RESUMO
Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. ONE SENTENCE SUMMARY: A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.
RESUMO
The seasonal burden of influenza coupled with the pandemic outbreaks of more pathogenic strains underscore a critical need to understand the pathophysiology of influenza injury in the lung. Interleukin-22 (IL-22) is a promising cytokine that is critical in protecting the lung during infection. This cytokine is strongly regulated by the soluble receptor IL-22-binding protein (IL-22BP), which is constitutively expressed in the lungs where it inhibits IL-22 activity. The IL-22/IL-22BP axis is thought to prevent chronic exposure of epithelial cells to IL-22. However, the importance of this axis is not understood during an infection such as influenza. Here we demonstrate through the use of IL-22BP-knockout mice (il-22ra2-/-) that a pro-IL-22 environment reduces pulmonary inflammation during H1N1 (PR8/34 H1N1) infection and protects the lung by promoting tight junction formation. We confirmed these results in normal human bronchial epithelial cells in vitro demonstrating improved membrane resistance and induction of the tight junction proteins Cldn4, Tjp1, and Tjp2. Importantly, we show that administering recombinant IL-22 in vivo reduces inflammation and fluid leak into the lung. Taken together, our results demonstrate the IL-22/IL-22BP axis is a potential targetable pathway for reducing influenza-induced pneumonia.
Assuntos
Inflamação/terapia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/terapia , Interleucinas/metabolismo , Células Matadoras Naturais/imunologia , Pulmão/imunologia , Infecções por Orthomyxoviridae/terapia , Receptores de Interleucina/metabolismo , Junções Íntimas/patologia , Animais , Células Cultivadas , Humanos , Inflamação/imunologia , Influenza Humana/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Infecções por Orthomyxoviridae/imunologia , Pneumonia , Receptores de Interleucina/genética , Transdução de Sinais , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Interleucina 22RESUMO
Lung epithelial cells are increasingly recognized to be active effectors of microbial defense, contributing to both innate and adaptive immune function in the lower respiratory tract. As immune sentinels, lung epithelial cells detect diverse pathogens through an ample repertoire of membrane-bound, endosomal, and cytosolic pattern-recognition receptors (PRRs). The highly plastic epithelial barrier responds to detected threats via modulation of paracellular flux, intercellular communications, mucin production, and periciliary fluid composition. Epithelial PRR stimulation also induces production of cytokines that recruit and sculpt leukocyte-mediated responses, and promotes epithelial generation of antimicrobial effector molecules that are directly microbicidal. The epithelium can alternately enhance tolerance to pathogens, preventing tissue damage through PRR-induced inhibitory signals, opsonization of pathogen-associated molecular patterns, and attenuation of injurious leukocyte responses. The inducibility of these protective responses has prompted attempts to therapeutically harness epithelial defense mechanisms to protect against pneumonias. Recent reports describe successful strategies for manipulation of epithelial defenses to protect against a wide range of respiratory pathogens. The lung epithelium is capable of both significant antimicrobial responses that reduce pathogen burdens and tolerance mechanisms that attenuate immunopathology. This manuscript reviews inducible lung epithelial defense mechanisms that offer opportunities for therapeutic manipulation to protect vulnerable populations against pneumonia.
Assuntos
Pneumonia/terapia , Receptores de Reconhecimento de Padrão/metabolismo , Mucosa Respiratória/imunologia , Animais , Anti-Infecciosos/metabolismo , Humanos , Tolerância Imunológica , Imunidade , Hospedeiro Imunocomprometido , Pneumonia/imunologia , Receptores de Reconhecimento de Padrão/genética , Regulação para CimaRESUMO
Approximately 2 billion people are infected with Mycobacterium tuberculosis (Mtb), resulting in 1.4 million deaths every year. Among Mtb-infected individuals, clinical isolates belonging to the W-Beijing lineage are increasingly prevalent, associated with drug resistance, and cause severe disease immunopathology in animal models. Therefore, it is exceedingly important to identify the immune mechanisms that mediate protection against rapidly emerging Mtb strains, such as W-Beijing lineage. IL-22 is a member of the IL-10 family of cytokines with both protective and pathological functions at mucosal surfaces. Thus far, collective data show that IL-22 deficient mice are not more susceptible to aerosolized infection with less virulent Mtb strains. Thus, in this study we addressed the functional role for the IL-22 pathway in immunity to emerging Mtb isolates, using W-Beijing lineage member, Mtb HN878 as a prototype. We show that Mtb HN878 stimulates IL-22 production in TLR2 dependent manner and IL-22 mediates protective immunity during chronic stages of Mtb HN878 infection in mice. Interestingly, IL-22-dependent pathways in both epithelial cells and macrophages mediate protective mechanisms for Mtb HN878 control. Thus, our results project a new protective role for IL-22 in emerging Mtb infections.
Assuntos
Células Epiteliais/imunologia , Interleucinas/metabolismo , Pulmão/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Células Cultivadas , Doença Crônica , Resistência a Medicamentos , Humanos , Imunidade nas Mucosas , Interleucinas/genética , Pulmão/microbiologia , Pulmão/patologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Interleucina 22RESUMO
Bacterial pneumonia and tracheobronchitis are diagnosed frequently following lung transplantation. The diseases share clinical signs of inflammation and are often difficult to differentiate based on culture results. Microbiome and host immune-response signatures that distinguish between pneumonia and tracheobronchitis are undefined. Using a retrospective study design, we selected 49 bronchoalveolar lavage fluid samples from 16 lung transplant recipients associated with pneumonia (n = 8), tracheobronchitis (n = 12) or colonization without respiratory infection (n = 29). We ensured an even distribution of Pseudomonas aeruginosa or Staphylococcus aureus culture-positive samples across the groups. Bayesian regression analysis identified non-culture-based signatures comprising 16S ribosomal RNA microbiome profiles, cytokine levels and clinical variables that characterized the three diagnoses. Relative to samples associated with colonization, those from pneumonia had significantly lower microbial diversity, decreased levels of several bacterial genera and prominent multifunctional cytokine responses. In contrast, tracheobronchitis was characterized by high microbial diversity and multifunctional cytokine responses that differed from those of pneumonia-colonization comparisons. The dissimilar microbiomes and cytokine responses underlying bacterial pneumonia and tracheobronchitis following lung transplantation suggest that the diseases result from different pathogenic processes. Microbiomes and cytokine responses had complementary features, suggesting that they are closely interconnected in the pathogenesis of both diseases.
Assuntos
Bronquite/diagnóstico , Líquido da Lavagem Broncoalveolar/microbiologia , Citocinas/metabolismo , Transplante de Pulmão/efeitos adversos , Microbiota , Pneumonia Bacteriana/diagnóstico , Traqueíte/diagnóstico , Adulto , Idoso , Teorema de Bayes , Bronquite/etiologia , Bronquite/metabolismo , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Bacteriana/etiologia , Pneumonia Bacteriana/metabolismo , Estudos Retrospectivos , Traqueíte/etiologia , Traqueíte/metabolismo , TransplantadosRESUMO
Asthma is a common respiratory disease affecting â¼300 million people worldwide. Airway inflammation is thought to contribute to asthma pathogenesis, but the direct relationship between inflammation and airway hyperresponsiveness (AHR) remains unclear. This study investigates the role of inflammation in a steroid-insensitive, severe allergic airway disease model and in severe asthmatics stratified by inflammatory profile. First, we used the T-helper (T(H))-17 cells adoptive transfer mouse model of asthma to induce pulmonary inflammation, which was lessened by tumor necrosis factor (TNF)-α neutralization or neutrophil depletion. Although decreased airspace inflammation following TNFα neutralization and neutrophil depletion rescued lung compliance, neither intervention improved AHR to methacholine, and tissue inflammation remained elevated when compared with control. Further, sputum samples were collected and analyzed from 41 severe asthmatics. In severe asthmatics with elevated levels of sputum neutrophils, but low levels of eosinophils, increased inflammatory markers did not correlate with worsened lung function. This subset of asthmatics also had significantly higher levels of T(H)17-related cytokines in their sputum compared with severe asthmatics with other inflammatory phenotypes. Overall, this work suggests that lung compliance may be linked with cellular inflammation in the airspace, whereas T-cell-driven AHR may be associated with tissue inflammation and other pulmonary factors.
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Asma/complicações , Inflamação/complicações , Pulmão/fisiologia , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Animais , Asma/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/imunologia , Broncoconstritores/farmacologia , Criança , Citocinas/imunologia , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Escarro/imunologiaRESUMO
Mesothelioma is an incurable cancer of the pleura with a life expectancy of less than 1 year. On the basis of in vivo efficacy seen with the herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene-modified PA1STK cell line and ganciclovir (GCV) in a murine model of mesothelioma, a first in humans, clinical trial was designed for this therapeutic concept. The study was a phase I clinical trial using direct infusion of escalating doses of HSVtk suicide gene-modified PA1STK cells directly into tumor-associated pleural effusions followed by 7 days of intravenous GCV infusion. Therapeutic levels of GCV in both serum and pleura were achieved within 1 h, and GCV trough levels remained above the therapeutic threshold for the duration of GCV treatment. The treatment was well tolerated without any Grade 3 or 4 toxicity observed. Significant inductions of both Th1 and Th2 cytokines up to 20-fold over baseline were observed. No significant differences were seen between serum and pleura cytokine profiles, with the exception of interleukin-10, which was consistently elevated in the pleura specimens. No objective radiographic responses were observed. The data indicate significant immunological responses and validate the principal anti-tumor mechanisms observed in preclinical models of mesothelioma in a human clinical trial.
Assuntos
Ganciclovir/uso terapêutico , Genes Transgênicos Suicidas/genética , Terapia Genética , Mesotelioma/terapia , Doenças Pleurais/terapia , Timidina Quinase/genética , Timidina Quinase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Ganciclovir/administração & dosagem , Ganciclovir/efeitos adversos , Herpesvirus Humano 1/genética , Humanos , Infusões Intravenosas , Interleucina-10/análise , Interleucina-10/metabolismo , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Doenças Pleurais/tratamento farmacológico , Doenças Pleurais/patologia , Equilíbrio Th1-Th2RESUMO
T helper type 17 (Th17) cells are a distinct lineage of T cells that produce the effector molecules IL-17, IL-17F, IL-21, and IL-22. Although the role of Th17 cells in autoimmunity is well documented, there is growing evidence that the Th17 lineage and other interleukin (IL)-17-producing cells are critical for host defense against bacterial, fungal, and viral infections at mucosal surfaces. Here we summarize recent progress in our understanding of the function of IL-17-producing cells as a bridge between innate and adaptive immunity against infectious diseases at the mucosa.
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Imunidade Inata/imunologia , Infecções/imunologia , Cooperação Linfocítica/imunologia , Mucosa/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linhagem da Célula , Humanos , Síndromes de Imunodeficiência/imunologia , Imunoterapia , Interleucinas/metabolismo , Camundongos , Camundongos Knockout , Modelos Imunológicos , Receptores de Interleucina-17/deficiência , Receptores de Interleucina-17/fisiologia , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
IL-22 is an IL-10 family cytokine member that was recently discovered to be produced by Th17 cells. Current studies have revealed that IL-22 targets cells of the digestive, skin, and respiratory organ systems and plays an important role in mucosal immunity. The IL-22 receptor (IL-22R) is expressed exclusively in these tissues, thereby allowing the cytokine to mediate epithelial innate immunity in response to a variety of pathogens. Although recent studies have shown the importance of IL-22 in host defense against Gram-negative bacterial organisms (in gut and lung), there is evidence that IL-22 also plays a role in autoimmune disease, such as psoriasis. IL-22 therefore, not unlike other cytokines, has complex pro-inflammatory, anti-inflammatory, and autoimmune effects which continue to be under further investigation. This review will focus on what is known about IL-22 and its function in mucosal host defense.
Assuntos
Imunidade nas Mucosas/imunologia , Interleucinas/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucinas/biossíntese , Modelos Biológicos , Interleucina 22RESUMO
Cystic Fibrosis (CF) is the most common lethal genetic disease in the Caucasian population and typically results in the development of bronchial inflammation, bronchiectasis, the progressive loss of lung function and ultimately death. Recently it has been shown that products of the Th(17) subset of T-cells, specifically, IL-17A and IL-17F are elevated in the sputum of CF patients. This review will go over experimental evidence supporting a role for the IL- 23/IL-17 axis in CF lung inflammation.
Assuntos
Fibrose Cística/diagnóstico , Fibrose Cística/patologia , Animais , Brônquios/imunologia , Brônquios/patologia , Bronquiectasia/diagnóstico , Cartilagem/patologia , Fibrose Cística/genética , Humanos , Sistema Imunitário/patologia , Inflamação , Interleucina-17/biossíntese , Articulações/patologia , Pulmão/patologia , Modelos Biológicos , Linfócitos T/metabolismo , Linfócitos T/microbiologiaRESUMO
OBJECTIVE: It has previously been shown that the onset and the degree of joint inflammation during immune complex (IC)-mediated arthritis depend on Fcgamma receptor type III (FcgammaRIII). Local adenoviral overexpression of interferon-gamma (IFNgamma) in the knee joint prior to onset of IC-mediated arthritis aggravated severe cartilage destruction. In FcgammaRI(-/-) mice, however, chondrocyte death was not enhanced by IFNgamma, whereas matrix metalloproteinase (MMP)-mediated aggrecan breakdown was markedly elevated, suggesting a role for the activating FcgammaRIII in the latter process. We undertook this study to determine the role of FcgammaRIII in joint inflammation and severe cartilage destruction in IFNgamma-stimulated IC-mediated arthritis, using FcgammaRIII(-/-) mice. METHODS: FcgammaRIII(-/-) and wild-type (WT) mice were injected in the knee joint with recombinant adenovirus encoding murine IFNgamma (AdIFNgamma) or with adenovirus encoding enhanced green fluorescent protein 1 day prior to induction of IC-mediated arthritis. Histologic sections were obtained 3 days after arthritis onset to study inflammation and cartilage damage. MMP-mediated expression of the VDIPEN neoepitope was detected by immunolocalization. Chemokine and FcgammaR expression levels were determined in synovial washouts and synovium, respectively. RESULTS: Injection of AdIFNgamma in naive knee joints markedly increased levels of messenger RNA for FcgammaRI, FcgammaRII, and FcgammaRIII. Upon IFNgamma overexpression prior to induction of IC-mediated arthritis, joint inflammation was similar in FcgammaRIII(-/-) and WT mice. The percentage of macrophages in the knee joint was increased, which correlated with high concentrations of the macrophage attractant macrophage inflammatory protein 1alpha. Furthermore, IFNgamma induced 2-fold and 3-fold increases in chondrocyte death in WT controls and FcgammaRIII(-/-) mice, respectively. Notably, VDIPEN expression also remained high in FcgammaRIII(-/-) mice. CONCLUSION: IFNgamma bypasses the dependence on FcgammaRIII in the development of IC-mediated arthritis. Furthermore, both FcgammaRI and FcgammaRIII can mediate MMP-dependent cartilage matrix destruction.
Assuntos
Artrite Reumatoide/imunologia , Morte Celular/imunologia , Condrócitos/imunologia , Interferon gama/biossíntese , Receptores de IgG/imunologia , Agrecanas , Animais , Cartilagem/imunologia , Cartilagem/fisiopatologia , Proteínas da Matriz Extracelular/imunologia , Doenças do Complexo Imune/imunologia , Interferon gama/imunologia , Lectinas Tipo C , Metaloproteinases da Matriz/imunologia , Camundongos , Modelos Animais , Proteoglicanas/imunologiaRESUMO
Marine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 x 10(-2)% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500-600 reactions per day.
Assuntos
DNA/sangue , Reação em Cadeia da Polimerase/métodos , Animais , Quimera , Feminino , Transplante de Células-Tronco Hematopoéticas , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Caracteres Sexuais , Cromossomo YRESUMO
Retrovirus-derived vectors are currently the preferred vectors used for human gene therapy protocols. Serious safety concerns persist, however, which are specifically related to the formation of a replication-competent virus, and no synthesis method currently employed precludes its formation with certainty. For many cell types, a low transduction efficiency results in insufficient therapeutic benefit. We describe the development of a molecular conjugate system, which permits transient chemical modification of a retrovirus with polylysine. This modification not only introduces additional safety features over standard unmodified retrovirus vectors, but also provides enhanced transduction efficiency.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , Polilisina/química , Retroviridae/química , Retroviridae/genética , Transdução Genética , Animais , Humanos , Polilisina/metabolismo , Retroviridae/metabolismoRESUMO
AIM: Protein tyrosine phosphatase-1B (PTP-1B) is an intracellular PTP known to dephosphorylate and inactivate upstream tyrosine phosphoproteins in the insulin signalling cascade. We and others reported increased abundance of catalytically impaired PTP-1B in tissue lysates from obese human subjects with and without type 2 diabetes, while genetic knockout of PTP-1B improves insulin sensitivity and prevents nutritionally mediated insulin resistance and obesity. The aim of the present work was to further elucidate the role of PTP-1B in glucose metabolism in vivo. METHODS: We used adenoviral constructs incorporating cDNAs for either wild-type (W/T) or a catalytically inactive C(215)S (C/S) mutant PTP-1B to achieve liver-selective PTP-1B overexpression in young Sprague-Dawley rats using tail vein injection, based on the high degree of hepatotropism of adenovirus 5 (Ad5). An Ad5-lacZ construct encoding beta-galactosidase was used as a control for viral effects alone. A hyperinsulinaemic euglycaemic clamp was used to study whole body glucose disposal and endogenous glucose production rates. RESULTS: Control studies in HIRcB cells confirmed catalytic activity and inactivity of W/T and C/S respectively. Mean PTP-1B abundance was 2.24 +/- 0.02- and 2.33 +/- 0.04-fold of saline-treated control in liver lysates of W/T and C/S rats respectively. Liver selective overexpression was confirmed by analysis of tissue lysates from liver, fat and muscle tissues. Ad5 treatment did not result in a statistically or clinically significant liver injury, as determined by serum alanine aminotransferase and histological examination. Seven days post injection, no significant difference in rate of weight gain, fasting blood glucose or insulin levels were seen in any group. Similarly, under steady-state glucose clamp conditions, glucose disposal rate (R(d)), endogenous glucose production rate (EGP) and serum insulin levels were similar in all groups. CONCLUSION: We conclude that moderate medium-term overabundance, to a degree resembling that seen in insulin-resistant states, of PTP-1B in liver tissue does not alter insulin action on glucose metabolism and that the major site of action of PTP-1B is presumably at insulin-responsive target tissue or tissues other than the liver.
Assuntos
Adenoviridae/genética , Resistência à Insulina , Fígado/enzimologia , Fígado/virologia , Proteínas Tirosina Fosfatases/biossíntese , Adenoviridae/enzimologia , Infecções por Adenoviridae/enzimologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/fisiopatologia , Animais , Peso Corporal/genética , Linhagem Celular , Linhagem Celular Transformada , Técnica Clamp de Glucose , Humanos , Hiperinsulinismo/enzimologia , Resistência à Insulina/genética , Fígado/patologia , Testes de Função Hepática , Masculino , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Coloração e Rotulagem , Fatores de TempoRESUMO
Many mutant mice deficient in leukocyte adhesion molecules display altered hematopoiesis and neutrophilia. This study investigated whether peripheral blood neutrophil concentrations in these mice are elevated as a result of accumulation of neutrophils in the circulation or altered hematopoiesis mediated by a disrupted regulatory feedback loop. Chimeric mice were generated by transplanting various ratios of CD18(+/+) and CD18(-/-) unfractionated bone marrow cells into lethally irradiated wild-type mice, resulting in approximately 0%, 10%, 50%, 90%, or 100% CD18 null neutrophils in the blood. The presence of only 10% CD18(+/+) neutrophils was sufficient to prevent the severe neutrophilia seen in mice reconstituted with CD18(-/-) bone marrow cells. These data show that the neutrophilia in CD18(-/-) mice is not caused by enhanced neutrophil survival or the inability of neutrophils to leave the vascular compartment. In CD18(-/-), CD18(-/-)E(-/-), CD18(-/-)P(-/-), EP(-/-), and EPI(-/-) mice, levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-17 (IL-17) were elevated in proportion to the neutrophilia seen in these mice, regardless of the underlying mutation. Antibiotic treatment or the propensity to develop skin lesions did not correlate with neutrophil counts. Blocking IL-17 or G-CSF function in vivo significantly reduced neutrophil counts in severely neutrophilic mice by approximately 50% (P <.05) or 70% (P <.01), respectively. These data show that peripheral blood neutrophil numbers are regulated by a feedback loop involving G-CSF and IL-17 and that this feedback loop is disrupted when neutrophils cannot migrate into peripheral tissues.
Assuntos
Moléculas de Adesão Celular/deficiência , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/fisiologia , Hematopoese , Interleucina-17/farmacologia , Animais , Transplante de Medula Óssea , Antígenos CD18/análise , Antígenos CD18/fisiologia , Selectina E/análise , Selectina E/fisiologia , Retroalimentação , Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/fisiologia , Molécula 1 de Adesão Intercelular/análise , Molécula 1 de Adesão Intercelular/fisiologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/sangue , Interleucina-17/fisiologia , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/química , Neutrófilos/citologia , Selectina-P/análise , Selectina-P/fisiologia , Quimeras de TransplanteRESUMO
Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. Moreover, deficient CD4+ T lymphocytes impair vaccination approaches to prevent opportunistic infection. Therefore, we investigated a CD4+ T cell-independent vaccine approach to a prototypic AIDS-defining infection, Pneumocystis carinii (PC) pneumonia. Here, we demonstrate that bone marrow-derived dendritic cells (DCs) expressing the murine CD40 ligand, when pulsed ex vivo by PC antigen, elicited significant titers of anti-PC IgG in CD4-deficient mice. Vaccinated animals demonstrated significant protection from PC infection, and this protection was the result of an effective humoral response, since adoptive transfer of CD4-depleted splenocytes or serum conferred this protection to CD4-deficient mice. Western blot analysis of PC antigen revealed that DC-vaccinated, CD4-deficient mice predominantly reacted to a 55-kDa PC antigen. These studies show promise for advances in CD4-independent vaccination against HIV-related pathogens.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas Fúngicas/administração & dosagem , Pneumocystis/imunologia , Pneumonia por Pneumocystis/prevenção & controle , Transferência Adotiva , Animais , Sequência de Bases , Primers do DNA , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit(+) human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.
Assuntos
Antígenos CD34/análise , Transferência Genética Horizontal , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Adenoviridae/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Vetores Genéticos , Humanos , Receptores Virais/análise , Retroviridae/genéticaRESUMO
Bacterial pneumonia remains an important cause of morbidity and mortality worldwide, especially in immune-compromised patients. Cytokines and chemokines are critical molecules expressed in response to invading pathogens and are necessary for normal lung bacterial host defenses. Here we show that interleukin (IL)-17, a novel cytokine produced largely by CD4+ T cells, is produced in a compartmentalized fashion in the lung after challenge with Klebsiella pneumoniae. Moreover, overexpression of IL-17 in the pulmonary compartment using a recombinant adenovirus encoding murine IL-17 (AdIL-17) resulted in the local induction of tumor necrosis factor-alpha, IL-1beta, macrophage inflammatory protein-2, and granulocyte colony-stimulating factor (G-CSF); augmented polymorphonuclear leukocyte recruitment; and enhanced bacterial clearance and survival after challenge with K. pneumoniae. However, simultaneous treatment with AdIL-17 provided no survival benefit after intranasal K. pneumoniae challenge. These data show that IL-17 may have a role in priming for enhanced chemokine and G-CSF production in the context of lung infection and that optimally timed gene therapy with IL-17 may augment host defense against bacterial pneumonia.
Assuntos
Interleucina-17/metabolismo , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/metabolismo , Pulmão/imunologia , Pneumonia Bacteriana/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Quimiocina CXCL2 , Quimiocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-17/genética , Pulmão/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Autologous and allogeneic bone marrow transplantations have evolved as important cancer therapy modalities. For both indications, peripheral blood has been shown to have distinct advantages over bone marrow as the stem cell source. Cytokine combinations for mobilization have enhanced stem cell yield and accelerated engraftment. However, novel mobilizing agents and strategies are needed to further improve clinical outcomes. Within the donor graft, the dynamic equilibrium between T cells and stem cells critically influences engraftment and transplantation results. IL-17 is a cytokine produced almost exclusively from activated T cells. IL-17 was expressed in vivo with adenovirus technology. Here, proof-of-principle studies demonstrate that IL-17 effectively mobilizes hemopoietic precursor cells (CFU-granulocyte-erythrocyte-macrophage-monocyte, CFU-high proliferative potential) and primitive hemopoietic stem cells (Lin(-/low)c-kit(+)Sca1(+)). Moreover, mouse IL-17 adenovirus-mobilized peripheral blood stem cells rescued lethally irradiated mice. Bone marrow was found to be 45-75% of donor origin at 1 year. In secondary recipients, donor-derived bone marrow cells ranged from 45 to 95%. These data show that IL-17 mobilizes stem cells in mice with short- and long-term reconstituting capacity. Additional comparative studies are needed as well as studies in tumor models to refine distinct potential clinical applications for IL-17-mobilized peripheral blood stem cells.
Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas , Interleucina-17/fisiologia , Animais , Divisão Celular/genética , Divisão Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Imunofenotipagem , Interleucina-17/administração & dosagem , Interleucina-17/genética , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação/imunologia , Células-Tronco/citologiaRESUMO
Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.