RESUMEN
Mesenchymal stromal cell (MSC) apoptosis is required for in vivo immunosuppression. However, the induction of apoptosis is heavily dependent on the recipient's immune system. In graft-versus-host disease (GvHD), patients who fail to respond to MSCs are in fact those whose immune cells are unable to induce MSC apoptosis ex vivo. The information is critical to explain why responses in clinical trials vary even though the same sources of MSC products are infused. More importantly, it highlights the need for an alternative MSC treatment for the nonresponders. By using a mouse model of ovalbumin (OVA)-induced allergic inflammation, we demonstrated that we could generate apoptotic MSCs (ApoMSCs) in vitro and use them to successfully reduce allergic airway inflammation. In order to address the logistics of their potential future clinical application, we have shown that ApoMSCs could be cryopreserved without impairing efficacy compared to freshly generated ApoMSCs. We have also highlighted that MSCs need to undergo complete apoptosis before cryopreservation to retain their immunosuppressive activity. The cryopreserved ApoMSCs could serve as a potential future off-the-shelf cellular product, in particular for patients who suffer from inflammatory conditions yet do not harbor the immune capacity to induce MSC apoptosis in vivo. Our data provide proof-of-concept that under laboratory conditions, ApoMSCs can be successfully frozen and thawed without affecting their anti-inflammatory activity, as tested in a murine model of allergic inflammation.
RESUMEN
Streptococcal pneumonia is a worldwide health problem that kills â¼2 million people each year, particularly young children, the elderly, and immunosuppressed individuals. Alveolar macrophages and neutrophils provide the early innate immune response to clear pneumococcus from infected lungs. However, the level of neutrophil involvement is context dependent, both in humans and in mouse models of the disease, influenced by factors such as bacterial load, age, and coinfections. Here, we show that the G protein-coupled receptor (GPCR) adaptor protein norbin (neurochondrin, NCDN), which was hitherto known as a regulator of neuronal function, is a suppressor of neutrophil-mediated innate immunity. Myeloid norbin deficiency improved the immunity of mice to pneumococcal infection by increasing the involvement of neutrophils in clearing the bacteria, without affecting neutrophil recruitment or causing autoinflammation. It also improved immunity during Escherichia coli-induced septic peritonitis. It increased the responsiveness of neutrophils to a range of stimuli, promoting their ability to kill bacteria in a reactive oxygen species-dependent manner, enhancing degranulation, phagocytosis, and the production of reactive oxygen species and neutrophil extracellular traps, raising the cell surface levels of selected GPCRs, and increasing GPCR-dependent Rac and Erk signaling. The Rac guanine-nucleotide exchange factor Prex1, a known effector of norbin, was dispensable for most of these effects, which suggested that norbin controls additional downstream targets. We identified the Rac guanine-nucleotide exchange factor Vav as one of these effectors. In summary, our study presents the GPCR adaptor protein norbin as an immune suppressor that limits the ability of neutrophils to clear bacterial infections.
Asunto(s)
Neutrófilos , Infecciones Neumocócicas , Animales , Ratones , Ratones Noqueados , Neuropéptidos , Receptores Acoplados a Proteínas GRESUMEN
Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1ß and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.
Asunto(s)
Antiinflamatorios/farmacología , Chaperonina 60/farmacología , Chaperoninas/farmacología , Infiltración Neutrófila/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Neumonía/prevención & control , Animales , Anexina A1/genética , Líquido del Lavado Bronquioalveolar/química , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citocinas/análisis , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrinas/biosíntesis , Interleucina-1beta/biosíntesis , Lipopolisacáridos/toxicidad , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/biosíntesis , Neutrófilos/inmunología , Receptor Toll-Like 4/genéticaRESUMEN
OBJECTIVES: Ischemia and reperfusion (I/R) in the intestine could lead to severe endothelial injury, compromising intestinal motility. Reportedly, estradiol can control local and systemic inflammation induced by I/R injury. Thus, we investigated the effects of estradiol treatment on local repercussions in an intestinal I/R model. METHODS: Rats were subjected to ischemia via the occlusion of the superior mesenteric artery (45 min) followed by reperfusion (2h). Thirty minutes after ischemia induction (E30), 17ß-estradiol (E2) was administered as a single dose (280 µg/kg, intravenous). Sham-operated animals were used as controls. RESULTS: I/R injury decreased intestinal motility and increased intestinal permeability, accompanied by reduced mesenteric endothelial nitric oxide synthase (eNOS) and endothelin (ET) protein expression. Additionally, the levels of serum injury markers and inflammatory mediators were elevated. Estradiol treatment improved intestinal motility, reduced intestinal permeability, and increased eNOS and ET expression. Levels of injury markers and inflammatory mediators were also reduced following estradiol treatment. CONCLUSION: Collectively, our findings indicate that estradiol treatment can modulate the deleterious intestinal effects of I/R injury. Thus, estradiol mediates the improvement in gut barrier functions and prevents intestinal dysfunction, which may reduce the systemic inflammatory response.
Asunto(s)
Estradiol , Daño por Reperfusión , Animales , Estradiol/farmacología , Estrógenos , Intestinos , Isquemia , Masculino , Permeabilidad , Ratas , Reperfusión , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/prevención & controlRESUMEN
Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.
Asunto(s)
Asma/inmunología , Plaquetas/inmunología , Hiperreactividad Bronquial/inmunología , Pulmón/inmunología , Activación Plaquetaria/inmunología , Receptores CCR3/inmunología , Adolescente , Adulto , Anciano , Alérgenos/administración & dosificación , Animales , Antígenos Dermatofagoides/administración & dosificación , Proteínas de Artrópodos/administración & dosificación , Asma/genética , Asma/mortalidad , Asma/patología , Plaquetas/efectos de los fármacos , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/patología , Niño , Cisteína Endopeptidasas/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Persona de Mediana Edad , Activación Plaquetaria/efectos de los fármacos , Pyroglyphidae/química , Pyroglyphidae/inmunología , Receptores CCR3/genética , Receptores CCR4/genética , Receptores CCR4/inmunología , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Transducción de Señal , Análisis de SupervivenciaRESUMEN
OBJECTIVES: Ischemia and reperfusion (I/R) in the intestine could lead to severe endothelial injury, compromising intestinal motility. Reportedly, estradiol can control local and systemic inflammation induced by I/R injury. Thus, we investigated the effects of estradiol treatment on local repercussions in an intestinal I/R model. METHODS: Rats were subjected to ischemia via the occlusion of the superior mesenteric artery (45 min) followed by reperfusion (2h). Thirty minutes after ischemia induction (E30), 17β-estradiol (E2) was administered as a single dose (280 μg/kg, intravenous). Sham-operated animals were used as controls. RESULTS: I/R injury decreased intestinal motility and increased intestinal permeability, accompanied by reduced mesenteric endothelial nitric oxide synthase (eNOS) and endothelin (ET) protein expression. Additionally, the levels of serum injury markers and inflammatory mediators were elevated. Estradiol treatment improved intestinal motility, reduced intestinal permeability, and increased eNOS and ET expression. Levels of injury markers and inflammatory mediators were also reduced following estradiol treatment. CONCLUSION: Collectively, our findings indicate that estradiol treatment can modulate the deleterious intestinal effects of I/R injury. Thus, estradiol mediates the improvement in gut barrier functions and prevents intestinal dysfunction, which may reduce the systemic inflammatory response.
Asunto(s)
Animales , Masculino , Ratas , Daño por Reperfusión/prevención & control , Daño por Reperfusión/tratamiento farmacológico , Estradiol/farmacología , Permeabilidad , Reperfusión , Estrógenos , Intestinos , IsquemiaRESUMEN
Chronic obstructive pulmonary disease (COPD) is related to smoking and anti-inflammatory therapy is indicated. Among the mediators with anti-inflammatory properties, we highlight piperlongumine (PL), an alkaloid/amide of Piper longum. Here we evaluated the PL administration on an experimental model of respiratory inflammation resulting from exposure to cigarette smoke. Male Balb/c mice were exposed to burning of 10 commercial cigarettes, 2x/day, for five weeks on specific equipment. PL efficacy was evaluated in control, exposed to smoke without treatment and PL treated (2.0 mg/kg, 3x/week) groups. Animals were weighed and plethysmographic analyses performed at the end of the exposure protocol. Inflammatory cells were evaluated in the bronchoalveolar lavage (BAL) and hemoglobin and glucose in the blood. Lung fragments were processed for histopathological studies and AnxA1, COX-2, NF-kB and neutrophil elastase expressions. Plethysmography revealed that PL maintained pulmonary frequency, volume and ventilation parameters similar to controls, with respiratory volume reduction compared to untreated animals. Final weight was reduced in both exposed groups. PL decreased hemoglobin concentration, attenuated the reduction of glucose levels and reduced influx of lymphocytes, neutrophils and macrophages in BAL. Histopathologically occured infiltration of inflammatory cells, increase of the interalveolar septa and intra-alveolar spaces in untreated animals. But, PL administration recovered lung tissues and, immunohistochemically, promoted increased expression of AnxA1 and reduction of COX-2, NF-kB and neutrophil elastase. Together the results indicate that PL attenuates systemic and pulmonary inflammatory changes, partially by modulating the expression the endogenous AnxA1, and may represent a promising therapy in preventing the inflammation induced by cigarette smoke.
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Antiinflamatorios/farmacología , Dioxolanos/farmacología , Inflamación/inducido químicamente , Lesión Pulmonar/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Fumar Tabaco/efectos adversos , Animales , Anexina A1/metabolismo , Ciclooxigenasa 2/metabolismo , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Lesión Pulmonar/fisiopatología , Linfocitos/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Neutrófilos , Fumar Tabaco/metabolismo , Fumar Tabaco/patología , Fumar Tabaco/fisiopatologíaRESUMEN
Organ donor's age negatively influences graft survival of organs, increasing risk of complications. Aging occurs in both men and women; however, the menopause marks a decrease in sex hormones and a sudden increase in the process of vascular aging. We investigated sex hormones' influence on the lung inflammatory process induced by BD in female rats. Wistar rats were grouped as: female rats from high estradiol to heat period (non-OVx) and ovariectomized (OVx) female rats. Ovariectomy was carried out 10 days before BD. BD was induced using intracranial balloon rapid inflation. Serum hormones and inflammatory mediators were quantified, leukocytes and platelets counted and lung samples were collected for RT-PCR, immunohistochemical, and histological analysis. Female sex hormones and corticosterone were reduced 6 h after BD in non-OVx group. The infiltration of leukocytes in female non-OVx lungs was higher compared to OVx. G-CSF, VEGF, and CINC-1 were found increased in non-OVx group serum in comparison to OVx. Lung mediators were increased in non-OVx rats compared to controls. The acute reduction of sex hormones induced by BD appears to have a worse effect on lung inflammation than a reduction that has happened over a prolonged period of time, allowing a physiological adaptation prior to BD.
Asunto(s)
Muerte Encefálica , Neumonía , Animales , Estradiol , Femenino , Hormonas Esteroides Gonadales , Neumonía/etiología , Ratas , Ratas WistarRESUMEN
BACKGROUND: Despite the overwhelming evidence showing the influence of sex or obesity in the development of respiratory diseases in humans and animals, the mechanisms by which these combined two factors influence allergic asthma are not well understood. OBJECTIVE: We have investigated the interaction between sex and weight gain in an experimental model of lung allergic inflammation induced by chicken egg ovalbumin (OVA) in mice. METHODS: Animals were fed a high-fat diet for 8 weeks and then sensitized and challenged with OVA. RESULTS: Our results demonstrate that in comparison with males, high-fat diet (HFD) allergic female mice exhibit a reduction in the number of leucocytes in the lung lumen when challenged with OVA and, in contrast, an accumulation of these cells in the lung tissue. In addition, we also observed that allergic HFD female mice presented a robust lung remodelling in comparison with HFD males, evidenced by higher deposition of collagen in the airways and TGF-ß in lung fluid. Measuring epithelial adhesion molecule expression, we observed that female mice presented a significantly lower expression of CD103 than males in BAL cells, regardless of the diet. Similarly, HFD female mice express lower levels of EpCAM in lung tissue in comparison with males and lean females. Levels of A20/TNFAIP3 expression in lung tissue demonstrated that HFD female mice express lower levels of these regulatory factors than all the other groups. However, this reduction was not accompanied by an increase in activated NF-κB. CONCLUSIONS: Our results present evidence that the interaction between sex and weight gain alters the progression of allergic asthma in mice with females developing airway remodelling at a much earlier stage than males. CLINICAL RELEVANCE: These data may contribute to a better understanding of the clinical differences in the development and severity of allergic asthma observed between men and women of reproductive age.
Asunto(s)
Asma , Dieta Alta en Grasa/efectos adversos , Obesidad , Caracteres Sexuales , Animales , Asma/inducido químicamente , Asma/inmunología , Asma/patología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Masculino , Ratones , Obesidad/inducido químicamente , Obesidad/inmunología , Obesidad/patología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: We have previously demonstrated that Mycobacteria tuberculosis chaperonin 60.1 inhibits leucocyte diapedesis and bronchial hyperresponsiveness in a murine model of allergic lung inflammation. METHODS: In the present study, we have investigated the effect of a shorter peptide sequence derived from Cpn 60.1, named IRL201104, on allergic lung inflammation induced by ovalbumin (OVA) in mice and by house dust mite (HDM) in guinea pigs, as well as investigating the action of IRL201104 on human cells in vitro. RESULTS: Pre-treatment of mice or guinea pigs with IRL201104 inhibits the infiltration of eosinophils to the lung, cytokine release, and in guinea pig skin, inhibits allergen-induced vascular permeability. The protective effect of intranasal IRL201104 against OVA-induced eosinophilia persisted for up to 20 days post-treatment. Moreover, OVA-sensitized mice treated intranasally with 20 ng/kg of IRL201104 show a significant increase in the expression of the anti-inflammatory molecule ubiquitin A20 and significant inhibition of the activation of NF-κB in lung tissue. Our results also show that A20 expression was significantly reduced in blood leucocytes and ASM obtained from patients with asthma compared to cells obtained from healthy subjects which were restored after incubation with IRL201104 in vitro, when added alone, or in combination with LPS or TNF-α in ASM. CONCLUSIONS: Our results suggest that a peptide derived from mycobacterial Cpn60.1 has a long-lasting anti-inflammatory and immunomodulatory activity which may help explain some of the protective effects of TB against allergic diseases.
Asunto(s)
Antiinflamatorios/farmacología , Asma/inmunología , Proteínas Bacterianas/farmacología , Chaperonina 60/farmacología , Mycobacterium tuberculosis/química , Péptidos/farmacología , Animales , Antiinflamatorios/química , Asma/tratamiento farmacológico , Asma/patología , Proteínas Bacterianas/química , Líquido del Lavado Bronquioalveolar , Chaperonina 60/química , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Cobayas , Humanos , Pulmón , Ratones , Ratones Endogámicos BALB C , Péptidos/químicaRESUMEN
RATIONAL: Acute lung injury (ALI) is a common complication after intestinal ischemia and reperfusion (I/R) injury that can lead to acute respiratory distress syndrome (ARDS). We have previously demonstrated that females are protected against lung damage induced by intestinal I/R through an estrogen mediated mechanism. OBJECTIVES: To investigate the effect of obesity on ALI induced by intestinal I/R in female mice. METHODS: C57Bl/6 female mice were fed with a standard low-fat diet (SD) or a high-fat diet (HFD) for 9 weeks. Intestinal I/R injury was induced by a 45â¯min occlusion of the mesenteric artery followed by 2 and 24â¯h of reperfusion. RESULTS: Significant increase in lung myeloperoxidase expression (MPO) and neutrophil numbers of SD and HFD mice occurred at 2â¯h and 24â¯h of reperfusion. Furthermore, HFD mice presented a significant increase in lung eosinophil peroxidase (EPO) expression and eosinophil numbers compared to SD mice. Lung wet/dry weight ratio was significantly greater in HFD mice at 2 and 24â¯h of reperfusion, accompanied by a significant increase in the expression of inducible NO in the lung tissue and a significant decrease in arterial oxygen saturation at 24â¯h of reperfusion relative to SD mice. CONCLUSION: Obesity predisposes female mice to increased pulmonary oedema and deterioration in gas exchange, which is accompanied by an increase in iNOS expression in the lung.
Asunto(s)
Lesión Pulmonar Aguda/etiología , Obesidad/complicaciones , Edema Pulmonar/etiología , Daño por Reperfusión/complicaciones , Lesión Pulmonar Aguda/fisiopatología , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Intestinos/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Peroxidasa/metabolismo , Intercambio Gaseoso Pulmonar/fisiología , Factores SexualesRESUMEN
Platelet activation occurs in patients with allergic inflammation, and platelets can be activated directly by allergen via an IgE-dependent process. Platelets have been shown to activate APCs such as CD11c+ dendritic cells in vitro. Although CD11c+ dendritic cells are a requisite for allergen sensitization, the role of platelets in this process is unknown. In this study, we investigated whether platelets were necessary for allergen sensitization. Balb/c mice sensitized to ovalbumin were exposed to subsequent aerosolized allergen (ovalbumin challenge). We analyzed lung CD11c+ cell activation, colocalization with platelets, and some other indices of inflammation. The role of platelets at the time of allergen sensitization was assessed through platelet depletion experiments restricted to the period of sensitization. Platelets colocalized with airway CD11c+ cells, and this association increased after allergen sensitization as well as after subsequent allergen exposure. Temporary platelet depletion (>95%) at the time of allergen sensitization led to a suppression of IgE and IL-4 synthesis and to a decrease in the pulmonary recruitment of eosinophils, macrophages, and lymphocytes after subsequent allergen exposure. Furthermore, in mice previously depleted of platelets at the time of sensitization, the recovered platelet population was shown to have reduced expression of FcεRI. Pulmonary CD11c+ cell recruitment was suppressed in these mice after allergen challenge, suggesting that the migration of CD11c+ cells in vivo may be dependent on direct platelet recognition of allergen. We conclude that platelets are necessary for efficient host sensitization to allergen. This propagates the subsequent inflammatory response during secondary allergen exposure and increases platelet association with airway CD11c+ cells.
Asunto(s)
Alérgenos/inmunología , Plaquetas/inmunología , Inmunización , Animales , Antígeno CD11c/metabolismo , Femenino , Inmunoglobulina E/biosíntesis , Interleucina-4/biosíntesis , Leucocitos/patología , Pulmón/patología , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Receptores de IgE/metabolismo , Trombocitopenia/inmunología , Trombocitopenia/patología , Factores de TiempoRESUMEN
Mesenchymal stem cells (MSCs) show considerable promise as a cellular immunotherapy for the treatment of a number of autoimmune and inflammatory disorders. However, the precise physiologically and therapeutically relevant mechanism(s) by which MSCs mediate immune modulation remains elusive. Dental pulp stem cells are a readily available source of MSCs that have been reported to show similar immune modulation in vitro as bone marrow MSCs. To test their potential in vivo, we used a clinically relevant humanized mouse model of GvHD in which only human T cells engraft. In this model, we found no effects on either T-cell proliferation, T-cell phenotype or disease progression. To determine if this lack of efficacy was related to a failure of engraftment or persistence of the cells, we used viability dependent radioactive cell tracking and showed that no cells were detectable after 24-h postinjection. Given the apparent failure of MSC to survive following intravenous injection, we hypothesized that their apoptosis may account for the widely reported therapeutic effect in numerous experimental models in vivo. To address this, we employed a well-established model of allergic airway inflammation to compare the efficacy of live and apoptotic MSCs in a fully immunocompetent model. In this model, both live and apoptotic dental pulp MSCs induced a robust immune suppressive reaction that was substantially greater with apoptotic cells. We propose that the mechanism of immune modulation following systemic application of MSCs is a result of cell entrapment and apoptosis occurring in the lungs.
Asunto(s)
Apoptosis , Pulpa Dental/inmunología , Inmunomodulación , Trasplante de Células Madre Mesenquimatosas , Linfocitos T/inmunología , Animales , Proliferación Celular , Células Cultivadas , Pulpa Dental/patología , Modelos Animales de Enfermedad , Femenino , Enfermedad Injerto contra Huésped/terapia , Pulmón/patología , Ratones , Ratones Endogámicos , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/terapia , Linfocitos T/fisiologíaRESUMEN
BACKGROUND: Lung inflammation is one of the main consequences of intestinal ischemia reperfusion (intestinal IR) and, in severe cases, can lead to acute respiratory distress syndrome and death. We have previously demonstrated that estradiol exerts a protective effect on lung edema and cytokine release caused by intestinal IR in male rats. MATERIALS AND METHODS: We investigated the role of estradiol on the generation of interleukin (IL)-1ß, IL-10, vascular endothelial growth factor (VEGF), and cytokine-induced neutrophil chemoattractant 1 (CINC-1) in a female rat model of intestinal IR. Blood and bone marrow leukocytes were also quantified. Seven-days-ovariectomized rats were subjected to intestinal IR by occlusion of the superior mesenteric artery for 45 min. After reperfusion of the tissue for 2 h, the rats were sacrificed. Lung tissue was collected, cultured for 24 h and assayed. RESULTS: We observed a significant increase in serum levels of IL-10, CINC-1, uric acid and circulating, but not bone marrow, leukocyte numbers. In addition, intestinal IR induced a significant increase in the ex-vivo lung levels of IL-1ß, IL-10, and VEGF. Treatment with 17ß-estradiol before the induction of intestinal IR prevented the systemic release of IL-10, CINC-1, and uric acid, but it did not affect the leukocytosis. In addition, 17ß-estradiol significantly prevented the ex-vivo release of IL-1ß and VEGF from lung tissue. CONCLUSIONS: We demonstrated that intestinal IR interferes with lung homeostasis, priming the tissue to generate proinflammatory mediators for at least 24 h postischemia. Furthermore, our data confirm that the inflammatory responses caused by intestinal IR are estradiol mediated.
Asunto(s)
Estradiol/fisiología , Enfermedades Intestinales/complicaciones , Intestinos/irrigación sanguínea , Enfermedades Pulmonares/etiología , Daño por Reperfusión/complicaciones , Animales , Citocinas/sangre , Femenino , Enfermedades Intestinales/sangre , Recuento de Leucocitos , Pulmón/metabolismo , Enfermedades Pulmonares/sangre , Ratas Wistar , Daño por Reperfusión/sangre , Ácido Úrico/sangreRESUMEN
Glucocorticosteroids are widely used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). However, there are growing concerns about the side effect profile of this class of drug, particularly an increased risk of pneumonia. Over the last two decades there have been many attempts to find drugs to allow a reduction of glucocorticosteroids, including xanthines such as theophylline. Use of xanthines has been shown to lead to a reduction in the requirement for glucocorticosteroids, although xanthines also have a narrow therapeutic window limiting their wider use. Doxofylline is another xanthine that has been shown to be of clinical benefit in patients with asthma or COPD, but to have a wider therapeutic window than theophylline. In the present study we have demonstrated that doxofylline produces a clear steroid sparing effect in both an allergic and a non-allergic model of lung inflammation. Thus, we have shown that concomitant treatment with a low dose of doxofylline and a low dose of the glucocorticosteroid dexamethasone (that alone had no effect) significantly reduced both allergen-induced eosinophil infiltration into the lungs of allergic mice, and lipopolysaccharide (LPS)-induced neutrophil infiltration into the lung, equivalent to a higher dose of each drug. Our results suggest that doxofylline demonstrates significant anti-inflammatory activity in the lung which can result in significant steroid sparing activity.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Neumonía/tratamiento farmacológico , Teofilina/análogos & derivados , Animales , Broncodilatadores/farmacología , Dexametasona/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Eosinófilos/metabolismo , Femenino , Glucocorticoides/administración & dosificación , Lipopolisacáridos/toxicidad , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Neumonía/fisiopatología , Teofilina/farmacologíaRESUMEN
The immunosuppressive activity of mesenchymal stromal cells (MSCs) is well documented. However, the therapeutic benefit is completely unpredictable, thus raising concerns about MSC efficacy. One of the affecting factors is the unresolved conundrum that, despite being immunosuppressive, MSCs are undetectable after administration. Therefore, understanding the fate of infused MSCs could help predict clinical responses. Using a murine model of graft-versus-host disease (GvHD), we demonstrate that MSCs are actively induced to undergo perforin-dependent apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. When examining patients with GvHD who received MSCs, we found a striking parallel, whereby only those with high cytotoxic activity against MSCs responded to MSC infusion, whereas those with low activity did not. The need for recipient cytotoxic cell activity could be replaced by the infusion of apoptotic MSCs generated ex vivo. After infusion, recipient phagocytes engulf apoptotic MSCs and produce indoleamine 2,3-dioxygenase, which is ultimately necessary for effecting immunosuppression. Therefore, we propose the innovative concept that patients should be stratified for MSC treatment according to their ability to kill MSCs or that all patients could be treated with ex vivo apoptotic MSCs.
Asunto(s)
Apoptosis/fisiología , Células Madre Mesenquimatosas/citología , Animales , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/fisiopatología , Humanos , Inmunomodulación/genética , Inmunomodulación/fisiología , Terapia de Inmunosupresión/métodos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiologíaRESUMEN
OBJECTIVE: Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. METHODS: Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund's adjuvant (CFA)-induced unilateral arthritis model, assessed over 14â days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. RESULTS: At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. CONCLUSIONS: Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions.
Asunto(s)
Artritis Experimental/metabolismo , Hiperalgesia/metabolismo , Sinovitis/metabolismo , Canales Catiónicos TRPC/fisiología , Anciano , Anciano de 80 o más Años , Animales , Artritis Experimental/complicaciones , Artritis Experimental/patología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Conducta Animal , Edema/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Hiperalgesia/etiología , Hiperalgesia/patología , Indoles/farmacología , Masculino , Ratones Noqueados , Persona de Mediana Edad , Neovascularización Patológica/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Piperidinas/farmacología , ARN Mensajero/genética , Membrana Sinovial/irrigación sanguínea , Sinovitis/etiología , Sinovitis/patología , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/biosíntesis , Canales Catiónicos TRPC/deficiencia , Canales Catiónicos TRPC/genéticaRESUMEN
The properties of commercially prepared heparin as an anticoagulant and antithrombotic agent in medicine are better understood than is the physiological role of heparin in its native form, where it is uniquely found in the secretory granules of mast cells. In the present study we have isolated and characterised the glycosaminoglycans (GAGs) released from degranulating rat peritoneal mast cells. Analysis of the GAGs by NMR spectroscopy showed the presence of both heparin and the galactosaminoglycan dermatan sulphate; heparinase digestion profiles and measurements of anticoagulant activity were consistent with this finding. The rat peritoneal mast cell GAGs significantly inhibited accumulation of leukocytes in the rat peritoneal cavity in response to IL-1ß (p < 0.05, n = 6/group), and inhibited adhesion and diapedesis of leukocytes in the inflamed rat cremasteric microcirculation in response to LPS (p < 0.001, n = 4/group). FTIR spectra of human umbilical vein endothelial cells (HUVECs) were altered by treatment of the cells with heparin degrading enzymes, and restored by the addition of exogenous heparin. In conclusion, we have shown that rat peritoneal mast cells contain a mixture of GAGs that possess anticoagulant and anti-inflammatory properties.
Asunto(s)
Anticoagulantes/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Mastocitos/metabolismo , Animales , Antiinflamatorios/metabolismo , Dermatán Sulfato/metabolismo , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Masculino , Microcirculación/fisiología , Cavidad Peritoneal/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
We have investigated whether the mechanism by which the non-anticoagulant N-acetyl-de-O-sulfated-heparin (NSH) inhibits leukocyte infiltration is mediated by an effect on platelet function. We show that oral treatment with two doses of NSH significantly inhibits eosinophil and neutrophil recruitment into the lungs. Intravital microscopy analysis shows that NSH inhibits leukocyte and platelet diapedesis in the microcirculation of the cremaster muscle and in the trachea. More importantly, there were significantly lower numbers of leukocytes recruited into the lung in response to LPS in thrombocytopenic mice when transfused with platelets pretreated with NSH in vitro when compared with mice transfused with untreated platelets. Using intravital analysis of the microvasculature of the cremaster muscle, we have demonstrated that the reinfusion of activated platelets significantly re-established leukocyte diapedesis in response to LPS but that this effect was not observed when platelets were pretreated in vitro with NSH. Finally, we investigated whether the effect of NSH altered the expression of adhesion molecules on the surface of platelets and leukocytes in blood samples collected from mice treated orally with NSH. Our results demonstrate that NSH significantly inhibited the detection of P-selectin as evaluated by flow cytometry, confirming that part of the antiinflammatory action of NSH is via an effect on platelet function.
RESUMEN
Lipid nanocapsules (LNCs) are semi-rigid spherical capsules with a triglyceride core that present a promising formulation option for the pulmonary delivery of drugs with poor aqueous solubility. Whilst the biodistribution of LNCs of different size has been studied following intravenous administration, the fate of LNCs following pulmonary delivery has not been reported. We investigated quantitatively whether lung inflammation affects the clearance of 50nm lipid nanocapsules, or is exacerbated by their pulmonary administration. Studies were conducted in mice with lipopolysaccharide-induced lung inflammation compared to healthy controls. Particle deposition and nanocapsule clearance kinetics were measured by single photon emission computed tomography/computed tomography (SPECT/CT) imaging over 48 h. A significantly lower lung dose of (111)In-LNC50 was achieved in the lipopolysaccharide (LPS)-treated animals compared with healthy controls (p<0.001). When normalised to the delivered lung dose, the clearance kinetics of (111)In-LNC50 from the lungs fit a first order model with an elimination half-life of 10.5±0.9h (R(2)=0.995) and 10.6±0.3h (R(2)=1.000) for healthy and inflamed lungs respectively (n=3). In contrast, (111)In-diethylene triamine pentaacetic acid (DTPA), a small hydrophilic molecule, was cleared rapidly from the lungs with the majority of the dose absorbed within 20min of administration. Biodistribution to lungs, stomach-intestine, liver, trachea-throat and blood at the end of the imaging period was unaltered by lung inflammation. This study demonstrated that lung clearance and whole body distribution of lipid nanocapsules were unaffected by the presence of acute lung inflammation.