RESUMO
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the generation of anti-DNA autoantibodies due to exposure of immune cells to excessive amounts of extracellular DNA. Lack of P-selectin in mice induces the development of a lupus-like syndrome and patients with cutaneous lupus have reduced P-selectin expression in skin vessels. Using flow cytometry we analyzed in healthy donors and patients the expression of P-selectin Glycoprotein Ligand-1 (PSGL-1) in circulating neutrophils and the implication of PSGL-1/P-selectin interaction in neutrophil extracellular traps (NETs) generation. We found a statistical significance that neutrophils from active SLE patients have a reduced expression of PSGL-1 and low levels of PSGL-1 in neutrophils from SLE patients associated with the presence of anti-dsDNA antibodies, clinical lung involvement, Raynaud's phenomenon, and positive lupus anticoagulant. PSGL-1 is present along the DNA in the NET. In healthy donors, neutrophil interaction with immobilized P-selectin triggers Syk activation, increases the NETs percentage and reduces the amount of DNA extruded in the NETs. In active SLE patients, neutrophil interaction with P-selectin does not activate Syk or reduce the amount of DNA extruded in the NETs, that might contribute to increase the extracellular level of DNA and hence, to disease pathogenesis.
Assuntos
Doenças Autoimunes , Armadilhas Extracelulares , Lúpus Eritematoso Sistêmico , Animais , Camundongos , Doenças Autoimunes/metabolismo , DNA/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Selectina-P/metabolismo , HumanosRESUMO
Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Raios Ultravioleta/efeitos adversos , Proteínas Quinases Ativadas por AMP , Animais , Animais Recém-Nascidos , Apoptose/genética , Apoptose/efeitos da radiação , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Modelos Animais de Doenças , Fator de Crescimento de Hepatócito/genética , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Camundongos Transgênicos , Neoplasias de Células Escamosas/etiologia , Neoplasias de Células Escamosas/patologia , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
The mechanisms that work alongside BRAFV600E oncogene in melanoma development, in addition to ultraviolet (UV) radiation (UVR), are of great interest. Analysis of human melanoma tumors [data from The Cancer Genome Atlas (TCGA)] revealed that 50% or more of the samples expressed no or low amounts of serine/threonine protein kinase STK11 (also known as LKB1) protein. Here, we report that, in a mouse model, concomitant neonatal BrafV600E activation and Lkb1 tumor suppressor ablation in melanocytes led to full melanoma development. A single postnatal dose of UVB radiation had no effect on melanoma onset in Lkb1-depleted mice compared with BrafV600E-irradiated mice, but increased tumor multiplicity. In concordance with these findings and previous reports, Lkb1-null irradiated mice exhibited deficient DNA damage repair (DDR). Histologically, tumors lacking Lkb1 were enriched in neural-like tumor morphology. Genetic profiling and gene set enrichment analyses of tumor sample mutated genes indicated that loss of Lkb1 promoted the selection of altered genes associated with neural differentiation processes. Thus, these results suggest that the loss of Lkb1 cooperates with BrafV600E and UVR, impairing the DDR and increasing melanoma multiplicity and neural-like dedifferentiation.
RESUMO
Background: Early diagnosis and treatment of Systemic lupus erythematosus (SLE) and Systemic sclerosis (SSc) present significant challenges for clinicians. Although various studies have observed changes in serum levels of selectins between healthy donors and patients with autoimmune diseases, including SLE and SSc, their potential as biomarkers has not been thoroughly explored. We aimed to investigate serum profiles of PSGL-1 (sPSGL-1), ADAM8 (sADAM8) and P-, E- and L-selectins (sP-, sE- and sL-selectins) in defined SLE and SSc patient cohorts to identify disease-associated molecular patterns. Methods: We collected blood samples from 64 SLE patients, 58 SSc patients, and 81 healthy donors (HD). Levels of sPSGL-1, sADAM8 and selectins were analyzed by ELISA and leukocyte membrane expression of L-selectin and ADAM8 by flow cytometry. Results: Compared to HD, SLE and SSc patients exhibited elevated sE-selectin and reduced sL-selectin levels. Additionally, SLE patients exhibited elevated sPSGL-1 and sADAM8 levels. Compared to SSc, SLE patients had decreased sL-selectin and increased sADAM8 levels. Furthermore, L-selectin membrane expression was lower in SLE and SSc leukocytes than in HD leukocytes, and ADAM8 membrane expression was lower in SLE neutrophils compared to SSc neutrophils. These alterations associated with some clinical characteristics of each disease. Using logistic regression analysis, the sL-selectin/sADAM8 ratio in SLE, and a combination of sL-selectin/sE-selectin and sE-selectin/sPSGL-1 ratios in SSc were identified and cross-validated as potential serum markers to discriminate these patients from HD. Compared to available diagnostic biomarkers for each disease, both sL-selectin/sADAM8 ratio for SLE and combined ratios for SSc provided higher sensitivity (98% SLE and and 67% SSc correctly classified patients). Importantly, the sADAM8/% ADAM8(+) neutrophils ratio discriminated between SSc and SLE patients with the same sensitivity and specificity than current disease-specific biomarkers. Conclusion: SLE and SSc present specific profiles of sPSGL-1, sE-, sL-selectins, sADAM8 and neutrophil membrane expression which are potentially relevant to their pathogenesis and might aid in their early diagnosis.
Assuntos
Proteínas ADAM , Biomarcadores , Lúpus Eritematoso Sistêmico , Glicoproteínas de Membrana , Proteínas de Membrana , Escleroderma Sistêmico , Humanos , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Feminino , Biomarcadores/sangue , Masculino , Proteínas ADAM/sangue , Adulto , Pessoa de Meia-Idade , Glicoproteínas de Membrana/sangue , Proteínas de Membrana/sangue , IdosoRESUMO
BACKGROUND AND PURPOSE: Interstitial lung disease (ILD) is the main cause of mortality in systemic sclerosis (SSc), and current therapies available are of low efficacy or high toxicity. Thus, the identification of innovative less toxic and high efficacy therapeutic approaches to ILD treatment is an urgent need. The interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin initiates leukocyte extravasation and deletion of the corresponding gene (Selplg) induces a SSc-like syndrome with high incidence of ILD in aged mice. EXPERIMENTAL APPROACH: Aged PSGL-1 KO (Selplg-/- ) mice were used to assess the therapeutic effects of nanotherapy with everolimus, included in liposomes decorated with high MW hyaluronic acid (LipHA+Ev) and administered intratracheally to specifically target CD44-expressing lung cells. KEY RESULTS: PSGL-1 KO mice had increased numbers of CD45+ and CD45- cells, including alveolar and interstitial macrophages, eosinophils, granulocytes and NK cells, and myofibroblasts in bronchoalveolar lavage (BAL). CD45+ and CD45- cells expressing pro-inflammatory and pro-fibrotic cytokines were also increased. Lungs from PSGL-1 KO mice showed increased immune cell infiltration and apoptosis and exacerbated interstitial and peribronchial fibrosis. Targeted nanotherapy with LipHA+Ev decreased the myofibroblasts in BAL, cells producing proinflammatory and profibrotic cytokines, and the degree of lung inflammation at histology. LipHA+Ev treatment also decreased the severity of peribronchial and interstitial lung fibrosis, from moderate to mild levels. CONCLUSIONS AND IMPLICATIONS: In PSGL-1 KO mice, targeted nanotherapy with LipHA+Ev was an effective treatment for SSc-ILD, reducing the number of inflammatory and fibrotic cells in BAL and reducing inflammation and fibrosis in lungs.
Assuntos
Doenças Pulmonares Intersticiais , Fibrose Pulmonar , Escleroderma Sistêmico , Animais , Citocinas , Everolimo/farmacologia , Everolimo/uso terapêutico , Fibrose , Inflamação/patologia , Pulmão/patologia , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/etiologia , Glicoproteínas de Membrana , Camundongos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/genética , Escleroderma Sistêmico/patologiaRESUMO
PSGL-1 is expressed in all plasma cells, but only in a small percentage of circulating B cells. Patients with systemic sclerosis (SSc) show reduced expression of PSGL-1 in B cells and increased prevalence of pulmonary arterial hypertension. PSGL-1 deficiency leads to a SSc-like syndrome and SSc-associated pulmonary hypertension in female mice. In this work, the expression of PSGL-1 was assessed during murine B cell development in the bone marrow and in several peripheral and spleen B cell subsets. The impact of PSGL-1 absence on B cell biology was also evaluated. Interestingly, the percentage of PSGL-1 expressing cells and PSGL-1 expression levels decreased in the transition from common lymphoid progenitors to immature B cells. PSGL-1-/- mice showed reduced frequencies of peripheral B cells and reduced B cell lineage-committed precursors in the bone marrow. In the spleen of WT mice, the highest percentages of PSGL-1+ populations were shown by Breg (90%), B1a (34.7%), and B1b (19.1%), while only 2.5-8% of B2 cells expressed PSGL-1; however, within B2 cells, the class-switched subsets showed the highest percentages of PSGL-1+ cells. Interestingly, PSGL-1-/- mice had increased IgG+ and IgD+ subsets and decreased IgA+ population. Of note, the percentage of PSGL-1+ cells was increased in all the B cell subclasses studied in peritoneal fluid. Furthermore, PSGL-1 engagement during in vitro activation with anti-IgM and anti-CD40 antibodies of human peripheral B cells, blocked IL-10 expression by activated human B cells. Remarkably, PSGL-1 expression in circulating plasma cells was reduced in pulmonary arterial hypertension patients. In summary, although the expression of PSGL-1 in mature B cells is low, the lack of PSGL-1 compromises normal B cell development and it may also play a role in the maturation and activation of peripheral naïve B cells.
Assuntos
Linfócitos B/imunologia , Glicoproteínas de Membrana/imunologia , Hipertensão Arterial Pulmonar/imunologia , Idoso , Animais , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Cavidade Peritoneal/citologia , Baço/citologia , Baço/imunologiaRESUMO
OBJECTIVE: Pulmonary arterial hypertension (PAH), one of the major complications of systemic sclerosis (SSc), is a rare disease with unknown etiopathogenesis and noncurative treatments. As mice deficient in P-selectin glycoprotein ligand 1 (PSGL-1) develop a spontaneous SSc-like syndrome, we undertook this study to analyze whether they develop PAH and to examine the molecular mechanisms involved. METHODS: Doppler echocardiography was used to estimate pulmonary pressure, immunohistochemistry was used to assess vascular remodeling, and myography of dissected pulmonary artery rings was used to analyze vascular reactivity. Angiotensin II (Ang II) levels were quantified by enzyme-linked immunosorbent assay, and Western blotting was used to measure Ang II type 1 receptor (AT1 R), AT2 R, endothelial cell nitric oxide synthase (eNOS), and phosphorylated eNOS expression in lung lysates. Flow cytometry allowed us to determine cytokine production by immune cells and NO production by endothelial cells. In all cases, there were 4-8 mice per experimental group. RESULTS: PSGL-1-/- mice showed lung vessel wall remodeling and a reduced mean ± SD expression of pulmonary AT2 R (expression ratio [relative to ß-actin] in female mice age >18 months: wild-type mice 0.799 ± 0.508 versus knockout mice 0.346 ± 0.229). With aging, female PSGL-1-/- mice had impaired up-regulation of estrogen receptor α (ERα) and developed lung vascular endothelial dysfunction coinciding with an increase in mean ± SEM pulmonary Ang II levels (wild-type 48.70 ± 5.13 pg/gm lung tissue versus knockout 78.02 ± 28.09 pg/gm lung tissue) and a decrease in eNOS phosphorylation, leading to reduced endothelial NO production. These events led to a reduction in the pulmonary artery acceleration time:ejection time ratio in 33% of aged female PSGL-1-/- mice, indicating pulmonary hypertension. Importantly, we found expanded populations of interferon-γ-producing PSGL-1-/- T cells and B cells and a reduced presence of regulatory T cells. CONCLUSION: The absence of PSGL-1 induces a reduction in Treg cells, NO production, and ERα expression and causes an increase in Ang II in the lungs of female mice, favoring the development of PAH.
Assuntos
Hipertensão Pulmonar/genética , Glicoproteínas de Membrana/deficiência , Escleroderma Sistêmico/genética , Angiotensina II/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/biossíntese , Remodelação Vascular/genéticaRESUMO
Elucidating the contribution of somatic mutations to cancer is essential for personalized medicine. STK11 (LKB1) appears to be inactivated in human cancer. However, somatic missense mutations also occur, and the role/s of these alterations to this disease remain unknown. Here, we investigated the contribution of four missense LKB1 somatic mutations in tumor biology. Three out of the four mutants lost their tumor suppressor capabilities and showed deficient kinase activity. The remaining mutant retained the enzymatic activity of wild type LKB1, but induced increased cell motility. Mechanistically, LKB1 mutants resulted in differential gene expression of genes encoding vesicle trafficking regulating molecules, adhesion molecules and cytokines. The differentially regulated genes correlated with protein networks identified through comparative secretome analysis. Notably, three mutant isoforms promoted tumor growth, and one induced inflammation-like features together with dysregulated levels of cytokines. These findings uncover oncogenic roles of LKB1 somatic mutations, and will aid in further understanding their contributions to cancer development and progression.
Assuntos
Biomarcadores Tumorais/genética , Movimento Celular , Inflamação/patologia , Neoplasias Pulmonares/patologia , Melanoma/patologia , Mutação de Sentido Incorreto , Proteínas Serina-Treonina Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Camundongos Nus , Fosforilação , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intestinal helminth infections are common and of paramount economic importance in domestic animals. Available chemotherapy is limited and anthelmintic resistance is widespread in some hosts. This scenario favors the exploration of alternative methods of control, among them immune modulators. The effect of Escherichia coli LPS+Propionibacterium acnes on a primary infection of Heligmosomoides polygyrus (Trichostongyloidea) in mice has been tested. Nematode infection induced a rise of specific IgG1, both serum and intestinal, and a significant reduction in the unspecific (ConA) lymphoproliferative response. Treatment with the immune modulator (days -2, 0, 7 and 14 post infection) elicited an apparent delay of larval intramucosal development. Moreover cumulative nematode egg shedding in treated mice was significantly lower (p=0.0041). Preliminary results point toward the interest of immune modulators to control intestinal helminths.
Assuntos
Formação de Anticorpos/imunologia , Lipopolissacarídeos/farmacologia , Propionibacterium acnes/imunologia , Infecções por Strongylida/imunologia , Animais , Anticorpos Anti-Helmínticos/análise , Anticorpos Anti-Helmínticos/sangue , Formação de Anticorpos/efeitos dos fármacos , Fezes/parasitologia , Heligmosomatoidea/imunologia , Fatores Imunológicos/farmacologia , Imunomodulação/imunologia , Intestinos/parasitologia , Intestinos/patologia , Larva , Camundongos Endogâmicos BALB C , Contagem de Ovos de Parasitas , Carga ParasitáriaRESUMO
Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAF(V600E)) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAF(V600E)-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAF(V600E) oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAF(V600E) leads to lung adenoma development. Importantly, expression of BRAF(V600E) concomitant with the loss of only a single-copy of Lkb1, overcomes senencence-like features of BRAF(V600E)-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAF(V600E) mutated lung adenomas in human cancer patients.