RESUMEN
Ulcerative colitis (UC) is a chronic, relapsing and debilitating idiopathic inflammation, with variable and complex pathophysiologies. Our objective was to elucidate patterns of gene expression underlying the progression of UC disease. Single endoscopic pinch FFPE biopsies (n = 41) were sampled at both active and inactive stages at the same site in individual UC patients and compared with each other and with non-inflammatory bowel disease healthy controls. Gene expression results were validated by quantitative reverse transcriptase-PCR (QRT-PCR), and results at the protein level were validated by immunohistochemistry and western blot. Analysis of microarray results demonstrated that UC patients in remission display an intermediate gene expression phenotype between active UC patients and controls. It is clear that UC active site recovery does not revert fully back to a healthy control phenotype. Both UC active and inactive tissue displayed evidence, at both the gene expression and protein level, of a positive precancerous state as indicated by increases in the expression of Chitinase 3-Like-1, and the colorectal cancer metastasis marker MMP1. A key distinguishing feature between active and inactive UC, however, was the mobilization of marker genes and proteins for the Epithelial Mesenchymal Transition (EMT) pathway only in active UC. Analysis of the gene expression signatures associated with UC remission identified multiple pathways which appear to be permanently dysregulated in UC patients at formerly active sites in spite of clear histological recovery. Among these pathways, the EMT pathway was specifically up-regulated only in active UC emphasizing the potential for cancer progression in these patients.
Asunto(s)
Colitis Ulcerosa/metabolismo , Transición Epitelial-Mesenquimal , Proteínas de la Matriz Extracelular/biosíntesis , Regulación de la Expresión Génica , Metaloproteinasa 1 de la Matriz/biosíntesis , Adulto , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Masculino , Metaloproteinasa 1 de la Matriz/genética , Persona de Mediana EdadRESUMEN
Resistin-like molecule α (RELMα) has mitogenic, angiogenic, vasoconstrictive, and chemokine-like properties and is highly relevant in lung pathology. Here, we used RELMα knockout (Retnla(-/-)) mice to investigate the role of RELMα in pulmonary vascular remodeling after intermittent ovalbumin (OVA) challenge. We compared saline- and OVA-exposed wild-type (WT) mice and found that OVA induced significant increases in right ventricular systolic pressure, cardiac hypertrophy, pulmonary vascular remodeling of intra-alveolar arteries, goblet cell hyperplasia in airway epithelium, and intensive lung inflammation, especially perivascular inflammation. Genetic ablation of Retnla prevented the OVA-induced increase in pulmonary pressure and cardiac hypertrophy seen in WT mice. Histological analysis showed that Retnla(-/-) mice exhibited less vessel muscularization, less perivascular inflammation, reduced medial thickness of intra-alveolar vessels, and fewer goblet cells in upper airway epithelium (250-600 µm) than did WT animals after OVA challenge. Gene expression profiles showed that genes associated with vascular remodeling, including those related to muscle protein, contractile fibers, and actin cytoskeleton, were expressed at a lower level in OVA-challenged Retnla(-/-) mice than in similarly treated WT mice. In addition, bronchoalveolar lavage from OVA-challenged Retnla(-/-) mice had lower levels of cytokines, such as IL-1ß, -1 receptor antagonist, and -16, chemokine (C-X-C motif) ligand 1, -2, -9, -10, and -13, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, TIMP metallopeptidase inhibitor-1, and triggering receptor expressed on myeloid cells-1, than did that from WT mice when analyzed by cytokine array dot blots. Retnla knockout inhibited the OVA-induced T helper 17 response but not the T helper 2 response. Altogether, our results suggest that RELMα is involved in immune response-induced pulmonary vascular remodeling and the associated increase in inflammation typically observed after OVA challenge.
Asunto(s)
Hipertensión Pulmonar/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Remodelación Vascular/inmunología , Alérgenos/inmunología , Animales , Citocinas/metabolismo , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/genética , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Noqueados , Ovalbúmina/inmunologíaRESUMEN
Over expression of hepcidin antimicrobial peptide is a common feature of iron-restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP-011, a "murinized" ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, ß-thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor-ß superfamily members. We found that erythropoietin and RAP-011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP-011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin-treated mice exhibited iron-restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP-011-treated mice did not exhibit the same degree of iron-restricted erythropoiesis. In conclusion, we have demonstrated that RAP-011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP-011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP-011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron-restricted erythropoiesis.
Asunto(s)
Eritropoyesis/efectos de los fármacos , Hemoglobinas/análisis , Hepcidinas/genética , Hierro/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Receptores de Activinas Tipo II/química , Animales , Transporte Biológico , Recuento de Células Sanguíneas , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Femenino , Inmunoglobulina G/química , Hierro/sangre , Ligandos , Ratones Endogámicos C57BL , Ratones Transgénicos , Bazo/metabolismoRESUMEN
BACKGROUND: Sepsis is a leading cause of mortality in intensive care units worldwide. A better understanding of the blood systems response to sepsis should expedite the identification of biomarkers for early diagnosis and therapeutic interventions. METHODS: We analyzed microarray studies whose data is available from the GEO repository and which were performed on the whole blood of septic patients and normal controls. RESULTS: We identified 6 cohorts consisting of 450 individuals (sepsis = 323, control = 127) providing genome-wide messenger RNA (mRNA) expression data. Through meta-analysis we found the "Lysosome" and "Cytoskeleton" pathways were upregulated in human sepsis patients relative to controls, in addition to previously known signaling pathways (including MAPK, TLR). The key regulatory genes in the "Lysosome" pathway include lysosomal acid hydrolases (e.g., protease cathepsin A, D) as well as the major (LAMP1, 2) and minor (SORT1, LAPTM4B) membrane proteins. In contrast, pathways related to "Ribosome", "Spliceosome" and "Cell adhesion molecules" were found to be downregulated, along with known pathways for immune dysfunction. Overall, our study revealed distinct mRNA activation profiles and protein-protein interaction networks in blood of human sepsis. CONCLUSIONS: Our findings suggest that aberrant mRNA expression in the lysosome and cytoskeleton pathways may play a pivotal role in the molecular pathobiology of human sepsis.
Asunto(s)
Citoesqueleto/metabolismo , Lisosomas/metabolismo , Transcriptoma , Humanos , Sistema de Señalización de MAP Quinasas , Sepsis/sangre , Transducción de SeñalRESUMEN
Elucidating the molecular pathways active in pathologic tissues has important implications for defining disease subsets, selecting therapy, and monitoring disease activity. The development of therapeutics directed at IFN-α or IFN-γ makes the discovery of probes that report precisely on the activity of different IFN pathways a high priority. We show that, although type I and II IFNs induce the expression of a largely overlapping group of molecules, precise probes of IFN-γ activity can be defined. Used in combination, these probes show prominent IFN-γ effects in Sjögren syndrome (SS) tissues. In contrast, dermatomyositis muscle shows a dominant type I IFN pattern. Interestingly, heterogeneity of IFN signatures exists in patients with SS, with some patients demonstrating a predominant type I pattern. The biochemical patterns largely distinguish the target tissues in patients with SS from those with dermatomyositis and provide a relative weighting of the effects of distinct IFN pathways in specific biopsies. In SS, type I and II IFN effects are localized to the same epithelial cells, surrounded by inflammatory cells expressing IFN-γ-induced proteins, suggesting reinforcing interactions. Precise probes of the different IFN pathways active in tissues of complex rheumatic diseases will be critical to classify disease, elucidate pathogenesis, and select therapy.
Asunto(s)
Interferón gamma/fisiología , Enfermedades Reumáticas/fisiopatología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Interferón gamma/metabolismo , Glándulas Salivales/citología , Glándulas Salivales/metabolismoRESUMEN
BACKGROUND: Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing. OBJECTIVE: We evaluated 6706 cis-acting expression-associated variants (eSNPs) identified through a genome-wide eQTL survey of CD4(+) lymphocytes for association with asthma. METHODS: eSNPs were tested for association with asthma in 359 asthmatic patients and 846 control subjects from the Childhood Asthma Management Program, with verification by using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by using formaldehyde-assisted isolation of regulatory elements (FAIRE) quantitative PCR and chromatin immunoprecipitation PCR in lung-derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes. RESULTS: Cis-acting eSNPs demonstrated associations with asthma in both cohorts. We confirmed the previously reported association of ORMDL3/GSDMB variants with asthma (combined P = 2.9 × 10(-8)). Reproducible associations were also observed for eSNPs in 3 additional genes: fatty acid desaturase 2 (FADS2; P = .002), N-acetyl-α-D-galactosaminidase (NAGA; P = .0002), and Factor XIII, A1 (F13A1; P = .0001). Subsequently, we demonstrated that FADS2 mRNA is increased in CD4(+) lymphocytes in asthmatic patients and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity. CONCLUSIONS: Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.
Asunto(s)
Asma , Linfocitos T CD4-Positivos/inmunología , Ácido Graso Desaturasas , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , alfa-N-Acetilgalactosaminidasa , Asma/epidemiología , Asma/genética , Asma/inmunología , Asma/patología , Linfocitos T CD4-Positivos/patología , Niño , Preescolar , Costa Rica , Método Doble Ciego , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/inmunología , Femenino , Humanos , Masculino , alfa-N-Acetilgalactosaminidasa/genética , alfa-N-Acetilgalactosaminidasa/inmunologíaRESUMEN
Pulmonary hypertension (PH) is characterized by elevated pulmonary artery pressure that leads to progressive right heart failure and ultimately death. Injury to endothelium and consequent wound repair cascades have been suggested to trigger pulmonary vascular remodeling, such as that observed during PH. The relationship between injury to endothelium and disease pathogenesis in this disorder remains poorly understood. We and others have shown that, in mice, hypoxia-induced mitogenic factor (HIMF, also known as FIZZ1 or RELMα) plays a critical role in the pathogenesis of lung inflammation and the development of PH. In this study, we dissected the mechanism by which HIMF and its human homolog resistin (hRETN) induce pulmonary endothelial cell (EC) apoptosis and subsequent lung inflammation-mediated PH, which exhibits many of the hallmarks of the human disease. Systemic administration of HIMF caused increases in EC apoptosis and interleukin (IL)-4-dependent vascular inflammatory marker expression in mouse lung during the early inflammation phase. In vitro, HIMF, hRETN, and IL-4 activated pulmonary microvascular ECs (PMVECs) by increasing angiopoietin-2 expression and induced PMVEC apoptosis. In addition, the conditioned medium from hRETN-treated ECs had elevated levels of endothelin-1 and caused significant increases in pulmonary vascular smooth muscle cell proliferation. Last, HIMF treatment caused development of PH that was characterized by pulmonary vascular remodeling and right heart failure in wild-type mice but not in IL-4 knockout mice. These data suggest that HIMF contributes to activation of vascular inflammation at least in part by inducing EC apoptosis in the lung. These events lead to subsequent PH.
Asunto(s)
Apoptosis/fisiología , Células Endoteliales/metabolismo , Hipertensión Pulmonar/metabolismo , Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucina-4/metabolismo , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Células Endoteliales/citología , Hipertensión Pulmonar/patología , Interleucina-4/genética , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía/metabolismo , Neumonía/patologíaRESUMEN
Increased hepcidin antimicrobial peptide correlates with hypoferremia and anemia in various disease states, but its requirement for anemia of inflammation has not been adequately demonstrated. Anemia of inflammation is usually described as normocytic and normochromic, while diseases associated with over expression of hepcidin, alone, are often microcytic and hypochromic. These differences in erythrocyte parameters suggest anemia in many inflammatory states may not be fully explained by hepcidin-mediated iron sequestration. We used turpentine-induced sterile abscesses to model chronic inflammation in mice with targeted disruption of Hepcidin 1 [Hepc1 (-/-)] or its positive regulator, Interleukin-6 [IL-6 (-/-)], to determine whether these genes are required for features characteristic of anemia of inflammation. Although hemoglobin levels did not decline in Hepc1 (-/-) mice with sterile abscesses, erythrocyte numbers were significantly reduced compared to untreated Hepc1 (-/-) mice. In contrast, both hemoglobin concentration and erythrocyte number declined significantly in wild type and IL-6 (-/-) mice with sterile abscesses. Both Hepc1 (-/-) and IL-6 (-/-) mice had increased erythrocyte mean cell volume and mean cell hemoglobin following sterile abscesses, while wild types had no change. Thus, IL-6 (-/-) mice with sterile abscesses exhibit an intermediate phenotype between wild type and Hepc1 (-/-). Our results demonstrate the requirement of Hepc1 for the development of anemia in this rodent model. Simultaneously, our results demonstrate hepcidin-independent effects of inflammation on the suppression of erythropoiesis. Our results suggest chronic anemia associated with inflammation may benefit from interventions protecting erythrocyte number in addition to anti-hepcidin interventions aimed at enhancing iron availability.
Asunto(s)
Anemia/sangre , Eritropoyesis/fisiología , Hepcidinas/sangre , Inflamación/sangre , Anemia/patología , Animales , Modelos Animales de Enfermedad , Femenino , Inmunofenotipificación , Inflamación/patología , Hierro/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Patients with genetic defects of the cyclic (c) adenosine-monophosphate (AMP)-signaling pathway and those with neonatal-onset multisystem inflammatory disease (NOMID) develop tumor-like lesions of the long bones. The molecular basis of this similarity is unknown. NOMID is caused by inappropriate caspase-1 activity, which in turn activates the inflammasome. The present study demonstrates that NOMID bone lesions are derived from the same osteoblast progenitor cells that form fibroblastoid tumors in mice and humans with defects that lead to increased cAMP-dependent protein kinase A (PKA) signaling. NOMID tumor cells showed high PKA activity, and an increase in their cAMP signaling led to PKA-specific activation of caspase-1. Increased PKA led to inflammation-independent activation of caspase-1 via over-expression of the proto-oncogene (and early osteoblast factor) Ets-1. In NOMID tumor cells, as in cells with defective PKA regulation, increased prostaglandin E2 (PGE2) led to increased cAMP levels and activation of Wnt signaling, like in other states of inappropriate PKA activity. Caspase-1 and PGE2 inhibition led to a decrease in cell proliferation of both NOMID and cells with abnormal PKA. These data reveal a previously unsuspected link between abnormal cAMP signaling and defective regulation of the inflammasome and suggest that caspase-1 and PGE2 inhibition may be therapeutic targets in bone lesions associated with defects of these two pathways.
Asunto(s)
Síndromes Periódicos Asociados a Criopirina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Osteoblastos/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Células Madre/metabolismo , Animales , Huesos/patología , Caspasa 1/genética , Células Cultivadas , Síndromes Periódicos Asociados a Criopirina/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Dinoprostona/genética , Dinoprostona/metabolismo , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Ratones , Proto-Oncogenes Mas , Proteína Proto-Oncogénica c-ets-1/genética , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Células del Estroma/metabolismo , Activación Transcripcional/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
Anemia is common in older adults and associated with adverse health outcomes in epidemiological studies. A thorough understanding of the complex pathophysiological mechanisms driving anemia in the elderly is lacking; but inflammation, iron restriction, and impaired erythroid maturation are thought to influence the phenotype. We hypothesized that interleukin-6 contributes to this anemia, given its pro-inflammatory activities, its ability to induce hepcidin antimicrobial peptide, and its negative impact on several tissues in older adults. We tested this hypothesis by comparing changes in indices of inflammation, iron metabolism and erythropoiesis in aged C57BL/6 mice to aged mice with targeted deletions of interleukin-6 or hepcidin antimicrobial peptide. Circulating neutrophil and monocyte numbers and inflammatory cytokines increased with age. Decline in hemoglobin concentration and red blood cell number indicated that C57BL/6, interleukin-6 knockout mice, and hepcidin antimicrobial peptide knockout mice all demonstrated impaired erythropoiesis by 24 months. However, the interleukin-6 knock out genotype and the hepcidin antimicrobial peptide knock out genotype resulted in improved erythropoiesis in aged mice. Increased erythropoietic activity in the spleen suggested that the erythroid compartment was stressed in aged C57BL/6 mice compared to aged interleukin-6 knockout mice. Our data suggest C57BL/6 mice are an appropriate mammalian model for the study of anemia with age. Furthermore, although interleukin-6 and hepcidin antimicrobial peptide are not required, they can participate in the development of anemia in aging mice, and could be targeted, pre-clinically, with existing interventions to determine the feasibility of such agents for the treatment of anemia in older adults.
Asunto(s)
Envejecimiento/genética , Envejecimiento/metabolismo , Anemia/sangre , Anemia/genética , Hepcidinas/fisiología , Interleucina-6/fisiología , Animales , Femenino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de la EspecieRESUMEN
BACKGROUND: Both chronic hypoxia and allergic inflammation induce vascular remodeling in the lung, but only chronic hypoxia appears to cause PH. We investigate the nature of the vascular remodeling and the expression and role of hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) in explaining this differential response. METHODS: We induced pulmonary vascular remodeling through either chronic hypoxia or antigen sensitization and challenge. Mice were evaluated for markers of PH and pulmonary vascular remodeling throughout the lung vascular bed as well as HIMF expression and genomic analysis of whole lung. RESULTS: Chronic hypoxia increased both mean pulmonary artery pressure (mPAP) and right ventricular (RV) hypertrophy; these changes were associated with increased muscularization and thickening of small pulmonary vessels throughout the lung vascular bed. Allergic inflammation, by contrast, had minimal effect on mPAP and produced no RV hypertrophy. Only peribronchial vessels were significantly thickened, and vessels within the lung periphery did not become muscularized. Genomic analysis revealed that HIMF was the most consistently upregulated gene in the lungs following both chronic hypoxia and antigen challenge. HIMF was upregulated in the airway epithelial and inflammatory cells in both models, but only chronic hypoxia induced HIMF upregulation in vascular tissue. CONCLUSIONS: The results show that pulmonary vascular remodeling in mice induced by chronic hypoxia or antigen challenge is associated with marked increases in HIMF expression. The lack of HIMF expression in the vasculature of the lung and no vascular remodeling in the peripheral resistance vessels of the lung is likely to account for the failure to develop PH in the allergic inflammation model.
Asunto(s)
Antígenos , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neumonía/complicaciones , Arteria Pulmonar/metabolismo , Células Th2/inmunología , Animales , Presión Arterial , Aspergillus/inmunología , Enfermedad Crónica , Modelos Animales de Enfermedad , Hipertensión Pulmonar Primaria Familiar , Perfilación de la Expresión Génica , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/etiología , Hipertrofia Ventricular Derecha/inmunología , Hipertrofia Ventricular Derecha/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Neumonía/inmunología , Neumonía/patología , Arteria Pulmonar/inmunología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Regulación hacia ArribaRESUMEN
HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements and related motifs present in the 3'untranslated region (UTR) of mRNAs. We postulate that HuR critically regulates the epithelial response by associating with multiple ARE-bearing, functionally related inflammatory transcripts. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNF-α plus IFN-γ, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein complexes from resting and cytokine-treated cells were immunoprecipitated using anti-HuR and isotype-control Ab, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1, and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control immunoprecipitation. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene ribonucleoprotein-immunoprecipitation and shown to be 3'UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic. Conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. HuR-mediated regulation in airway epithelium appears broader than previously appreciated, coordinating numerous inflammatory genes through multiple posttranscriptional mechanisms.
Asunto(s)
Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Quimiocinas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Mucosa Respiratoria/inmunología , Proteínas Quinasas Activadas por AMP/fisiología , Biotinilación , Bronquios/inmunología , Bronquios/metabolismo , Bronquios/patología , Línea Celular Transformada , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Quimiocinas/genética , Proteínas ELAV , Proteína 1 Similar a ELAV , Humanos , Mediadores de Inflamación/fisiología , Unión Proteica/genética , Unión Proteica/inmunología , Estabilidad del ARN/genética , Estabilidad del ARN/inmunología , Reproducibilidad de los Resultados , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal/genética , Transducción de Señal/inmunología , Tráquea/inmunología , Tráquea/metabolismo , Tráquea/patología , Transcripción Genética/inmunologíaRESUMEN
Although T cells have been shown to play a direct role in kidney ischemia-reperfusion injury (IRI), little is known about the underlying mechanisms. We hypothesized that studying the transcriptional responses in kidney-infiltrating T cells would help elucidate novel therapeutic targets for kidney IRI. Unilateral renal pedicle clamping for 45 min was performed in male C57BL/6 mice, and CD3(+) T cells were isolated from the kidney and purified. Transcriptional activities of T cell were measured by array-based PCR compared between ischemic kidneys and contralateral nonischemic kidneys. Among total of 89 genes analyzed, 24, 22, 24, and 37 genes were significantly changed at 6 h, day 3, day 10, and day 28 after IRI. Genes associated with cytokines, chemokines, and costimulatory molecules were upregulated. Pathway analysis identified CC motif chemokine receptor 5 (CCR5) as a candidate pathophysiological pathway. CCR5 upregulation was validated at the protein level, and CCR5 blockade improved renal function after kidney IRI. Using discovery techniques to identify transcriptional responses in purified kidney-infiltrating cells enabled the elucidation of novel mechanisms and therapeutic targets for IRI.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Riñón/lesiones , Riñón/patología , Receptores CCR5/metabolismo , Daño por Reperfusión/fisiopatología , Linfocitos T/metabolismo , Animales , Anticuerpos , Complejo CD3/genética , Complejo CD3/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Análisis por Conglomerados , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Riñón/citología , Enfermedades Renales/metabolismo , Enfermedades Renales/fisiopatología , Masculino , Ratones , Receptores CCR5/genética , Daño por Reperfusión/metabolismo , Organismos Libres de Patógenos EspecíficosRESUMEN
PRKAR1A inactivation leads to dysregulated cAMP signaling and Carney complex (CNC) in humans, a syndrome associated with skin, endocrine and other tumors. The CNC phenotype is not easily explained by the ubiquitous cAMP signaling defect; furthermore, Prkar1a(+/-) mice did not develop skin and other CNC tumors. To identify whether a Prkar1a defect is truly a generic but weak tumorigenic signal that depends on tissue-specific or other factors, we investigated Prkar1a(+/-) mice when bred within the Rb1(+/-) or Trp53(+/-) backgrounds, or treated with a two-step skin carcinogenesis protocol. Prkar1a(+/-) Trp53(+/-) mice developed more sarcomas than Trp53(+/-) mice (P < 0.05) and Prkar1a(+/-) Rb1(+/-) mice grew more (and larger) pituitary and thyroid tumors than Rb1(+/-) mice. All mice with double heterozygosity had significantly reduced life-spans compared with their single-heterozygous counterparts. Prkar1a(+/-) mice also developed more papillomas than wild-type animals. A whole-genome transcriptome profiling of tumors produced by all three models identified Wnt signaling as the main pathway activated by abnormal cAMP signaling, along with cell cycle abnormalities; all changes were confirmed by qRT-PCR array and immunohistochemistry. siRNA down-regulation of Ctnnb1, E2f1 or Cdk4 inhibited proliferation of human adrenal cells bearing a PRKAR1A-inactivating mutation and Prkar1a(+/-) mouse embryonic fibroblasts and arrested both cell lines at the G0/G1 phase of the cell cycle. In conclusion, Prkar1a haploinsufficiency is a relatively weak tumorigenic signal that can act synergistically with other tumor suppressor gene defects or chemicals to induce tumors, mostly through Wnt-signaling activation and cell cycle dysregulation, consistent with studies in human neoplasms carrying PRKAR1A defects.
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Ciclo Celular , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Neoplasias/genética , Neoplasias/patología , Proteína de Retinoblastoma/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteínas Wnt/metabolismo , Animales , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Haploidia , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Neoplasias/fisiopatología , Procesos Neoplásicos , Papiloma/inducido químicamente , Papiloma/genética , Papiloma/metabolismo , Papiloma/fisiopatología , Proteína de Retinoblastoma/metabolismo , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/fisiopatología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Wnt/genéticaRESUMEN
BACKGROUND: We and others have shown previously that over-expression of hepcidin antimicrobial peptide, independently of inflammation, induces several features of anemia of inflammation and chronic disease, including hypoferremia, sequestration of iron stores and iron-restricted erythropoiesis. Because the iron-restricted erythropoiesis evident in hepcidin transgenic mice differs from the normocytic, normochromic anemia most often observed in anemia of inflammation, we tested the hypothesis that chronic inflammation may contribute additional features to anemia of inflammation which continue to impair erythropoiesis following the acute phase of inflammation in which hepcidin is active. DESIGN AND METHODS: We compared erythropoiesis and iron handling in mice with turpentine-induced sterile abscesses with erythropoiesis and iron handling in hepcidin transgenic mice. We compared erythrocyte indices, expression of genes in the hepcidin regulatory pathway, tissue iron distribution, expression of heme and iron transport genes in splenic macrophages, the phenotype of erythroid maturation and chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester fluorescence. RESULTS: Mice with sterile abscesses exhibited an intense, acute inflammatory phase followed by a mild to moderate chronic inflammatory phase. We found that erythrocytes in mice with sterile abscesses were normocytic and normochromic in contrast to those in hepcidin transgenic mice. We also observed that although hypoferremia resolved in the late phases of inflammation, erythropoiesis remained suppressed, with evidence of inefficient maturation of erythroid precursors in the bone marrow of mice with sterile abscesses. Finally, we observed increased oxidative stress in erythroid progenitors and circulating erythrocytes of mice with sterile abscesses which was not evident in hepcidin transgenic mice. CONCLUSIONS: Our results suggest that chronic inflammation inhibits late stages of erythroid production in the turpentine-induced sterile abscess model and induces features of impaired erythropoiesis which are distinct from those in hepcidin transgenic mice.
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Péptidos Catiónicos Antimicrobianos/biosíntesis , Células Precursoras Eritroides/metabolismo , Eritropoyesis , Animales , Péptidos Catiónicos Antimicrobianos/genética , Enfermedad Crónica , Células Precursoras Eritroides/patología , Hepcidinas , Inflamación/sangre , Inflamación/inducido químicamente , Inflamación/genética , Mediadores de Inflamación/sangre , Irritantes/efectos adversos , Irritantes/farmacología , Ratones , Ratones Transgénicos , Trementina/efectos adversos , Trementina/farmacologíaRESUMEN
BACKGROUND: Studies suggested that microRNAs influence cellular activities in the uterus including cell differentiation and embryo implantation. In assisted reproduction cycles, luteal phase support, given to improve endometrial characteristics and to facilitate the implantation process, has been a standard practice. The effect of different types of luteal phase support using steroid hormones in relation to endometrial miRNA profiles during the peri-implantation period has not seen described. This study was designed to evaluate the expression of miRNAs during the luteal phase following controlled ovarian stimulation for IVF and the influence of different luteal phase support protocols on miRNA profiles. METHODS: The study was approved by the Johns Hopkins Hospital Institutional Review Board. Endometrial biopsies were obtained on the day of oocyte retrieval from 9 oocyte donors (group I). An additional endometrial biopsy was obtained 3-5 days later (Group II) after the donors were randomized into three groups. Group IIa had no luteal-phase support, group IIb had luteal support with micronized progesterone (P), and Group IIc had luteal support with progesterone plus 17-beta-estradiol (P + E). Total RNA was isolated and microarray analysis was performed using an Illumina miRNA expression panel. RESULTS: A total of 526 miRNAs were identified. Out of those, 216 miRNAs were differentially regulated (p < 0.05) between the comparison groups. As compared to the day of retrieval, 19, 11 and 6 miRNAs were differentially regulated more than 2 fold in the groups of no support, in the P support only, and in the P + E support respectively, 3-5 days after retrieval. During the peri-implantation period (3-5 days after retrieval) the expression of 33 and 6 miRNAs increased, while the expression of 3 and 0 miRNAs decreased, in the P alone and in the P + E group respectively as compared to the no steroid supplementation group. CONCLUSION: Luteal support following COS has a profound influence on miRNA profiles. Up or down regulation of miRNAs after P or P + E support suggest a role(s) of luteal support in the peri-implantation uterus in IVF cycles through the regulation of associated target genes.
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Mantenimiento del Cuerpo Lúteo/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Endometrio/efectos de los fármacos , Fase Luteínica/efectos de los fármacos , MicroARNs/metabolismo , Inducción de la Ovulación , Regulación hacia Arriba/efectos de los fármacos , Adulto , Mantenimiento del Cuerpo Lúteo/metabolismo , Endometrio/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Femenino , Perfilación de la Expresión Génica , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Antagonistas de Hormonas/farmacología , Humanos , Fase Luteínica/metabolismo , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Donación de Oocito , Embarazo , Progesterona/farmacología , Progestinas/farmacología , Donantes de Tejidos , Adulto JovenRESUMEN
Hypoxia-induced mitogenic factor (HIMF), also known as found in inflammatory zone 1 and resistin-like molecule α, belongs to a novel class of cysteine-rich secreted proteins. It exhibits mitogenic and chemotactic properties during pulmonary hypertension-associated vascular remodeling, as well as fibrogenic properties during pulmonary fibrosis. HIMF expression in the lung was reported to be regulated by Th2 cytokines (IL-4 and IL-13) via the transcription factor STAT6 pathway in a bleomycin-induced pulmonary fibrosis model. However, in this study, we found that in the hypoxia-induced pulmonary hypertension model, lung HIMF expression is increased in IL-4 and STAT6 knockout (KO) mice to the same degree as in wild-type (WT) mice, suggesting that induction of HIMF expression does not require Th2 regulation in this model. We also found that HIMF-induced proliferative activity, hypertrophy, collagen, and extracellular matrix deposition in the pulmonary arteries are significantly less in IL-4 KO mice than in WT mice. In addition, HIMF-induced production of angiogenic factors/chemokines, such as vascular endothelial growth factor, MCP-1, and stromal-derived factor-1, in the lung resident cells, as well as macrophage infiltration, were significantly suppressed in the lungs of IL-4 KO mice. We also show that IL-4 was significantly increased in the lungs of HIMF-treated WT mice. Our in vitro studies using pulmonary microvascular endothelial cells revealed that HIMF stimulated cell proliferation, vascular endothelial growth factor expression, and MCP-1 production in a manner that is dependent on the IL-4/IL-4Rα system. These findings suggest that IL-4 signaling may play a significant role in HIMF-induced lung inflammation and vascular remodeling.
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Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucina-4/metabolismo , Neumonía/metabolismo , Transducción de Señal/inmunología , Animales , Movimiento Celular , Proliferación Celular , Células Endoteliales/inmunología , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Hipertensión Pulmonar/metabolismo , Immunoblotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/inmunología , Interleucina-4/inmunología , Pulmón/irrigación sanguínea , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Neumonía/inmunología , Neumonía/patología , Fibrosis Pulmonar/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. OBJECTIVE: We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1). METHODS: Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illumina's BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD. RESULTS: We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with T(H)2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations. CONCLUSION: Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.
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Dermatitis Atópica/patología , Uniones Estrechas/inmunología , Adulto , Edad de Inicio , Células Cultivadas , Claudina-1 , Dermatitis Atópica/epidemiología , Dermatitis Atópica/genética , Dermatitis Atópica/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido SimpleRESUMEN
A major complication in COVID-19 infection consists in the onset of acute respiratory distress fueled by a dysregulation of the host immune network that leads to a run-away cytokine storm. Here, we present an in silico approach that captures the host immune system's complex regulatory dynamics, allowing us to identify and rank candidate drugs and drug pairs that engage with minimal subsets of immune mediators such that their downstream interactions effectively disrupt the signaling cascades driving cytokine storm. Drug-target regulatory interactions are extracted from peer-reviewed literature using automated text-mining for over 5000 compounds associated with COVID-induced cytokine storm and elements of the underlying biology. The targets and mode of action of each compound, as well as combinations of compounds, were scored against their functional alignment with sets of competing model-predicted optimal intervention strategies, as well as the availability of like-acting compounds and known off-target effects. Top-ranking individual compounds identified included a number of known immune suppressors such as calcineurin and mTOR inhibitors as well as compounds less frequently associated for their immune-modulatory effects, including antimicrobials, statins, and cholinergic agonists. Pairwise combinations of drugs targeting distinct biological pathways tended to perform significantly better than single drugs with dexamethasone emerging as a frequent high-ranking companion. While these predicted drug combinations aim to disrupt COVID-induced acute respiratory distress syndrome, the approach itself can be applied more broadly to other diseases and may provide a standard tool for drug discovery initiatives in evaluating alternative targets and repurposing approved drugs.
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Tratamiento Farmacológico de COVID-19 , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Calcineurina , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Dexametasona , Humanos , SARS-CoV-2RESUMEN
BACKGROUND: Despite advances in screening and local therapy, prostate cancer remains the second most common cause of cancer related death among American men, with those having high grade disease being at highest risk for prostate cancer mortality. Here we identify the genes and cellular pathways that distinguish high grade from low grade pathologically localized prostate cancer. METHODS: Cancer cells from low grade (Gleason 3 + 3 = 6) or high grade (4 + 4 = 8) tumors of men with localized disease were isolated by laser capture micro-dissection. Expression profiling was conducted across 18,344 unique annotated genes and data were analyzed using packages from the R/Bioconductor project to determine differential gene expression and perform gene set enrichment analysis. Publically available expression data was retrieved and analyzed individually in the same manner and in cross platform meta-analyses. RESULTS: Six hundred seventy genes were differentially expressed between Gleason sum 6 and 8 tumors with a false discovery rate of <5% (P < 0.0014) including genes previously shown to mediate prostate cancer survival, proliferation, and metastasis. Functional themes associated with Gleason grade included developmental processes, signal transduction, chemokine and embryonic stem cell pathways with specific enrichment of the androgen receptor, EGFR, TNF-alpha, and Notch signaling cascades. CONCLUSIONS: In addition to androgen receptor signaling, growth factor, and cytokine mediated pathways are active in clinically localized high grade prostate cancer. The availability of therapeutics that selectively target these pathways encourages the development of clinical trials for their selective use in the neoadjuvant or adjuvant setting in men at high risk for disease progression.