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1.
Immunity ; 57(6): 1243-1259.e8, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38744291

RESUMO

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.


Assuntos
Acetilcolina , Cloretos , Células Epiteliais , Mucosa Intestinal , Animais , Acetilcolina/metabolismo , Camundongos , Cloretos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Intestino Delgado/imunologia , Intestino Delgado/parasitologia , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células em Tufo
2.
bioRxiv ; 2023 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-36993541

RESUMO

Tuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.

3.
J Allergy Clin Immunol ; 151(6): 1484-1493, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36708815

RESUMO

BACKGROUND: Mast cells (MCs) within the airway epithelium in asthma are closely related to airway dysfunction, but cross talk between airway epithelial cells (AECs) and MCs in asthma remains incompletely understood. Human rhinovirus (RV) infections are key triggers for asthma progression, and AECs from individuals with asthma may have dysregulated antiviral responses. OBJECTIVE: We utilized primary AECs in an ex vivo coculture model system to examine cross talk between AECs and MCs after epithelial rhinovirus infection. METHODS: Primary AECs were obtained from 11 children with asthma and 10 healthy children, differentiated at air-liquid interface, and cultured in the presence of laboratory of allergic diseases 2 (LAD2) MCs. AECs were infected with rhinovirus serogroup A 16 (RV16) for 48 hours. RNA isolated from both AECs and MCs underwent RNA sequencing. Direct effects of epithelial-derived interferons on LAD2 MCs were examined by real-time quantitative PCR. RESULTS: MCs increased expression of proinflammatory and antiviral genes in AECs. AECs demonstrated a robust antiviral response after RV16 infection that resulted in significant changes in MC gene expression, including upregulation of genes involved in antiviral responses, leukocyte activation, and type 2 inflammation. Subsequent ex vivo modeling demonstrated that IFN-ß induces MC type 2 gene expression. The effects of AEC donor phenotype were small relative to the effects of viral infection and the presence of MCs. CONCLUSIONS: There is significant cross talk between AECs and MCs, which are present in the epithelium in asthma. Epithelial-derived interferons not only play a role in viral suppression but also further alter MC immune responses including specific type 2 genes.


Assuntos
Asma , Infecções por Enterovirus , Infecções por Picornaviridae , Criança , Humanos , Interferons , Rhinovirus/fisiologia , Mastócitos/metabolismo , Epitélio/metabolismo , Células Epiteliais , Antivirais/farmacologia , Imunidade
4.
Sci Rep ; 12(1): 6972, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35484173

RESUMO

Common alphacoronaviruses and human rhinoviruses (HRV) induce type I and III interferon (IFN) responses important to limiting viral replication in the airway epithelium. In contrast, highly pathogenic betacoronaviruses including SARS-CoV-2 may evade or antagonize RNA-induced IFN I/III responses. In airway epithelial cells (AECs) from children and older adults we compared IFN I/III responses to SARS-CoV-2 and HRV-16, and assessed whether pre-infection with HRV-16, or pretreatment with recombinant IFN-ß or IFN-λ, modified SARS-CoV-2 replication. Bronchial AECs from children (ages 6-18 years) and older adults (ages 60-75 years) were differentiated ex vivo to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 or HRV-16, and RNA and protein was harvested from cell lysates 96 h. following infection and supernatant was collected 48 and 96 h. following infection. In additional experiments cultures were pre-infected with HRV-16, or pre-treated with recombinant IFN-ß1 or IFN-λ2 before SARS-CoV-2 infection. In a subset of experiments a range of infectious concentrations of HRV-16, SARS-CoV-2 WA-01, SARS-CoV-2 Delta variant, and SARS-CoV-2 Omicron variant were studied. Despite significant between-donor heterogeneity SARS-CoV-2 replicated 100 times more efficiently than HRV-16. IFNB1, INFL2, and CXCL10 gene expression and protein production following HRV-16 infection was significantly greater than following SARS-CoV-2. IFN gene expression and protein production were inversely correlated with SARS-CoV-2 replication. Treatment of cultures with recombinant IFNß1 or IFNλ2, or pre-infection of cultures with HRV-16, markedly reduced SARS-CoV-2 replication. In addition to marked between-donor heterogeneity in IFN responses and viral replication, SARS-CoV-2 (WA-01, Delta, and Omicron variants) elicits a less robust IFN response in primary AEC cultures than does rhinovirus, and heterologous rhinovirus infection, or treatment with recombinant IFN-ß1 or IFN-λ2, reduces SARS-CoV-2 replication, although to a lesser degree for the Delta and Omicron variants.


Assuntos
Tratamento Farmacológico da COVID-19 , Interferons , Adolescente , Idoso , Antivirais , Criança , Humanos , Interferons/farmacologia , Pessoa de Meia-Idade , RNA , Rhinovirus , SARS-CoV-2
5.
Front Pharmacol ; 12: 765951, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34867390

RESUMO

Rationale: SARS-CoV-2 gains entrance to airway epithelial cells (AECs) through binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) on the cell surface. However, ACE2 also converts angiotensin II into angiotensin-(1-7) and counterbalances the renin-angiotensin-aldosterone system, with resultant protective effects in the cardiovascular system. Some data suggest that two common antihypertension medications (angiotensin II receptor antagonists, ARBs; and angiotensin-converting-enzyme inhibitors, ACEIs) may increase ACE2 expression in heart and kidney cells, fueling debate about how these widely used medications may modulate SARS-CoV-2 infectivity and risk of COVID-19. Aim: Determine whether exposure of bronchial AECs to the ARB losartan or the ACEI captopril modulate expression of ACE2 by AECs, SARS CoV2 replication, or expression of proinflammatory cytokines and type I and III interferon (IFN) responses. Methods: Primary bronchial AECs from children and adults (n = 19; Ages 8-75 yrs) were differentiated ex vivo at an air-liquid interface to generate organotypic cultures. Cultures were treated with captopril (1 µM) or losartan (2 µM) with culture media changes starting 72 h before infection with SARS-CoV-2. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 isolate USA-WA1/2020 at a multiplicity of infection (MOI) of 0.5. At 96 h following infection, RNA and protein were isolated. SARS-CoV-2 replication in cultures was assessed with quantitative PCR (qPCR). ACE2, IL-6, IL-1B, IFNB1, and IFNL2 expression were assessed by qPCR. Results: Neither captopril nor losartan treatment significantly changed ACE2, IL-6, IL-1B, IFNB1, or IFNL2 expression by AECs as compared to SARS-CoV-2 infected AEC cultures without captopril or losartan treatment. At 96 h following infection, SARS-CoV-2 copy number/ng RNA was not significantly different between untreated AEC cultures, cultures treated with captopril, or cultures treated with losartan. Conclusion: These findings suggest that at the level of the airway epithelium neither the ACEI captopril or ARB losartan significantly modify expression of the SARS-CoV-2 entry factor ACE2, nor does either medication increase replication SARS-CoV-2 replication. This ex vivo data is reassuring and is consistent with evolving clinical data suggesting ACEIs and ARBs do not increase the risk for poor prognosis with COVID-19 and may actually reduce the risk of COVID-19 disease.

6.
bioRxiv ; 2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34845445

RESUMO

INTRODUCTION: Common alphacoronaviruses and human rhinoviruses (HRV) induce type I and III interferon (IFN) responses important to limiting viral replication in the airway epithelium. In contrast, highly pathogenic betacoronaviruses including SARS-CoV-2 may evade or antagonize RNA-induced IFN I/III responses. METHODS: In airway epithelial cells (AECs) from children and older adults we compared IFN I/III responses to SARS-CoV-2 and HRV-16, and assessed whether pre-infection with HRV-16, or pretreatment with recombinant IFN-ß or IFN-λ, modified SARS-CoV-2 replication. Bronchial AECs from children (ages 6-18 yrs.) and older adults (ages 60-75 yrs.) were differentiated ex vivo to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 or HRV-16, and RNA and protein was harvested from cell lysates 96 hrs. following infection and supernatant was collected 48 and 96 hrs. following infection. In additional experiments cultures were pre-infected with HRV-16, or pre-treated with recombinant IFN-ß1 or IFN-λ2 before SARS-CoV-2 infection. RESULTS: Despite significant between-donor heterogeneity SARS-CoV-2 replicated 100 times more efficiently than HRV-16. IFNB1, INFL2, and CXCL10 gene expression and protein production following HRV-16 infection was significantly greater than following SARS-CoV-2. IFN gene expression and protein production were inversely correlated with SARS-CoV-2 replication. Treatment of cultures with recombinant IFNß1 or IFNλ2, or pre-infection of cultures with HRV-16, markedly reduced SARS-CoV-2 replication. DISCUSSION: In addition to marked between-donor heterogeneity in IFN responses and viral replication, SARS-CoV-2 elicits a less robust IFN response in primary AEC cultures than does rhinovirus, and heterologous rhinovirus infection, or treatment with recombinant IFN-ß1 or IFN-λ2, markedly reduces SARS-CoV-2 replication.

7.
Clin Chem ; 68(1): 143-152, 2021 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-34286830

RESUMO

BACKGROUND: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. METHODS: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. CONCLUSION: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19 , COVID-19/diagnóstico , Técnicas de Laboratório Clínico , Humanos , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Manejo de Espécimes
8.
J Exp Med ; 218(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33724365

RESUMO

SAMD9L is an interferon-induced tumor suppressor implicated in a spectrum of multisystem disorders, including risk for myeloid malignancies and immune deficiency. We identified a heterozygous de novo frameshift variant in SAMD9L in an infant with B cell aplasia and clinical autoinflammatory features who died from respiratory failure with chronic rhinovirus infection. Autopsy demonstrated absent bone marrow and peripheral B cells as well as selective loss of Langerhans and Purkinje cells. The frameshift variant led to expression of a truncated protein with interferon treatment. This protein exhibited a gain-of-function phenotype, resulting in interference in global protein synthesis via inhibition of translational elongation. Using a mutational scan, we identified a region within SAMD9L where stop-gain variants trigger a similar translational arrest. SAMD9L variants that globally suppress translation had no effect or increased mRNA transcription. The complex-reported phenotype likely reflects lineage-dominant sensitivities to this translation block. Taken together, our findings indicate that interferon-triggered SAMD9L gain-of-function variants globally suppress translation.


Assuntos
Mutação da Fase de Leitura , Regulação da Expressão Gênica/genética , Mutação em Linhagem Germinativa , Biossíntese de Proteínas/genética , Proteínas Supressoras de Tumor/genética , Células A549 , Linfócitos B/metabolismo , Linfócitos B/patologia , Evolução Fatal , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Heterozigoto , Humanos , Recém-Nascido , Interferons/farmacologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequenciamento Completo do Genoma
9.
Methods Protoc ; 4(1)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33430421

RESUMO

Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2.

10.
bioRxiv ; 2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32511368

RESUMO

BACKGROUND: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse transcription PCR (RT-qPCR) (1). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce (2). To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. METHODS: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral activation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/uL, 100% sensitivity, and 99.4% specificity when compared side-by-side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. CONCLUSION: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

11.
J Biol Chem ; 296: 100076, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33187989

RESUMO

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however, whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production, we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) coculture model. RSV infection of BEC/HLF cocultures led to decreased hyaluronidase expression by HLFs, increased accumulation of HA, and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however, BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared with control mice, which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared with SPC-Cre(-) RSV-infected littermates. Taken together, these data demonstrate that altered extracellular matrix accumulation of HA occurs following RSV infection and may contribute to airway inflammation. In addition, loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment.


Assuntos
Ácido Hialurônico/biossíntese , Pulmão/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Versicanas/genética , Animais , Líquido da Lavagem Broncoalveolar , Técnicas de Cocultura , Células Epiteliais/metabolismo , Humanos , Hialuronan Sintases/genética , Hialuronoglucosaminidase/genética , Pulmão/citologia , Pulmão/enzimologia , Camundongos , Células U937
12.
J Immunol ; 205(11): 3050-3057, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33097575

RESUMO

Early life respiratory syncytial virus (RSV) infection has been linked to the onset of asthma. Despite this association, our knowledge of the progression of the initial viral infection is limited, and no safe or effective vaccine currently exists. Bronchioalveolar lavage, whole-lung cellular isolation, and gene expression analysis were performed on 3-wk- (juvenile) and 8-wk-old (adult) RSV-infected C57BL/6 mice to investigate age-related differences in immunologic responses; juvenile mice displayed a sustained myeloid infiltrate (including monocytes and neutrophils) with increased RNA expression of Ccl2, Ccl3, and Ccl4, when compared with adult mice, at 72 h postinfection. Juvenile mice demonstrated αSma expression (indicative of myofibroblast activity), increased hyaluronan deposition in the lung parenchyma (attributed to asthma progression), and a lack of CD64 upregulation on the surface of monocytes (which, in conjunction with serum amyloid P, is responsible for clearing residual hyaluronan and cellular debris). RSV infection of human airway epithelial cell, human lung fibroblast, and U937 monocyte cocultures (at air-liquid interface) displayed similar CCL expression and suggested matrix metalloproteinase-7 and MMP9 as possible extracellular matrix modifiers. These mouse data, in conjunction with our findings in human monocytes, suggest that the sustained influx of myeloid cells in the lungs of juvenile mice during acute RSV infection could potentiate extracellular matrix remodeling, facilitating conditions that support the development of asthma.


Assuntos
Matriz Extracelular/imunologia , Células Mieloides/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Adolescente , Animais , Asma/imunologia , Asma/virologia , Lavagem Broncoalveolar/métodos , Linhagem Celular Tumoral , Criança , Células Epiteliais/imunologia , Células Epiteliais/virologia , Matriz Extracelular/virologia , Feminino , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Pulmão/imunologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/virologia , Células Mieloides/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Células U937
15.
Front Immunol ; 10: 3159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32047499

RESUMO

Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.


Assuntos
Matriz Extracelular/imunologia , Fibroblastos , Ácido Hialurônico/metabolismo , Mastócitos , Infecções por Vírus Respiratório Sincicial/imunologia , Adesão Celular/imunologia , Células Cultivadas , Quimases/biossíntese , Técnicas de Cocultura , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Humanos , Pulmão/imunologia , Pulmão/virologia , Mastócitos/imunologia , Mastócitos/metabolismo , Vírus Sincicial Respiratório Humano , Triptases/biossíntese
16.
Sci Rep ; 8(1): 15768, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30361541

RESUMO

Airway remodeling may contribute to decreased lung function in asthmatic children. Bronchial epithelial cells (BECs) may regulate fibroblast expression of extracellular matrix (ECM) constituents and fibroblast-to-myofibroblast transition (FMT). Our objective was to determine if human lung fibroblast (HLF) expression of collagen I (COL1A1), hyaluronan synthase 2 (HAS2), and the FMT marker alpha-smooth muscle actin (α-SMA) by HLFs conditioned by BECs from asthmatic and healthy children correlate with lung function measures and exacerbation history among BEC donors. BECs from asthmatic (n = 23) and healthy children (n = 15) were differentiated at an air-liquid interface (ALI) and then co-cultured with HLFs for 96 hours. Expression of COL1A1, HAS2, and α-SMA by HLFs was determined by quantitative polymerase chain reaction (qPCR). FMT was quantified by measuring HLF cytoskeletal α-SMA by flow cytometry. Pro-collagen Iα1, hyaluronan (HA), and PGE2 were measured in BEC-HLF supernatant. Correlations between lung function measures of BEC donors, and COL1A1, HAS2, and α-SMA gene expression, as well as supernatant concentrations of HA, pro-collagen Iα1, hyaluronan (HA), and PGE2 were assessed. We observed that expression of α-SMA and COL1A1 by HLFs co-cultured with asthmatic BECs was negatively correlated with BEC donor lung function. BEC-HLF supernatant concentrations of pro-collagen Iα1 were negatively correlated, and PGE2 concentrations positively correlated, with asthmatic BEC donor lung function. Expression of HAS2, but not α-SMA or COL1A1, was greater by HLFs co-cultured with asthmatic BECs from donors with a history of severe exacerbations than by HLFs co-cultured with BECs from donors who lacked a history of severe exacerbations. In conclusion, α-SMA and COL1A1 expression by HLFs co-cultured with BECs from asthmatic children were negatively correlated with lung function measures, supporting our hypothesis that epithelial regulation of HLFs and airway deposition of ECM constituents by HLFs contributes to lung function deficits among asthmatic children. Furthermore, epithelial regulation of airway HAS2 may influence the susceptibility of children with asthma to experience severe exacerbations. Finally, epithelial-derived PGE2 is a potential regulator of airway FMT and HLF production of collagen I that should be investigated further in future studies.


Assuntos
Asma/genética , Asma/fisiopatologia , Brônquios/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Doadores de Tecidos , Actinas/metabolismo , Adolescente , Asma/patologia , Criança , Colágeno Tipo I/metabolismo , Citoesqueleto/metabolismo , Dinoprostona/metabolismo , Feminino , Humanos , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Masculino
17.
Respir Res ; 19(1): 146, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30071849

RESUMO

BACKGROUND: Airway inflammation is a hallmark of asthma. Alterations in extracellular matrix (ECM) hyaluronan (HA) content have been shown to modulate the recruitment and retention of inflammatory cells. Bronchial epithelial cells (BECs) regulate the activity of human lung fibroblasts (HLFs); however, their contribution in regulating HLF production of HA in asthma is unknown. In this study, we tested the hypothesis that BECs from asthmatic children promote the generation of a pro-inflammatory, HA-enriched ECM by HLFs, which promotes the retention of leukocytes. METHODS: BECs were obtained from well-characterized asthmatic and healthy children ages 6-18 years. HLFs were co-cultured with BECs for 96 h and samples were harvested for analysis of gene expression, synthesis and accumulation of HA, and subjected to a leukocyte adhesion assay with U937 monocytes. RESULTS: We observed increased expression of HA synthases HAS2 and HAS3 in HLFs co-cultured with asthmatic BECs. Furthermore, we demonstrated greater total accumulation and increased synthesis of HA by HLFs co-cultured with asthmatic BECs compared to healthy BEC/HLF co-cultures. ECM generated by HLFs co-cultured with asthmatic BECs displayed increased HA-dependent adhesion of leukocytes in a separate in vitro binding assay. CONCLUSIONS: Our findings demonstrate that BEC regulation of HA production by HLFs is altered in asthma, which may in turn promote the establishment of a more leukocyte-permissive ECM promoting airway inflammation in this disease.


Assuntos
Asma/metabolismo , Brônquios/metabolismo , Matriz Extracelular/metabolismo , Ácido Hialurônico/biossíntese , Leucócitos/metabolismo , Mucosa Respiratória/metabolismo , Adolescente , Brônquios/citologia , Criança , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Humanos , Pulmão/metabolismo , Masculino , Mucosa Respiratória/citologia , Células U937
18.
BMC Pulm Med ; 18(1): 91, 2018 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-29843677

RESUMO

BACKGROUND: An increasing number of studies using primary human bronchial epithelial cells (BECs) have reported intrinsic differences in the expression of several genes between cells from asthmatic and non-asthmatic donors. The stability of gene expression by primary BECs with increasing cell passage number has not been well characterized. METHODS: To determine if expression by primary BECs from asthmatic and non-asthmatic children of selected genes associated with airway remodeling, innate immune response, immunomodulatory factors, and markers of differentiated airway epithelium, are stable over increasing cell passage number, we studied gene expression patterns in passages 1, 2, 3, 4, and 5 BECs from asthmatic (n = 6) and healthy (n = 6) subjects that were differentiated at an air-liquid interface. RNA was harvested from BECs and RT-PCR was performed for TGFß1, TGFß2, activin A, FSTL3, MUC5AC, TSLP, IL-33, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1. RESULTS: Expression of TGFß1, TGFß2, activin A, FSTL3, MUC5AC, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1 by primary BECs from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3; however, gene expression at cell passages 4 and 5 was significantly greater and more variable compared to passage 1 BECs for many of these genes. IL-33 and FOXJ1 expression was also stable between passages 1 through 3, however, expression at passages 4 and 5 was significantly lower than by passage 1 BECs. TSLP, p63, and KRT5 expression was stable across BEC passages 1 through 5 for both asthmatic and healthy BECs. CONCLUSIONS: These observations illustrate the importance of using BECs from passage ≤3 when studying gene expression by asthmatic and non-asthmatic primary BECs and characterizing the expression pattern across increasing cell passage number for each new gene studied, as beyond passage 3 genes expressed by primary BECs appear to less accurately model in vivo airway epithelial gene expression.


Assuntos
Asma , Brônquios , Células Epiteliais , Adolescente , Remodelação das Vias Aéreas/fisiologia , Asma/diagnóstico , Asma/patologia , Asma/fisiopatologia , Barreira Alveolocapilar/metabolismo , Brônquios/metabolismo , Brônquios/patologia , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Criança , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Imunidade Inata/fisiologia , Fatores Imunológicos/metabolismo , Masculino , Comunicação Parácrina/fisiologia , Transcriptoma
19.
Am J Respir Cell Mol Biol ; 59(1): 104-113, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29394092

RESUMO

Bronchial epithelial cells (BECs) from healthy children inhibit human lung fibroblast (HLF) expression of collagen and fibroblast-to-myofibroblast transition (FMT), whereas asthmatic BECs do so less effectively, suggesting that diminished epithelial-derived regulatory factors contribute to airway remodeling. Preliminary data demonstrated that secretion of the activin A inhibitor follistatin-like 3 (FSTL3) by healthy BECs was greater than that by asthmatic BECs. We sought to determine the relative secretion of FSTL3 and activin A by asthmatic and healthy BECs, and whether FSTL3 inhibits FMT. To quantify the abundance of the total proteome FSTL3 and activin A in supernatants of differentiated BEC cultures from healthy children and children with asthma, we performed mass spectrometry and ELISA. HLFs were cocultured with primary BECs and then HLF expression of collagen I and α-smooth muscle actin (α-SMA) was quantified by qPCR, and FMT was quantified by flow cytometry. Loss-of-function studies were conducted using lentivirus-delivered shRNA. Using mass spectrometry and ELISA results from larger cohorts, we found that FSTL3 concentrations were greater in media conditioned by healthy BECs compared with asthmatic BECs (4,012 vs. 2,553 pg/ml; P = 0.002), and in media conditioned by asthmatic BECs from children with normal lung function relative to those with airflow obstruction (FEV1/FVC ratio < 0.8; n = 9; 3,026 vs. 1,922 pg/ml; P = 0.04). shRNA depletion of FSTL3 in BECs (n = 8) increased HLF collagen I expression by 92% (P = 0.001) and α-SMA expression by 88% (P = 0.02), and increased FMT by flow cytometry in cocultured HLFs, whereas shRNA depletion of activin A (n = 6) resulted in decreased α-SMA (22%; P = 0.01) expression and decreased FMT. Together, these results indicate that deficient FSTL3 expression by asthmatic BECs impairs epithelial regulation of HLFs and FMT.


Assuntos
Asma/patologia , Epitélio/metabolismo , Epitélio/patologia , Proteínas Relacionadas à Folistatina/deficiência , Pulmão/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Actinas/metabolismo , Ativinas/metabolismo , Adolescente , Sequência de Aminoácidos , Criança , Colágeno Tipo I/metabolismo , Células Epiteliais/metabolismo , Feminino , Proteínas Relacionadas à Folistatina/química , Proteínas Relacionadas à Folistatina/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Masculino , RNA Interferente Pequeno/metabolismo
20.
J Allergy Clin Immunol ; 142(2): 451-459, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29106997

RESUMO

BACKGROUND: Respiratory viral infection in early childhood, including that from respiratory syncytial virus (RSV), has been previously associated with the development of asthma. OBJECTIVE: We aimed to determine whether ex vivo RSV infection of bronchial epithelial cells (BECs) from children with asthma would induce specific gene expression patterns and whether such patterns were associated with lung function among BEC donors. METHODS: Primary BECs from carefully characterized children with asthma (n = 18) and matched healthy children without asthma (n = 8) were differentiated at an air-liquid interface for 21 days. Air-liquid interface cultures were infected with RSV for 96 hours and RNA was subsequently isolated from BECs. In each case, we analyzed gene expression using RNA sequencing and assessed differences between conditions by linear modeling of the data. BEC donors completed spirometry to measure lung function. RESULTS: RSV infection of BECs from subjects with asthma, compared with uninfected BECs from subjects with asthma, led to a significant increase in expression of 6199 genes. There was significantly greater expression of 195 genes in BECs from children with asthma and airway obstruction (FEV1/forced vital capacity < 0.85 and FEV1 < 100% predicted) than in BECs from children with asthma without obstruction, or in BECs from healthy children. These specific genes were found to be highly enriched for viral response genes induced in parallel with types I and III interferons. CONCLUSIONS: BECs from children with asthma and with obstructive physiology exhibit greater expression of types I and III interferons and interferon-stimulated genes than do cells from children with normal lung function, and expression of interferon-associated genes correlates with the degree of airway obstruction. These findings suggest that an exaggerated interferon response to viral infection by airway epithelial cells may be a mechanism leading to lung function decline in a subset of children with asthma.


Assuntos
Asma/imunologia , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Pulmão/fisiologia , Mucosa Respiratória/fisiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Adolescente , Asma/complicações , Células Cultivadas , Criança , Feminino , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon gama/genética , Masculino , Infecções por Vírus Respiratório Sincicial/complicações , Análise de Sequência de RNA , Espirometria , Transcriptoma
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