association. On the other hand, helix αB′ is important for homo-dimerization of Act1, implicating a dual ligand-binding model for SEFIR domain, with distinct structural motifs participating in either homotypic or heterotypic interactions. Furthermore, although IL-17RB-SEFIR structure resembles closest to the Toll/Interleukin-1 receptor (TIR) domain of TLR10 with low sequence homology, substantial differences were observed at helices αC, αD and DD′ loop. This study provides the first structural view of the IL-17 receptor intracellular signaling, unraveling the mechanism for the specificity of SEFIR versus TIR domain in their respective signaling pathways.
Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.