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1.
J Trauma Acute Care Surg ; 97(1): 32-38, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38444065

RESUMO

INTRODUCTION: The endothelial glycocalyx on the luminal surface of endothelial cells contributes to the permeability barrier of the pulmonary vasculature. Dimethyl sulfoxide (DMSO) has a disordering effect on plasma membranes, which prevents the formation of ordered membrane domains important in the shedding of the endothelial glycocalyx. We hypothesized that DMSO would protect against protein leak by preserving the endothelial glycocalyx in a murine model of acute respiratory distress syndrome (ARDS). METHODS: C57BL/6 mice were given ARDS via intratracheally administered lipopolysaccharide (LPS). Dimethyl sulfoxide (220 mg/kg) was administered intravenously for 4 days. Animals were sacrificed postinjury day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts and protein content were quantified. Lung sections were stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify the endothelial glycocalyx. Human umbilical vein endothelial cells (HUVECs) were exposed to LPS. Endothelial glycocalyx was measured using fluorescein isothiocyanate-labeled wheat germ agglutinin, and co-immunoprecipitation was performed to measure interaction between sheddases and syndecan-1. RESULTS: Dimethyl sulfoxide treatment resulted in greater endothelial glycocalyx staining intensity in the lung when compared with sham (9,641 vs. 36,659 arbitrary units, p < 0.001). Total BAL cell counts were less for animals receiving DMSO (6.93 × 10 6 vs. 2.49 × 10 6 cells, p = 0.04). The treated group had less BAL macrophages (189.2 vs. 76.9 cells, p = 0.02) and lymphocytes (527.7 vs. 200.0 cells, p = 0.02). Interleukin-6 levels were lower in DMSO treated. Animals that received DMSO had less protein leak in BAL (1.48 vs. 1.08 µg/µL, p = 0.02). Dimethyl sulfoxide prevented LPS-induced endothelial glycocalyx loss in HUVECs and reduced the interaction between matrix metalloproteinase 16 and syndecan-1. CONCLUSION: Systemically administered DMSO protects the endothelial glycocalyx in the pulmonary vasculature, mitigating pulmonary capillary leak after acute lung injury. Dimethyl sulfoxide also results in decreased inflammatory response. Dimethyl sulfoxide reduced the interaction between matrix metalloproteinase 16 and syndecan-1 and prevented LPS-induced glycocalyx damage in HUVECs. Dimethyl sulfoxide may be a novel therapeutic for ARDS.


Assuntos
Lesão Pulmonar Aguda , Dimetil Sulfóxido , Modelos Animais de Doenças , Glicocálix , Camundongos Endogâmicos C57BL , Animais , Camundongos , Glicocálix/metabolismo , Glicocálix/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Lipopolissacarídeos , Masculino , Humanos , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos
2.
J Trauma Acute Care Surg ; 96(3): 386-393, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-37934622

RESUMO

BACKGROUND: Succinate is a proinflammatory citric acid cycle metabolite that accumulates in tissues during pathophysiological states. Oxidation of succinate after ischemia-reperfusion leads to reversal of the electron transport chain and generation of reactive oxygen species. Dimethyl malonate (DMM) is a competitive inhibitor of succinate dehydrogenase, which has been shown to reduce succinate accumulation. We hypothesized that DMM would protect against inflammation in a murine model of ARDS. METHODS: C57BL/6 mice were given ARDS via 67.7 µg of intratracheally administered lipopolysaccharide. Dimethyl malonate (50 mg/kg) was administered via tail vein injection 30 minutes after injury, then daily for 3 days. The animals were sacrificed on day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts were performed to examine cellular influx. Supernatant protein was quantified via Bradford protein assay. Animals receiving DMM (n = 8) were compared with those receiving sham injection (n = 8). Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify the endothelial glycocalyx (EGX). RESULTS: Total cell counts in BAL was less for animals receiving DMM (6.93 × 10 6 vs. 2.46 × 10 6 , p = 0.04). The DMM group had less BAL macrophages (168.6 vs. 85.1, p = 0.04) and lymphocytes (527.7 vs. 248.3; p = 0.04). Dimethyl malonate-treated animals had less protein leak in BAL than sham treated (1.48 vs. 1.15 µg/µl, p = 0.03). Treatment with DMM resulted in greater staining intensity of the EGX in the lung when compared with sham (12,016 vs. 15,186 arbitrary units, p = 0.03). Untreated animals had a greater degree of weight loss than treated animals (3.7% vs. 1.1%, p = 0.04). Dimethyl malonate prevented the upregulation of monocyte chemoattractant protein-1 (1.66 vs. 0.92 RE, p = 0.02) and ICAM-1 (1.40 vs. 1.01 RE, p = 0.05). CONCLUSION: Dimethyl malonate reduces lung inflammation and capillary leak in ARDS. This may be mediated by protection of the EGX and inhibition of monocyte chemoattractant protein-1 and ICAM-1. Dimethyl malonate may be a novel therapeutic for ARDS.


Assuntos
Quimiocina CCL2 , Malonatos , Síndrome do Desconforto Respiratório , Camundongos , Animais , Molécula 1 de Adesão Intercelular , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Pulmão/metabolismo , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/prevenção & controle , Succinatos
3.
Am J Pathol ; 194(3): 338-352, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38101567

RESUMO

The high mortality rates of acute lung injury and acute respiratory distress syndrome challenge the field to identify biomarkers and factors that can be exploited for therapeutic approaches. IL-22 is a cytokine that has antibacterial and reparative properties in the lung. However, it also can exacerbate inflammation and requires tight control by the extracellular inhibitory protein known as IL-22 binding protein (IL-22BP) (Il22ra2). This study showed the necessity of IL-22BP in controlling and preventing acute lung injury using IL-22BP knockout mice (Il22ra2-/-) in the bleomycin model of acute lung injury/acute respiratory distress syndrome. Il22ra2-/- mice had greater sensitivity (weight loss and death) and pulmonary inflammation in the acute phase (first 7 days) of the injury compared with wild-type C57Bl/6 controls. The inflammation was driven by excess IL-22 production, inducing the influx of pathogenic IL-17A+ γδ T cells to the lung. Interestingly, this inflammation was initiated in part by the noncanonical IL-22 signaling to macrophages, which express the IL-22 receptor (Il22ra1) in vivo after bleomycin challenge. This study further showed that IL-22 receptor alpha-1+ macrophages can be stimulated by IL-22 to produce a number of IL-17-inducing cytokines such as IL-1ß, IL-6, and transforming growth factor-ß1. Together, the results suggest that IL-22BP prevents IL-22 signaling to macrophages and reduces bleomycin-mediated lung injury.


Assuntos
Lesão Pulmonar Aguda , Lesão Pulmonar , Síndrome do Desconforto Respiratório , Animais , Camundongos , Lesão Pulmonar Aguda/patologia , Bleomicina/efeitos adversos , Citocinas/metabolismo , Inflamação/patologia , Interleucina 22 , Pulmão/patologia , Lesão Pulmonar/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome do Desconforto Respiratório/metabolismo
4.
Commun Biol ; 6(1): 1265, 2023 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-38092883

RESUMO

SARS-CoV-2 infection can cause persistent respiratory sequelae. However, the underlying mechanisms remain unclear. Here we report that sub-lethally infected K18-human ACE2 mice show patchy pneumonia associated with histiocytic inflammation and collagen deposition at 21 and 45 days post infection (DPI). Transcriptomic analyses revealed that compared to influenza-infected mice, SARS-CoV-2-infected mice had reduced interferon-gamma/alpha responses at 4 DPI and failed to induce keratin 5 (Krt5) at 6 DPI in lung, a marker of nascent pulmonary progenitor cells. Histologically, influenza- but not SARS-CoV-2-infected mice showed extensive Krt5+ "pods" structure co-stained with stem cell markers Trp63/NGFR proliferated in the pulmonary consolidation area at both 7 and 14 DPI, with regression at 21 DPI. These Krt5+ "pods" structures were not observed in the lungs of SARS-CoV-2-infected humans or nonhuman primates. These results suggest that SARS-CoV-2 infection fails to induce nascent Krt5+ cell proliferation in consolidated regions, leading to incomplete repair of the injured lung.


Assuntos
COVID-19 , Influenza Humana , Camundongos , Humanos , Animais , SARS-CoV-2 , Pulmão , Perfilação da Expressão Gênica
5.
Sci Adv ; 9(24): eadf6600, 2023 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-37315138

RESUMO

Acute hemorrhage commonly leads to coagulopathy and organ dysfunction or failure. Recent evidence suggests that damage to the endothelial glycocalyx contributes to these adverse outcomes. The physiological events mediating acute glycocalyx shedding are undefined, however. Here, we show that succinate accumulation within endothelial cells drives glycocalyx degradation through a membrane reorganization-mediated mechanism. We investigated this mechanism in a cultured endothelial cell hypoxia-reoxygenation model, in a rat model of hemorrhage, and in trauma patient plasma samples. We found that succinate metabolism by succinate dehydrogenase mediates glycocalyx damage through lipid oxidation and phospholipase A2-mediated membrane reorganization, promoting the interaction of matrix metalloproteinase 24 (MMP24) and MMP25 with glycocalyx constituents. In a rat hemorrhage model, inhibiting succinate metabolism or membrane reorganization prevented glycocalyx damage and coagulopathy. In patients with trauma, succinate levels were associated with glycocalyx damage and the development of coagulopathy, and the interaction of MMP24 and syndecan-1 was elevated compared to healthy controls.


Assuntos
Células Endoteliais , Hemorragia , Animais , Ratos , Metabolismo dos Lipídeos , Hipóxia , Succinatos , Ácido Succínico
6.
Shock ; 60(1): 56-63, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37086080

RESUMO

ABSTRACT: Introduction: Endothelial glycocalyx damage occurs in numerous pathological conditions and results in endotheliopathy. Extracellular vesicles, including exosomes and microvesicles, isolated from adipose-derived mesenchymal stem cells (ASCs) have therapeutic potential in multiple disease states; however, their role in preventing glycocalyx shedding has not been defined. We hypothesized that ASC-derived exosomes and microvesicles would protect the endothelial glycocalyx from damage by LPS injury in cultured endothelial cells. Methods : Exosomes and microvesicles were collected from ASC conditioned media by centrifugation (10,000 g for microvesicles, 100,000 g for exosomes). Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). LPS-injured cells (n = 578) were compared with HUVECS with concomitant LPS injury plus 1.0 µg/mL of exosomes (n = 540) or microvesicles (n = 510) for 24 hours. These two cohorts were compared with control HUVECs that received phosphate-buffered saline only (n = 786) and HUVECs exposed to exosomes (n = 505) or microvesicles (n = 500) alone. Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify EGX. Real-time quantitative reverse-transcription polymerase chain reaction was used on HUVECs cell lystate to quantify hyaluron synthase-1 (HAS1) expression. Results: Exosomes alone decreased endothelial glycocalyx staining intensity when compared with control (4.94 vs. 6.41 AU, P < 0.001), while microvesicles did not cause a change glycocalyx staining intensity (6.39 vs. 6.41, P = 0.99). LPS injury resulted in decreased glycocalyx intensity as compared with control (5.60 vs. 6.41, P < 0.001). Exosomes (6.85 vs. 5.60, P < 0.001) and microvesicles (6.35 vs. 5.60, P < 0.001) preserved endothelial glycocalyx staining intensity after LPS injury. HAS1 levels were found to be higher in the exosome (1.14 vs. 3.67 RE, P = 0.02) and microvesicle groups (1.14 vs. 3.59 RE, P = 0.02) when compared with LPS injury. Hyaluron synthase-2 and synthase-3 expressions were not different in the various experimental groups. Conclusions: Exosomes alone can damage the endothelial glycocalyx. However, in the presence of LPS injury, both exosomes and microvesicles protect the glycocalyx layer. This effect seems to be mediated by HAS1. Level of Evidence : Basic science study.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Humanos , Exossomos/metabolismo , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/metabolismo , Glicocálix , Células Endoteliais da Veia Umbilical Humana/metabolismo
7.
PLoS One ; 17(10): e0276232, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36260622

RESUMO

The endothelial glycocalyx (EGX) contributes to the permeability barrier of vessels and regulates the coagulation cascade. EGX damage, which occurs in numerous disease states, including sepsis and trauma, results in endotheliopathy. While influenza and other viral infections are known to cause endothelial dysfunction, their effect on the EGX has not been described. We hypothesized that the H1N1 influenza virus would cause EGX degradation. Human umbilical vein endothelial cells (HUVECs) were exposed to varying multiplicities of infection (MOI) of the H1N1 strain of influenza virus for 24 hours. A dose-dependent effect was examined by using an MOI of 5 (n = 541), 15 (n = 714), 30 (n = 596), and 60 (n = 653) and compared to a control (n = 607). Cells were fixed and stained with FITC-labelled wheat germ agglutinin to quantify EGX. There was no difference in EGX intensity after exposure to H1N1 at an MOI of 5 compared to control (6.20 vs. 6.56 Arbitrary Units (AU), p = 0.50). EGX intensity was decreased at an MOI of 15 compared to control (5.36 vs. 6.56 AU, p<0.001). The degree of EGX degradation was worse at higher doses of the H1N1 virus; however, the decrease in EGX intensity was maximized at an MOI of 30. Injury at MOI of 60 was not worse than MOI of 30. (4.17 vs. 4.47 AU, p = 0.13). The H1N1 virus induces endothelial dysfunction by causing EGX degradation in a dose-dependent fashion. Further studies are needed to characterize the role of this EGX damage in causing clinically significant lung injury during acute viral infection.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Doenças Vasculares , Humanos , Glicocálix/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Influenza Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana , Doenças Vasculares/metabolismo , Aglutininas do Germe de Trigo/metabolismo
9.
Sci Transl Med ; 13(616): eabj7843, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34669442

RESUMO

In 1967, two toddlers immunized with a formalin-inactivated vaccine against respiratory syncytial virus (FIRSV) in the United States died from enhanced RSV disease (ERD), a severe form of illness resulting from aberrant priming of the antiviral immune response during vaccination. Up to 80% of immunized children subsequently exposed to wild-type virus were hospitalized. These events hampered RSV vaccine development for decades. Here, we provide a characterization of the clinical, immunopathological, and transcriptional signature of fatal human ERD, outlining evidence for safety evaluation of RSV vaccines and a framework for understanding disease enhancement for pathogens in general.


Assuntos
Doenças Transmissíveis , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Pré-Escolar , Humanos , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sinciciais Respiratórios
10.
PLoS One ; 16(10): e0254985, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34597299

RESUMO

BACKGROUND: The goal of this study was to determine if IL-22:Fc would Acute Respiratory Distress Syndrome (ARDS). SUMMARY BACKGROUND DATA: No therapies exist for ARDS and treatment is purely supportive. Interleukin-22 (IL-22) plays an integral component in recovery of the lung from infection. IL-22:Fc is a recombinant protein with a human FC immunoglobulin that increases the half-life of IL-22. STUDY DESIGN: ARDS was induced in C57BL/6 mice with intra-tracheal lipopolysaccharide (LPS) at a dose of 33.3 or 100 ug. In the low-dose LPS group (LDG), IL-22:FC was administered via tail vein injection at 30 minutes (n = 9) and compared to sham (n = 9). In the high-dose LPS group (HDG), IL-22:FC was administered (n = 11) then compared to sham (n = 8). Euthanasia occurred after bronchioalveolar lavage (BAL) on post-injury day 4. RESULTS: In the LDG, IL-22:FC resulted in decreased protein leak (0.15 vs. 0.25 ug/uL, p = 0.02). BAL protein in animals receiving IL-22:Fc in the HDG was not different. For the HDG, animals receiving IL-22:Fc had lower BAL cell counts (539,636 vs 3,147,556 cells/uL, p = 0.02). For the HDG, IL-6 (110.6 vs. 527.1 pg/mL, p = 0.04), TNF-α (5.87 vs. 25.41 pg/mL, p = 0.04), and G-CSF (95.14 vs. 659.6, p = 0.01) levels were lower in the BAL fluid of IL-22:Fc treated animals compared to sham. CONCLUSIONS: IL-22:Fc decreases lung inflammation and lung capillary leak in ARDS. IL-22:Fc may be a novel therapy for ARDS.


Assuntos
Fragmentos Fc das Imunoglobulinas/farmacologia , Interleucinas/farmacologia , Lesão Pulmonar/tratamento farmacológico , Pneumonia/tratamento farmacológico , Síndrome do Desconforto Respiratório/tratamento farmacológico , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Lipopolissacarídeos/toxicidade , Lesão Pulmonar/patologia , Contagem de Linfócitos , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Pneumonia/patologia , Receptores de Interleucina/metabolismo , Proteínas Recombinantes/farmacologia , Síndrome do Desconforto Respiratório/patologia , Mucosa Respiratória/patologia , Interleucina 22
11.
Cell Rep ; 35(5): 109055, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33905739

RESUMO

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.


Assuntos
Células Epiteliais Alveolares/virologia , Tratamento Farmacológico da COVID-19 , COVID-19/patologia , Pulmão/virologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Adulto , Idoso , Alanina/análogos & derivados , Alanina/farmacologia , Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Pré-Escolar , Descoberta de Drogas/métodos , Células Epiteliais/virologia , Epitélio/metabolismo , Epitélio/virologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Cultura Primária de Células , Mucosa Respiratória/virologia , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
12.
J Surg Res ; 264: 51-57, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33773321

RESUMO

BACKGROUND: Fresh frozen plasma (FFP) contains proinflammatory mediators released from cellular debris during frozen storage. In addition, recent studies have shown that transfusion of never-frozen plasma (NFP), instead of FFP, may be superior in trauma patients. We hypothesized that FFP would have higher levels of inflammatory mediators when compared to NFP. MATERIALS AND METHODS: FFP (n = 8) and NFP (n = 8) samples were obtained from an urban, level 1 trauma center blood bank. The cytokines in these samples were compared using a Milliplex (Milliplex Sigma) human cytokine magnetic bead panel multiplex assay for 41 different biomarkers. RESULTS: Growth factors that were higher in NFP included platelet-derived growth factor-AA (PDGF-AA; 8.09 versus 108.00 pg/mL, P < 0.001) and PDGF-AB (0.00 versus 215.20, P= 0.004). Soluble CD40-ligand (sCD40L), a platelet activator and pro-coagulant, was higher in NFP (31.81 versus 80.45 pg/mL, P< 0.001). RANTES, a leukocyte chemotactic cytokine was higher in NFP (26.19 versus 1418.00 pg/mL, P< 0.001). Interleukin-4 (5.70 versus 0.00 pg/mL, P= 0.03) and IL-8 (2.20 versus 0.52 pg/ml, P= 0.03) levels were higher in were higher in FFP. CONCLUSIONS: Frozen storage of plasma may result in decrease of several growth factors and/or pro-coagulants found in NFP. In addition, the freezing and thawing process may induce release of pro-inflammatory chemokines. Further studies are needed to determine if these cytokines result in improved outcomes with NFP over FFP in transfusion of trauma patients.


Assuntos
Preservação de Sangue/efeitos adversos , Criopreservação , Citocinas/análise , Peptídeos e Proteínas de Sinalização Intercelular/análise , Plasma/química , Transfusão de Componentes Sanguíneos/métodos , Preservação de Sangue/métodos , Citocinas/imunologia , Humanos , Plasma/imunologia , Resultado do Tratamento , Ferimentos e Lesões/terapia
13.
J Immunol ; 206(7): 1540-1548, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33648937

RESUMO

IL-17A and IL-22 derived from Th17 cells play a significant role in mucosal immunity and inflammation. TGF-ß and IL-6 promote Th17 differentiation; however, these cytokines have multiple targets. The identification and screening of additional molecules that regulate IL-17A and IL-22 responses in certain inflammatory conditions is of great clinical significance. In this study, we show that CDDO-Im, a specific Nrf2 activator, promotes IL-17A and IL-22 responses in murine Th17 cells. In contrast, CDDO-Im inhibits IL-17A response in multiple sclerosis patient-derived PBMCs. However, Nrf2 specifically regulates IL-22 response in vivo. Nrf2 acts through the regulation of antioxidant response element (ARE) binding motifs in target genes to induce or repress transcription. Promoter analysis revealed that Il17a, Rorc, and Ahr genes have several ARE motifs. We showed that Nrf2 bound to ARE repressor (ARE-R2) of Rorc and inhibited Rorc-dependent IL-17A transactivation. The luciferase reporter assay data showed that CDDO-Im regulated Ahr promoter activity. Chromatin immunoprecipitation quantitative PCR data showed that Nrf2 bound to ARE of AhR. Finally, we confirmed that the CDDO-Im-mediated induction of IL-22 production in CD4+ T cells was abrogated in CD4-specific Ahr knockout mice (AhrCD4 ). CH-223191, a specific AhR antagonist, inhibits CDDO-Im-induced IL-22 production in CD4+ T cells, which further confirmed the AhR-dependent regulation. Collectively, our data showed that Nrf2 via AhR pathways regulated IL-22 response in CD4+ T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucinas/metabolismo , Esclerose Múltipla/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Células Th17/imunologia , Animais , Compostos Azo/metabolismo , Regulação da Expressão Gênica , Humanos , Imidazóis/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/metabolismo , Regiões Promotoras Genéticas/genética , Pirazóis/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Interleucina 22
14.
J Trauma Acute Care Surg ; 90(2): 337-345, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33502147

RESUMO

BACKGROUND: The endothelial glycocalyx (EG) on the luminal surface of endothelial cells contributes to the permeability barrier of vessels and prevents activation of the coagulation cascade. Endothelial glycocalyx damage, which occurs in the shock state, results in endotheliopathy. Interleukin (IL)-22 is a cytokine with both proinflammatory and anti-inflammatory properties, and how IL-22 affects the EG has not been studied. We hypothesized that IL-22:Fc, a recombinant fusion protein with human IL-22 and the Fc portion of human immunoglobulin G1 (which extends the protein half-life), would not affect EG shedding in endothelium after injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). Lipopolysaccharide-injured cells (n = 284) were compared with HUVECs with LPS injury plus 0.375 µg/mL of IL-22:Fc treatment (n = 293) for 12 hours. These two cohorts were compared with control HUVECs (n = 286) and HUVECs exposed to IL-22:Fc alone (n = 269). Cells were fixed and stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify EG. Total RNA was collected, and select messenger RNAs were quantified by real time - quantitative polymerase chain reaction (RT-qPCR) using SYBR green fluorescence. RESULTS: Exposure of HUVECs to LPS resulted in degradation of the EG compared with control (5.86 vs. 6.09 arbitrary unit [AU], p = 0.01). Interleukin-22:Fc alone also resulted in degradation of EG (5.08 vs. 6.09 AU, p = 0.01). Treatment with IL-22:Fc after LPS injury resulted in less degradation of EG compared with LPS injury alone (5.86 vs. 5.08 AU, p = 0.002). Expression of the IL-22Ra1 receptor was not different for IL-22:Fc treated compared with LPS injury only (0.69 vs. 0.86 relative expression, p = 0.10). Treatment with IL-22:Fc after LPS injury resulted in less matrix metalloproteinase 2 (0.79 vs. 1.70 relative expression, p = 0.005) and matrix metalloproteinase 14 (0.94 vs. 2.04 relative expression, p = 0.02). CONCLUSIONS: Interleukin-22:Fc alone induces EG degradation. However, IL-22:Fc treatment after LPS injury appears to mitigate EG degradation. This protective effect appears to be mediated via reduced expression of metalloproteinases.


Assuntos
Células Endoteliais , Glicocálix , Fragmentos Fc das Imunoglobulinas/farmacologia , Interleucinas/metabolismo , Lipopolissacarídeos/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Regulação para Baixo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Glicocálix/imunologia , Glicocálix/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina G , Metaloproteinase 2 da Matriz/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Interleucina 22
15.
Mucosal Immunol ; 12(5): 1231-1243, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31296910

RESUMO

Secondary bacterial pneumonia is a significant complication of severe influenza infection and Staphylococcus aureus and Streptococcus pneumoniae are the primary pathogens of interest. IL-22 promotes S. aureus and S. pneumoniae host defense in the lung through epithelial integrity and induction of antimicrobial peptides and is inhibited by the soluble decoy receptor IL-22-binding protein (IL-22BP). Little is known about the effect of the IL-22/IL-22BP regulatory pathway on lung infection, and it has not been studied in the setting of super-infection. We exposed wild-type and IL-22BP-/- mice to influenza A/PR/8/34 for 6 days prior to infection with S. aureus (USA300) S. pneumoniae. Super-infected IL-22BP-/- mice had decreased bacterial burden and improved survival compared to controls. IL-22BP-/- mice exhibited decreased inflammation, increased lipocalin 2 expression, and deletion of IL-22BP was associated with preserved epithelial barrier function with evidence of improved tight junction stability. Human bronchial epithelial cells treated with IL-22Fc showed evidence of improved tight junctions compared to untreated cells. This study revealed that IL-22BP-/- mice are protected during influenza, bacterial super-infection, suggesting that IL-22BP has a pro-inflammatory role and impairs epithelial barrier function likely through interaction with IL-22.


Assuntos
Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Transporte/metabolismo , Interleucinas/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Superinfecção , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/patologia , Carga Bacteriana , Barreira Alveolocapilar/metabolismo , Barreira Alveolocapilar/patologia , Barreira Alveolocapilar/virologia , Proteínas de Transporte/genética , Modelos Animais de Doenças , Expressão Gênica , Interleucinas/genética , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Permeabilidade , Ligação Proteica , Staphylococcus aureus , Streptococcus pneumoniae , Junções Íntimas , Interleucina 22
16.
Vaccine ; 37(6): 808-816, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30638799

RESUMO

Respiratory infections are a leading cause of morbidity and mortality globally. This is partially due to a lack of effective vaccines and a clear understanding of how vaccination route and formulation influence protective immunity in mucosal tissues such as the lung. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing acute pulmonary infections and is a leading cause of hospital-acquired and ventilator-associated pneumonia. With multidrug-resistant P. aeruginosa infections on the rise, the need for a vaccine against this pathogen is critical. Growing evidence suggests that a successful P. aeruginosa vaccine may require mucosal antibody and Th1- and Th17-type CD4+ T cells to prevent pulmonary infection. Intradermal immunization with adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT), can direct protective immune responses to mucosal tissues, including the lungs. We reasoned that intradermal immunization with P. aeruginosa outer membrane proteins (OMPs) adjuvanted with dmLT could drive neutralizing antibodies and migration of CD4+ T cells to the lungs and protect against P. aeruginosa pneumonia in a murine model. Here we show that mice immunized with OMPs and dmLT had significantly more antigen-specific IgG and Th1- and Th17-type CD4+ memory T cells in the pulmonary environment compared to control groups of mice. Furthermore, OMPs and dmLT immunized mice were significantly protected against an otherwise lethal lung infection. Protection was associated with early IFN-γ and IL-17 production in the lungs of immunized mice. These results indicate that intradermal immunization with dmLT can drive protective immunity to the lung mucosa and may be a viable vaccination strategy for a multitude of respiratory pathogens.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteínas da Membrana Bacteriana Externa/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Pneumonia Bacteriana/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Doença Aguda , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Proteínas da Membrana Bacteriana Externa/genética , Toxinas Bacterianas/genética , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Feminino , Imunoglobulina G/sangue , Memória Imunológica , Injeções Intradérmicas , Interferon gama/imunologia , Interleucina-17/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Vacinas contra Pseudomonas/administração & dosagem , Pseudomonas aeruginosa , Vacinação/métodos
17.
Biol Chem ; 400(4): 443-456, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29604208

RESUMO

Inhalation is required for respiration and life in all vertebrates. This process is not without risk, as it potentially exposes the host to environmental pathogens with every breath. This makes the upper respiratory tract one of the most common routes of infection and one of the leading causes of morbidity and mortality in the world. To combat this, the lung relies on the innate immune defenses. In contrast to the adaptive immune system, the innate immune system does not require sensitization, previous exposure or priming to attack foreign particles. In the lung, the innate immune response starts with the epithelial barrier and mucus production and is reinforced by phagocytic cells and T cells. These cells are vital for the production of cytokines, chemokines and anti-microbial peptides that are critical for clearance of infectious agents. In this review, we discuss all aspects of the innate immune response, with a special emphasis on ways to target aspects of the immune response to combat antibiotic resistant bacteria.


Assuntos
Sistema Imunitário/imunologia , Imunidade Inata/imunologia , Pneumopatias/imunologia , Animais , Humanos
18.
JCI Insight ; 3(12)2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29925696

RESUMO

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Doenças do Sistema Imunitário/imunologia , Infecções por Pneumocystis/imunologia , Animais , Anti-Infecciosos/metabolismo , Antifúngicos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Subunidade alfa de Receptor de Interleucina-21/genética , Subunidade alfa de Receptor de Interleucina-21/metabolismo , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumocystis/imunologia , Infecções por Pneumocystis/genética , Infecções por Pneumocystis/patologia , Fator de Transcrição STAT3 , Transdução de Sinais , Interleucina 22
19.
Am J Pathol ; 187(4): 851-863, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28193481

RESUMO

Seasonal and pandemic influenza is a cause of morbidity and mortality worldwide. Most people infected with influenza virus display mild-to-moderate disease phenotypes and recover within a few weeks. Influenza is known to cause persistent alveolitis in animal models; however, little is known about the molecular pathways involved in this phenotype. We challenged C57BL/6 mice with influenza A/PR/8/34 and examined lung pathologic processes and inflammation, as well as transcriptomic and epigenetic changes at 21 to 60 days after infection. Influenza induced persistent parenchymal lung inflammation, alveolar epithelial metaplasia, and epithelial endoplasmic reticulum stress that were evident after the clearance of virus and resolution of morbidity. Influenza infection induced robust changes in the lung transcriptome, including a significant impact on inflammatory and extracellular matrix protein expression. Despite the robust changes in lung gene expression, preceding influenza (21 days) did not exacerbate secondary Staphylococcus aureus infection. Finally, we examined the impact of influenza on miRNA expression in the lung and found an increase in miR-155. miR-155 knockout mice recovered from influenza infection faster than controls and had decreased lung inflammation and endoplasmic reticulum stress. These data illuminate the dynamic molecular changes in the lung in the weeks after influenza infection and characterize the repair process, identifying a novel role for miR-155.


Assuntos
Epigênese Genética , Pulmão/metabolismo , Pulmão/virologia , Infecções por Orthomyxoviridae/genética , Transcriptoma/genética , Cicatrização/genética , Animais , Progressão da Doença , Estresse do Retículo Endoplasmático/genética , Epitélio/patologia , Perfilação da Expressão Gênica , Inflamação/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/etiologia , Pneumonia/microbiologia , Linfócitos T/imunologia , Fatores de Tempo
20.
J Immunol ; 197(1): 97-107, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27217583

RESUMO

Commensal microbiota are critical for the development of local immune responses. In this article, we show that gut microbiota can regulate CD4 T cell polarization during pulmonary fungal infections. Vancomycin drinking water significantly decreased lung Th17 cell numbers during acute infection, demonstrating that Gram-positive commensals contribute to systemic inflammation. We next tested a role for RegIIIγ, an IL-22-inducible antimicrobial protein with specificity for Gram-positive bacteria. Following infection, increased accumulation of Th17 cells in the lungs of RegIIIγ(-/-) and Il22(-/-) mice was associated with intestinal segmented filamentous bacteria (SFB) colonization. Although gastrointestinal delivery of rRegIIIγ decreased lung inflammatory gene expression and protected Il22(-/-) mice from weight loss during infection, it had no direct effect on SFB colonization, fungal clearance, or lung Th17 immunity. We further show that vancomycin only decreased lung IL-17 production in mice colonized with SFB. To determine the link between gut microbiota and lung immunity, serum-transfer experiments revealed that IL-1R ligands increase the accumulation of lung Th17 cells. These data suggest that intestinal microbiota, including SFB, can regulate pulmonary adaptive immune responses.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Microbioma Gastrointestinal/imunologia , Bactérias Gram-Positivas/imunologia , Pulmão/imunologia , Células Th17/imunologia , Animais , Antibacterianos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Células Cultivadas , Imunidade/efeitos dos fármacos , Interleucinas/genética , Interleucinas/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas a Pancreatite , Proteínas/genética , Proteínas/metabolismo , Vancomicina/uso terapêutico , Interleucina 22
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