RESUMEN

Extracellular vesicles (EVs) are evaginations of the cytoplasmic membrane, containing nucleic acids, proteins, lipids, enzymes, and toxins. EVs participate in various bacterial physiological processes. Staphylococcus epidermidis interacts and communicates with the host skin. S. epidermidis' EVs may have an essential role in this communication mechanism, modulating the immunological environment. This work aimed to evaluate if S. epidermidis' EVs can modulate cytokine production by keratinocytes in vitro and in vivo using the imiquimod-induced psoriasis murine model. S. epidermidis' EVs were obtained from a commensal strain (ATC12228EVs) and a clinical isolated strain (983EVs). EVs from both origins induced IL-6 expression in HaCaT keratinocyte cultures; nevertheless, 983EVs promoted a higher expression of the pro-inflammatory cytokines VEGF-A, LL37, IL-8, and IL-17F than ATCC12228EVs. Moreover, in vivo imiquimod-induced psoriatic skin treated with ATCC12228EVs reduced the characteristic psoriatic skin features, such as acanthosis and cellular infiltrate, as well as VEGF-A, IL-6, KC, IL-23, IL-17F, IL-36γ, and IL-36R expression in a more efficient manner than 983EVs; however, in contrast, Foxp3 expression did not significantly change, and IL-36 receptor antagonist (IL-36Ra) was found to be increased. Our findings showed a distinctive immunological profile induction that is dependent on the clinical or commensal EV origin in a mice model of skin-like psoriasis. Characteristically, proteomics analysis showed differences in the EVs protein content, dependent on origin of the isolated EVs. Specifically, in ATCC12228EVs, we found the proteins glutamate dehydrogenase, ornithine carbamoyltransferase, arginine deiminase, carbamate kinase, catalase, superoxide dismutase, phenol-soluble ß1/ß2 modulin, and polyglycerol phosphate α-glucosyltransferase, which could be involved in the reduction of lesions in the murine imiquimod-induced psoriasis skin. Our results show that the commensal ATCC12228EVs have a greater protective/attenuating effect on the murine imiquimod-induced psoriasis by inducing IL-36Ra expression in comparison with EVs from a clinical isolate of S. epidermidis.

Asunto(s)

Vesículas Extracelulares/metabolismo , Psoriasis/terapia , Staphylococcus epidermidis/metabolismo , Animales , Antígenos Ly/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Vesículas Extracelulares/química , Vesículas Extracelulares/trasplante , Humanos , Imiquimod/toxicidad , Interleucina-1/antagonistas & inhibidores , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Infiltración Neutrófila , Psoriasis/inducido químicamente , Psoriasis/patología , Piel/metabolismo , Piel/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Neutrophils are essential to control several fungal infections. These cells are commonly known for their pro-inflammatory activities. However, some studies have demonstrated the anti-inflammatory properties of neutrophils during certain infectious diseases, culminating in the inhibition of T cell proliferation. Chromoblastomycosis (CBM) is a deep and progressive mycosis that affects thousands of people worldwide. Although neutrophil infiltrates are observed in the lesion histopathology, the fungus can overtake the immune system response and destroy the host-infected tissue. The present study demonstrated that neutropenic animals had an increase in the IL-6 production in the spleen and liver, followed by a lower fungal burden in these organs up to 14 days of infection. Neutropenic animals also showed a lower F. pedrosoi-specific antibody production 14-days post infection and higher T-cell proliferation in the in vitro experiments after stimulation with F. pedrosoi-purified proteins. Taken together, our results suggest that the presence of regulatory neutrophils in the mouse model of F. pedrosoi infection could act favoring the spread of the fungus and the chronicity of the infection. These findings shed light on the CBM treatment, which might target neutrophil polarization as a new therapy approach to treat CBM lesions.

Asunto(s)

Anticuerpos/efectos adversos , Antígenos Ly/inmunología , Cromoblastomicosis/inmunología , Fonsecaea/patogenicidad , Neutropenia/inmunología , Neutrófilos/metabolismo , Linfocitos T/metabolismo , Animales , Polaridad Celular , Proliferación Celular , Cromoblastomicosis/complicaciones , Modelos Animales de Enfermedad , Fonsecaea/inmunología , Humanos , Interleucina-6/metabolismo , Hígado/inmunología , Activación de Linfocitos , Ratones , Neutropenia/inducido químicamente , Bazo/inmunología

RESUMEN

Echinococcus granulosus is a cestode parasite which causes cystic echinococcosis disease. Previously we observed that vaccination with E. granulosus antigens from human hydatid cyst fluid (HCF) significantly inhibits colon cancer growth. In the present work, we evaluate the anti-tumor immune response induced by human HCF against LL/2 lung cancer in mice. HCF vaccination protected from tumor growth, both in prophylactic and therapeutic settings, and significantly increased mouse survival compared to control mice. Considering that tumor-associated carbohydrate antigens are expressed in E. granulosus, we oxidized terminal carbohydrates in HCF with sodium periodate. This treatment abrogates the anti-tumor activity induced by HCF vaccination. We found that HCF vaccination-induced IgG antibodies that recognize LL/2 tumor cells by flow cytometry. An antigen-specific immune response is induced with HCF vaccination in the tumor-draining lymph nodes and spleen characterized by the production of IL-5 and, in less extent, IFNÉ£. In the tumor microenvironment, we found that NK1.1 positive cells from HCF-treated mice showed higher expression of CD69 than control mice ones, indicating a higher level of activation. When we depleted these cells by administrating the NK-specific antibody NK1.1, a significantly decreased survival was observed in HCF-induced mice, suggesting that NK1.1+ cells mediate the anti-tumor protection induced by HCF. These results suggest that HCF can evoke an integrated anti-tumor immune response involving both, the innate and adaptive components, and provide novel insights into the understanding of the intricate relationship between HCF vaccination and tumor growth.

Asunto(s)

Antígenos Ly/inmunología , Equinococosis/inmunología , Echinococcus granulosus/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Animales , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Humanos , Inmunidad/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Microambiente Tumoral/inmunología

RESUMEN

BACKGROUND: The role of myeloid-derived suppressor cells (MDSCs) in patients with severe tuberculosis who suffer from uncontrolled pulmonary inflammation caused by hypervirulent mycobacterial infection remains unclear. METHODS: This issue was addressed using C57BL/6 mice infected with highly virulent Mycobacterium bovis strain MP287/03. RESULTS: CD11b+GR1int population increased in the bone marrow, blood and lungs during advanced disease. Pulmonary CD11b+GR1int (Ly6GintLy6Cint) cells showed granularity similar to neutrophils and expressed immature myeloid cell markers. These immature neutrophils harbored intracellular bacilli and were preferentially located in the alveoli. T-cell suppression occurred concomitantly with CD11b+GR1int cell accumulation in the lungs. Furthermore, lung and bone marrow GR1+ cells suppressed both T-cell proliferation and interferon γ production in vitro. Anti-GR1 therapy given when MDSCs infiltrated the lungs prevented expansion and fusion of primary pulmonary lesions and the development of intragranulomatous caseous necrosis, along with increased mouse survival and partial recovery of T-cell function. Lung bacterial load was reduced by anti-GR1 treatment, but mycobacteria released from the depleted cells proliferated extracellularly in the alveoli, forming cords and clumps. CONCLUSIONS: Granulocytic MDSCs massively infiltrate the lungs during infection with hypervirulent mycobacteria, promoting bacterial growth and the development of inflammatory and necrotic lesions, and are promising targets for host-directed therapies.

Asunto(s)

Granulocitos , Pulmón/metabolismo , Mycobacterium bovis , Células Supresoras de Origen Mieloide , Tuberculosis , Animales , Antígenos Ly , Médula Ósea , Antígeno CD11b , Proliferación Celular , Modelos Animales de Enfermedad , Granulocitos/inmunología , Inmunomodulación , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Mycobacterium bovis/patogenicidad , Células Mieloides , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/patología , Neutrófilos , Tuberculosis/patología

RESUMEN

Monocytes play key roles in the maintenance of homeostasis and in the control of the infection. Monocytes are recruited from the bone marrow to inflammatory sites and are essential for antimicrobial activity to limit tissue damage and promote adaptive T cell responses. Here, we investigated the role of Nuclear Factor of Activated T cells 1 (NFAT1) in the regulation of Ly6Chi inflammatory monocyte recruitment to the CNS upon T. gondii infection. We show that NFAT-1-deficient monocytes are unable to migrate to the CNS of T. gondii-infected mice. Moreover, NFAT1-/- mice are highly susceptible to chronic T. gondii infection due to a failure to control parasite replication in the CNS. The inhibition of Ly6Chi inflammatory monocyte recruitment to the CNS severely blocked CXCL10 production and consequently the migration of IFN-γ-producing CD4+ T cells. Moreover, the transfer of Ly6Chi monocytes to infected NFAT1-/- mice favored CD4+ T cell migration to the CNS and resulted in the inhibition of parasite replication and host defense. Together, these results demonstrated for the first time the contribution of NFAT1 to the regulation of Ly6Chi monocyte recruitment to the CNS and to resistance during chronic T. gondii infection.

Asunto(s)

Infecciones Parasitarias del Sistema Nervioso Central/inmunología , Quimiotaxis de Leucocito/inmunología , Monocitos/inmunología , Factores de Transcripción NFATC/inmunología , Toxoplasmosis Animal/inmunología , Animales , Antígenos Ly/inmunología , Ratones , Ratones Noqueados , Células TH1/inmunología , Toxoplasma/inmunología

RESUMEN

OBJECTIVE: Hyperhomocysteinemia (HHcy) is a potent risk factor for diabetic cardiovascular diseases. We have previously reported that hyperhomocysteinemia potentiates type 1 diabetes mellitus-induced inflammatory monocyte differentiation, vascular dysfunction, and atherosclerosis. However, the effects of hyperhomocysteinemia on vascular inflammation in type 2 diabetes mellitus (T2DM) and the underlying mechanism are unknown. Approach and Results: Here, we demonstrate that hyperhomocysteinemia was induced by a high methionine diet in control mice (homocysteine 129 µmol/L), which was further worsened in T2DM db/db mice (homocysteine 180 µmol/L) with aggravated insulin intolerance. Hyperhomocysteinemia potentiated T2DM-induced mononuclear cell, monocyte, inflammatory monocyte (CD11b+Ly6C+), and M1 macrophage differentiation in periphery and aorta, which were rescued by folic acid-based homocysteine-lowering therapy. Moreover, hyperhomocysteinemia exacerbated T2DM-impaired endothelial-dependent aortic relaxation to acetylcholine. Finally, transfusion of bone marrow cells depleted for Ly6C by Ly6c shRNA transduction improved insulin intolerance and endothelial-dependent aortic relaxation in hyperhomocysteinemia+T2DM mice. CONCLUSIONS: Hyperhomocysteinemia potentiated systemic and vessel wall inflammation and vascular dysfunction partially via inflammatory monocyte subset induction in T2DM. Inflammatory monocyte may be a novel therapeutic target for insulin resistance, inflammation, and cardiovascular complications in hyperhomocysteinemia+T2DM.

Asunto(s)

Antígenos Ly/genética , Aterosclerosis/complicaciones , Diabetes Mellitus Tipo 2/genética , Hiperhomocisteinemia/complicaciones , Monocitos/metabolismo , Enfermedades Vasculares/etiología , Animales , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Femenino , Hiperhomocisteinemia/genética , Insulina/uso terapéutico , Resistencia a la Insulina , Macrófagos/metabolismo , Ratones , Distribución Aleatoria , Factores de Riesgo , Sensibilidad y Especificidad , Enfermedades Vasculares/fisiopatología

RESUMEN

BACKGROUND: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. METHODS: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. RESULTS: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFß1 (p = 0.038). CONCLUSION: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Asunto(s)

Linfocitos T CD4-Positivos/citología , Lupus Eritematoso Sistémico/inmunología , Lavado Peritoneal , Bazo/citología , Linfocitos T Reguladores/citología , Animales , Antígenos CD/análisis , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos Ly/análisis , Antígenos Ly/inmunología , Antígenos CD28/análisis , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/inmunología , Femenino , Factores de Transcripción Forkhead/análisis , Factores de Transcripción Forkhead/inmunología , Inmunosupresores , Subunidad alfa del Receptor de Interleucina-2/análisis , Subunidad alfa del Receptor de Interleucina-2/inmunología , Lectinas Tipo C/análisis , Lectinas Tipo C/inmunología , Receptores de Lipopolisacáridos/análisis , Receptores de Lipopolisacáridos/inmunología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inducido químicamente , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Bazo/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Terpenos

RESUMEN

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Asunto(s)

Animales , Femenino , Ratones , Bazo/citología , Lavado Peritoneal , Linfocitos T CD4-Positivos/citología , Linfocitos T Reguladores/citología , Lupus Eritematoso Sistémico/inmunología , Bazo/inmunología , Terpenos , Linfocitos T CD4-Positivos/inmunología , Antígenos Ly/análisis , Antígenos Ly/inmunología , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos CD/análisis , Antígenos CD/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Antígenos CD28/análisis , Antígenos CD28/inmunología , Recuento de Linfocitos , Receptores de Lipopolisacáridos/análisis , Receptores de Lipopolisacáridos/inmunología , Lectinas Tipo C/análisis , Lectinas Tipo C/inmunología , Factores de Transcripción Forkhead/análisis , Factores de Transcripción Forkhead/inmunología , Subunidad alfa del Receptor de Interleucina-2/análisis , Subunidad alfa del Receptor de Interleucina-2/inmunología , Inmunosupresores , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inducido químicamente , Ratones Endogámicos BALB C

RESUMEN

Infection with Schistosoma mansoni causes a chronic parasitic disease that progress to severe liver and gastrointestinal damage, and eventually death. During its development into mammalian hosts, immature schistosomula transit through the lung vasculature before they reach the liver to mature into adult worms. A low grade inflammatory reaction is induced during this process. However, molecules that are required for efficient leukocyte accumulation in the lungs of S. mansoni-infected subjects are unknown. In addition, specific leukocyte subsets that mediate pulmonary response during S. mansoni migration through the lung remain to be elucidated. ß2 integrins are fundamental regulators of leukocyte trans-endothelial migration and function. Therefore, we investigated their role during experimental schistosomiasis. Mice that express low levels of CD18 (the common ß2 integrin subunit) and wild type C57BL/6 mice were subcutaneously infected with S. mansoni cercariae. Cellular profiles of lungs and livers were evaluated in different time points after infection by flow cytometry. Low levels of CD18 affected the accumulation of patrolling Ly6Clow, intermediate Ly6Cinter monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells in the lungs 7 days after infection. This correlated with increased TNF-α levels. Strikingly, low CD18 expression resulted in monocytopenia both in the peripheral blood and bone marrow during acute infection. After 48 days, S. mansoni worm burdens were higher in the hepatic portal system of CD18low mice, which also displayed reduced hepatic accumulation of patrolling Ly6Clow and intermediate Ly6Cinter, but not inflammatory Ly6Chigh monocytes. Higher parasite burden resulted in increased granulomatous lesions in the liver, increased egg deposition and enhanced mortality. Overall, our data point for a fundamental role of CD18 for monocyte hematopoiesis during infection, which promotes an efficient host response against experimental schistosomiasis.

Asunto(s)

Antígenos CD18/metabolismo , Leucocitos Mononucleares/fisiología , Pulmón/inmunología , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/inmunología , Animales , Antígenos Ly/metabolismo , Antígenos CD18/genética , Movimiento Celular , Resistencia a la Enfermedad , Hematopoyesis , Humanos , Inmunidad Innata , Pulmón/parasitología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Animales , Mutación/genética , Recuento de Huevos de Parásitos

RESUMEN

Fructose-rich diet (FRD) has been associated with obesity development, which is characterized by adipocytes hypertrophy and chronic low-grade inflammation. Interaction of adipocytes and immune cells plays a key role in adipose tissue (AT) alterations in obesity. We assessed the metabolic and immune impairments in AT in a murine obesity model induced by FRD at different periods. Adult Swiss mice were divided into groups of 6 and 10 weeks of fructose (FRD 6wk, FRD 10wk) or water intake (CTR 6wk, CTR 10wk). FRD induced increased in body weight, epidydimal AT mass, and plasmatic and liver Tg, and impaired insulin sensitivity. Also, hypertrophic adipocytes from FRD 6wk-10wk mice showed higher IL-6 when stimulated with LPS and leptin secretion. Several of these alterations worsened in FRD 10wk. Regarding AT inflammation, FRD mice have increased TNFα, IL-6 and IL1ß, and decrease in IL-10 and CD206 mRNA levels. Using CD11b, LY6C, CD11c and CD206 as macrophages markers, we identified for first time in AT M1 (M1a: Ly6C+/-CD11c+CD206- and M1b: Ly6C+/-CD11c+CD206+) and M2 subtypes (Ly6C+/-CD11c-CD206+). M1a phenotype increased from 6 weeks onward, while Ly6C+/- M1b phenotype increased only after 10 weeks. Finally, co-culture of RAW264.7 (monocytes cell line) and CTR or FRD adipocytes showed that FRD 10wk adipocytes increased IL-6 expression in non- or LPS-stimulated monocytes. Our results showed that AT dysfunction got worse as the period of fructose consumption was longer. Inflammatory macrophage subtypes increased depending on the period of FRD intake, and hypertrophic adipocytes were able to create an environment that favored M1 phenotype in vitro.

Asunto(s)

Adipocitos/efectos de los fármacos , Fructosa/efectos adversos , Macrófagos/fisiología , Adipocitos/patología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/fisiología , Animales , Antígenos Ly/metabolismo , Biomarcadores/metabolismo , Peso Corporal/efectos de los fármacos , Antígenos CD11/metabolismo , Antígeno CD11b/metabolismo , Interleucina-6/metabolismo , Lectinas Tipo C/metabolismo , Hígado/efectos de los fármacos , Hígado/fisiología , Macrófagos/efectos de los fármacos , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Ratones , Receptores de Superficie Celular/metabolismo

RESUMEN

UNLABELLED: Systemic administration of bone marrow-derived mononuclear cells (BMDMCs) or bone marrow-derived mesenchymal stromal cells (MSCs) reduces inflammation and airway hyperresponsiveness (AHR) in a murine model of Th2-mediated eosinophilic allergic airway inflammation. However, since BMDMCs are a heterogeneous population that includes MSCs, it is unclear whether the MSCs alone are responsible for the BMDMC effects. To determine which BMDMC population(s) is responsible for ameliorating AHR and lung inflammation in a model of mixed Th2-eosinophilic and Th17-neutrophilic allergic airway inflammation, reminiscent of severe clinical asthma, BMDMCs obtained from normal C57Bl/6 mice were serially depleted of CD45, CD34, CD11b, CD3, CD19, CD31, or Sca-1 positive cells. The different resulting cell populations were then assessed for ability to reduce lung inflammation and AHR in mixed Th2/Th17 allergic airway inflammation induced by mucosal sensitization to and challenge with Aspergillus hyphal extract (AHE) in syngeneic C56Bl/6 mice. BMDMCs depleted of either CD11b-positive (CD11b+) or Sca-1-positive (Sca-1+) cells were unable to ameliorate AHR or lung inflammation in this model. Depletion of the other cell types did not diminish the ameliorating effects of BMDMC administration. In conclusion, in the current model of allergic inflammation, CD11b+ cells (monocytes, macrophages, dendritic cells) and Sca-1+ cells (MSCs) are responsible for the beneficial effects of BMDMCs. SIGNIFICANCE: This study shows that bone marrow-derived mononuclear cells (BMDMCs) are as effective as bone marrow-derived mesenchymal stromal cells (MSCs) in ameliorating experimental asthma. It also demonstrates that not only MSCs present in the pool of BMDMCs are responsible for BMDMCs' beneficial effects but also monocytes, which are the most important cell population to trigger these effects. All of this is in the setting of a clinically relevant model of severe allergic airways inflammation and thus provides further support for potential clinical use of cell therapy using MSCs, BMDMCs, and also adult cells such as monocytes in patients with severe asthma.

Asunto(s)

Antígenos Ly/metabolismo , Antígeno CD11b/metabolismo , Inflamación/terapia , Proteínas de la Membrana/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Hipersensibilidad Respiratoria/terapia , Animales , Células de la Médula Ósea/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismo , Células Th17/inmunología , Células Th2/inmunología

RESUMEN

UNLABELLED: Dense fibrosis and a robust macrophage infiltrate are key therapeutic barriers in pancreatic ductal adenocarcinoma (PDAC). CD40 activation can circumvent these barriers by inducing macrophages, originating from peripheral blood monocytes, to deplete fibrosis. The precise mechanism and therapeutic implications of this antifibrotic activity, though, remain unclear. Here, we report that IFNγ and CCL2 released systemically in response to a CD40 agonist cooperate to redirect a subset of Ly6C(+)CCR2(+)monocytes/macrophages to infiltrate tumors and deplete fibrosis. Whereas CCL2 is required for Ly6C(+)monocyte/macrophage infiltration, IFNγ is necessary for tumor-infiltrating monocytes/macrophages to shift the profile of matrix metalloproteinases (MMP) in tumors, leading to MMP-dependent fibrosis degradation. In addition, MMP13-dependent loss of extracellular matrix components induced by a CD40 agonist increased PDAC sensitivity to chemotherapy. Our findings demonstrate that fibrosis in PDAC is a bidirectional process that can be rapidly altered by manipulating a subset of tumor-infiltrating monocytes, leading to enhanced chemotherapy efficacy. SIGNIFICANCE: We report that CD40 agonists improve chemotherapy efficacy in pancreatic carcinoma by redirecting tumor-infiltrating monocytes/macrophages to induce fibrosis degradation that is dependent on MMPs. These findings provide novel insight into the plasticity of monocytes/macrophages in cancer and their capacity to regulate fibrosis and modulate chemotherapy efficacy in pancreatic carcinoma.

Asunto(s)

Quimiocina CCL2/metabolismo , Interferón gamma/metabolismo , Monocitos/metabolismo , Monocitos/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Animales , Antígenos Ly/metabolismo , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Antígenos CD40/agonistas , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Fibrosis , Expresión Génica , Isoinjertos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Monocitos/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas

RESUMEN

Secondary infections due to post-sepsis immunosuppression are a major cause of death in patients with sepsis. Repetitive inoculation of increasing doses of lipopolysaccharide (LPS) into mice mimics the immunosuppression associated with sepsis. Myeloid-derived suppressor cells (MDSCs, Gr-1(+) CD11b(+)) are considered a major component of the immunosuppressive network, interfering with T-cell responses in many pathological conditions. We used LPS-immunosuppressed (IS) mice to address whether MDSCs acquired their suppressive ability in the bone marrow (BM) and whether they could migrate to lymph nodes (LNs) to exert their suppressive function. Our results showed that Gr-1(+) CD11b(+) cells of IS mice already had the potential to inhibit T-cell proliferation in the BM. Moreover, soluble factors present in the BM from IS mice were responsible for inducing this inhibitory ability in control BM cells. In addition, migration of Gr-1(+) CD11b(+) to LNs in vivo was maximal when cells obtained from the BM of IS mice were inoculated into an IS context. In this regard, we found chemoattractant activity in cell-free LN extracts (LNEs) from IS mice and an increased expression of the LN-homing chemokine receptor C-C chemokine receptor type 7 (CCR7) in IS BM Gr-1(+) CD11b(+) cells. These results indicate that Gr-1(+) CD11b(+) cells found in BM from IS mice acquire their suppressive activity in the same niche where they are generated, and migrate to LNs to exert their inhibitory role. A better understanding of MDSC generation and/or regulation of factors able to induce their inhibitory function may provide new and more effective tools for the treatment of sepsis-associated immunosuppression.

Asunto(s)

Antígenos Ly/inmunología , Células de la Médula Ósea/inmunología , Antígeno CD11b/inmunología , Quimiotaxis/efectos de los fármacos , Huésped Inmunocomprometido , Lipopolisacáridos , Ganglios Linfáticos/inmunología , Células Mieloides/inmunología , Sepsis/inmunología , Animales , Antígenos Ly/metabolismo , Células de la Médula Ósea/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Ganglios Linfáticos/metabolismo , Activación de Linfocitos , Ratones Endogámicos BALB C , Células Mieloides/metabolismo , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Sepsis/inducido químicamente , Sepsis/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo

RESUMEN

The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b(+)Gr-1(+), mostly CD11b(+)Ly6G(+)Ly6C(int) and CD11b(+)Ly6G(-)Ly6C(high) cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b(+)Ly6G(+)Ly6C(int) cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b(+)Ly6G(+)Ly6C(int) cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b(+)Gr-1(+) cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b(+)Gr-1(+) cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b(+)Ly6G(+)Ly6C(int) cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.

Asunto(s)

Antígenos Ly/inmunología , Neoplasias de la Mama/inmunología , Antígeno CD11b/inmunología , Células Asesinas Naturales/inmunología , Acetato de Medroxiprogesterona/farmacología , Células Mieloides/inmunología , Animales , Antígenos Ly/metabolismo , Antineoplásicos Hormonales/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Antígeno CD11b/metabolismo , Diferenciación Celular , Proliferación Celular , Citotoxicidad Inmunológica , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Receptores de Glucocorticoides/metabolismo , Factor de Transcripción STAT3/metabolismo , Células Tumorales Cultivadas

RESUMEN

BACKGROUND: The neuroinflammatory response aimed at clearance of herpes simplex virus-1 (HSV-1) plays a key role in the pathogenesis of neuroaxonal damage in herpetic encephalitis. Leukocytes activated in an adaptive immune response access brain tissue by passing through the blood-brain barrier. The chemokine CCL5/RANTES is involved in recruitment of these cells to the brain acting via the receptors CCR1, CCR3 and mainly CCR5. Here, we evaluated the role of CCR5 on traffic of leukocytes in the brain microvasculature, cellular and cytokines profile in a severe form of herpetic encephalitis. RESULTS: Wild type and mice lacking CCR5 (CCR5-/-) were inoculated intracerebrally with 104 PFU of neurotropic HSV-1. We evaluated the traffic of leukocytes in the brain microvasculature using intravital microscopy and the profile of cytokines by Enzyme-Linked Immunosorbent Assay at 1 day post infection. Flow cytometry and histopathological analyses were also carried out in brain tissue. Absence of CCR5 leads to lower viral load and an increased leukocyte adhesion in brain microvasculature, predominantly of neutrophils (CD11+ Ly6G+ cells). Moreover, there was a significant increase in the levels of MIP-1/CCL2, RANTES/CCL5, KC/CXCL1 and MIG/CXCL9 in the brain of infected CCR5-/- mice. CONCLUSIONS: These results suggest that the absence of CCR5 may boost the immune response with a high neutrophil recruitment which most likely helps in viral clearance. Nonetheless, the elevated immune response may be detrimental to the host.

Asunto(s)

Encefalitis por Herpes Simple/inmunología , Encefalitis por Herpes Simple/patología , Regulación de la Expresión Génica/genética , Infiltración Neutrófila/fisiología , Receptores CCR5/deficiencia , Animales , Antígenos Ly/metabolismo , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/virología , Adhesión Celular/genética , Adhesión Celular/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalitis por Herpes Simple/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Leucocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR5/genética

RESUMEN

Sca1 is a surface marker of haematopoietic stem cell but its role in erythropoiesis is still largely unknown. In this work we evaluated the ability of Sca1⁺ cells to differentiate into cells of the erythrocytic lineage. We performed FACS analysis of complete and purified Sca1⁺ bone marrow cells from C3H/HeNHsd mice and measured the expression of CD71 and Terr119 to evaluate the stages in erythroid development. Definitive erythropoiesis was evident within the complete bone marrow, while only proerythroblasts were found in Sca1⁺ cells, suggesting that Sca1 is a negative regulator of erythropoiesis. We also used FDCP-mix cells and their PU.1 and SCL transfectants. The PU.1 transfectant showed significantly increased expression of Sca1 and was not induced to differentiate into red blood cells, while the SCL transfectant showed significantly lower expression of Sca1 and produced red blood cells. The results of this study suggest that increased Sca1 expression on erythropoietic precursors inhibits erythroid differentiation.

Asunto(s)

Antígenos Ly/metabolismo , Células Eritroides/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoyesis , Células Madre Hematopoyéticas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Eritrocitos/metabolismo , Glicoforinas/biosíntesis , Ratones , Ratones Endogámicos C3H , Receptores de Transferrina

RESUMEN

BACKGROUND: Sca-1 is controversial as a mammary stem cell marker in the literature, which may be due to the different isolation protocols and culture media used in different laboratories. The object of our study is to establish the Medium to promote the proliferation of mammary stem cell and explore the possibility of Sca-1 as mammary stem cell marker. METHODS: We used BM medium supplemented with different concentration of 17Β-oestradiol and GH to find out MaECM medium which promoted the proliferation of mouse mammary epithelial cells and inhibited the growth of fibroblasts. Flow cytometry was used to isolate Sca-1(+) and Sca-1(-) cell populations from cultured mammary epithelial cells. Mammary fat pad transplantation and Mammosphere- forming assay were done to confirm the stem cell potential of Sca-1(+) cells. Differentiating culture was used to detect the differentiation potential of Sca-1(+) cells. Real-time PCR was carried out to analyse the expression of mammary stem cell-related genes in Sca-1(+) cells. RESULTS: We first selected the medium suitable for mammary stem cell growth. Stem cell medium BM was used to culture mammary organoids, which generated many fibroblasts. We established MaECM medium supplemented with oestrogen and growth hormone (GH), in which oestrogen promoted mammary epithelial cell proliferation and inhibited fibroblast growth, and GH obviously enhanced the effect of oestrogen on mammary epithelial cell proliferation. Flow cytometry showed that 50% of cells were Sca-1(+) under the culture of MaECM medium. We confirmed that Sca-1(+) cells regenerated mammary outgrowths when transplanted in vivo, formed mammospheres in vitro and differentiated into luminal epithelial cells with milk-secreting function and myoepithelial cells under Matrigel culture. Furthermore, gene expression analysis by Real-time PCR revealed that Sca-1(+) cells expressed markedly higher levels of mammary stem cell-related genes in comparison to Sca-1(-) cells. CONCLUSION: Our research demonstrates that Sca-1(+) mammary stem cells can be more easily isolated when cultured in the presence of oestrogen and GH.

Asunto(s)

Antígenos Ly/metabolismo , Estradiol/metabolismo , Hormona del Crecimiento/metabolismo , Glándulas Mamarias Animales/citología , Proteínas de la Membrana/metabolismo , Células Madre/citología , Animales , Antígenos Ly/genética , Diferenciación Celular , Línea Celular , Proliferación Celular , Medios de Cultivo , Femenino , Citometría de Flujo , Glándulas Mamarias Animales/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Madre/metabolismo

RESUMEN

Tumor growth occurs by the imbalance between cells with effector function and cells with suppressor/regulatory functions. To investigate this scenario we administered the chemical carcinogen Urethane in BALB/c mice and followed these animals during 120 days to observe lung tumor development. In another set of experiments the same protocol was performed with the harvest of spleen, lung and blood at 20 and 30 days after Urethane injection. The lung was used for histology, spleen cells were evaluated for IFN-γ production, and serum nitrite was measured as an indirect form of nitric oxide (NO) evaluation. The spleen and lung-infiltrating cells were evaluated by flow cytometry for CD11b+Gr-1+ myeloid suppressor cells and CD4+FoxP3+ T regulatory cells. Urethane led to lung nodules development after 120 days and the time point evaluation showed that splenocytes stimulated ex vivo expressed higher levels of IFN-γ 20 days after the chemical injection. Also, the level of nitric oxide in serum was higher after 20 days of Urethane injection. There was no statistical difference in spleen cells percentages for CD11b+Gr-1+ and CD4+Foxp3+ in all groups. However, lung-infiltrating cells presented early (20 days) a higher expression of CD11b+Gr-1+ suggesting suppression at this site. In conclusion, it was possible to observe two distinct events at the very early time point after Urethane injection. In periphery there was an increase at the effector immune response (as depicted by IFN-γ-producing cells) and in tumor development site there was an increase at the suppressor cell (CD11b+Gr-1+) phenotype. Suppressor/regulatory cells are targets for cancer therapy.

Asunto(s)

Neoplasias Pulmonares/inmunología , Células Mieloides/metabolismo , Neoplasias Experimentales/inmunología , Linfocitos T Reguladores/metabolismo , Inmunidad Adaptativa , Animales , Antígenos Ly/biosíntesis , Antígeno CD11b/biosíntesis , Antígenos CD4/biosíntesis , Carcinógenos/administración & dosificación , Factores de Transcripción Forkhead/biosíntesis , Humanos , Terapia de Inmunosupresión , Interferón gamma/metabolismo , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Células Mieloides/inmunología , Células Mieloides/patología , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Microambiente Tumoral , Uretano/administración & dosificación

RESUMEN

The NK1.1 molecule participates in NK, NKT, and T-cell activation, contributing to IFN-gamma production and cytotoxicity. To characterize the early immune response to Plasmodium chabaudi AS, spleen NK1.1(+) and NK1.1(-) T cells were compared in acutely infected C57BL/6 mice. The first parasitemia peak in C57BL/6 mice correlated with increase in CD4(+)NK1.1(+)TCR-alphabeta(+), CD8(+)NK1.1(+)TCR-alphabeta(+), and CD4(+)NK1.1(-)TCR-alphabeta(+) cell numbers per spleen, where a higher increment was observed for NK1.1(+) T cells compared to NK1.1(-) T cells. According to the ability to recognize the CD1d-alpha-GalCer tetramer, CD4(+)NK1.1(+) cells in 7-day infected mice were not predominantly invariant NKT cells. At that time, nearly all NK1.1(+) T cells and around 30% of NK1.1(-) T cells showed an experienced/activated (CD44(HI)CD69(HI)CD122(HI)) cell phenotype, with high expression of Fas and PD-L1 correlating with their low proliferative capacity. Moreover, whereas IFN-gamma production by CD4(+)NK1.1(+) cells peaked at day 4 p.i., the IFN-gamma response of CD4(+)NK1.1(-) cells continued to increase at day 5 of infection. We also observed, at day 7 p.i., 2-fold higher percentages of perforin(+) cells in CD8(+)NK1.1(+) cells compared to CD8(+)NK1.1(-) cells. These results indicate that spleen NK1.1(+) and NK1.1(-) T cells respond to acute P. chabaudi malaria with different kinetics in terms of activation, proliferation, and IFN-gamma production.

Asunto(s)

Malaria/inmunología , Células T Asesinas Naturales/metabolismo , Plasmodium chabaudi/inmunología , Bazo/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD/biosíntesis , Antígenos Ly/biosíntesis , Proliferación Celular , Inmunofenotipificación , Interferón gamma/metabolismo , Activación de Linfocitos , Malaria/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Subfamilia B de Receptores Similares a Lectina de Células NK/biosíntesis , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/parasitología , Células T Asesinas Naturales/patología , Plasmodium chabaudi/patogenicidad , Bazo/parasitología , Bazo/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/parasitología , Subgrupos de Linfocitos T/patología

RESUMEN

The role of natural killer (NK) T cells in the development of lupus-like disease in mice is still controversial. We treated NZB/W mice with anti-NK1.1 monoclonal antibodies (mAbs) and our results revealed that administration of either an irrelevant immunoglobulin G2a (IgG2a) mAb or an IgG2a anti-NK1.1 mAb increased the production of anti-dsDNA antibodies in young NZB/W mice. However, the continuous administration of an anti-NK1.1 mAb protected aged NZB/W mice from glomerular injury, leading to prolonged survival and stabilization of the proteinuria. Conversely, the administration of the control IgG2a mAb led to an aggravation of the lupus-like disease. Augmented titres of anti-dsDNA in NZB/W mice, upon IgG2a administration, correlated with the production of BAFF/BLyS by dendritic, B and T cells. Treatment with an anti-NK1.1 mAb reduced the levels of interleukin-16, produced by T cells, in spleen cell culture supernatants from aged NZB/W. Adoptive transfer of NK T cells from aged to young NZB/W accelerated the production of anti-dsDNA in recipient NZB/W mice, suggesting that NK T cells from aged NZB/W are endowed with a B-cell helper activity. In vitro studies, using purified NK T cells from aged NZB/W, showed that these cells provided helper B-cell activity for the production of anti-dsDNA. We concluded that NK T cells are involved in the progression of lupus-like disease in mature NZB/W mice and that immunoglobulin of the IgG2a isotype has an enhancing effect on antibody synthesis due to the induction of BAFF/BLyS, and therefore have a deleterious effect in the NZB/W mouse physiology.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Factor Activador de Células B/biosíntesis , Inmunoglobulina G/uso terapéutico , Nefritis Lúpica/prevención & control , Envejecimiento/inmunología , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Monoclonales/inmunología , Antígenos Ly/inmunología , Células Cultivadas , Progresión de la Enfermedad , Femenino , Inmunoglobulina G/inmunología , Interleucina-16/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/trasplante , Lipopolisacáridos/inmunología , Hígado/inmunología , Nefritis Lúpica/inmunología , Ratones , Ratones Endogámicos , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Índice de Severidad de la Enfermedad , Bazo/inmunología , Linfocitos T Colaboradores-Inductores/inmunología