RESUMEN
Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Nasofaringe/inmunología , Nariz/citología , Mucosa Respiratoria/inmunología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/sangre , COVID-19/inmunología , COVID-19/patología , Granulocitos/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Células Asesinas Naturales/inmunología , Células T de Memoria/inmunología , Monocitos/inmunología , Nasofaringe/citología , Nasofaringe/virología , Neutrófilos/inmunología , Nariz/inmunología , Nariz/virología , Estudios Prospectivos , Mucosa Respiratoria/citología , Mucosa Respiratoria/virologíaRESUMEN
Antimicrobial peptides (AMPs) expressed by epithelial and immune cells are largely described for the defense against invading microorganisms. Recently, their immunomodulatory functions have been highlighted in various contexts. However how AMPs expressed by non-immune cells might influence autoimmune responses in peripheral tissues, such as the pancreas, is unknown. Here, we found that insulin-secreting ß-cells produced the cathelicidin related antimicrobial peptide (CRAMP) and that this production was defective in non-obese diabetic (NOD) mice. CRAMP administrated to prediabetic NOD mice induced regulatory immune cells in the pancreatic islets, dampening the incidence of autoimmune diabetes. Additional investigation revealed that the production of CRAMP by ß-cells was controlled by short-chain fatty acids produced by the gut microbiota. Accordingly, gut microbiota manipulations in NOD mice modulated CRAMP production and inflammation in the pancreatic islets, revealing that the gut microbiota directly shape the pancreatic immune environment and autoimmune diabetes development.
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Catelicidinas/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/inmunología , Intestinos/inmunología , Microbiota/fisiología , Páncreas/inmunología , Animales , Péptidos Catiónicos Antimicrobianos , Catelicidinas/genética , Diabetes Mellitus Tipo 1/microbiología , Ácidos Grasos Volátiles/inmunología , Femenino , Intestinos/microbiología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Páncreas/microbiologíaRESUMEN
BACKGROUND: Acute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects. METHODS: Primary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air-liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and L-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, L-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge. RESULTS: CS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and L-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline. CONCLUSION: Altogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15-related pathways.
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Fumar Cigarrillos , Enfermedad Pulmonar Obstructiva Crónica , Virosis , Humanos , Interferones , Factor 15 de Diferenciación de Crecimiento , Fumar Cigarrillos/efectos adversos , LactatosRESUMEN
Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.
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Células Epiteliales Alveolares , Organoides , Células Epiteliales Alveolares/metabolismo , Células Cultivadas , Células Epiteliales , Humanos , Pulmón , Organoides/metabolismo , Alveolos PulmonaresRESUMEN
The lung epithelium has long been overlooked as a key player in tuberculosis disease. In addition to acting as a direct barrier to Mycobacterium tuberculosis (Mtb), epithelial cells (EC) of the airways and alveoli act as first responders during Mtb infections; they directly sense and respond to Mtb by producing mediators such as cytokines, chemokines and antimicrobials. Interactions of EC with innate and adaptive immune cells further shape the immune response against Mtb. These three essential components, epithelium, immune cells and Mtb, are rarely studied in conjunction, owing in part to difficulties in coculturing them. Recent advances in cell culture technologies offer the opportunity to model the lung microenvironment more closely. Herein, we discuss the interplay between lung EC, immune cells and Mtb and argue that modelling these interactions is of key importance to unravel early events during Mtb infection.
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Mycobacterium tuberculosis , Tuberculosis , Células Epiteliales , Humanos , Inmunidad Innata , Pulmón/microbiologíaRESUMEN
BACKGROUND: Despite the well-known detrimental effects of cigarette smoke (CS), little is known about the complex gene expression dynamics in the early stages after exposure. This study aims to investigate early transcriptomic responses following CS exposure of airway epithelial cells in culture and compare these to those found in human CS exposure studies. METHODS: Primary bronchial epithelial cells (PBEC) were differentiated at the air-liquid interface (ALI) and exposed to whole CS. Bulk RNA-sequencing was performed at 1 h, 4 h, and 24 h hereafter, followed by differential gene expression analysis. Results were additionally compared to data retrieved from human CS studies. RESULTS: ALI-PBEC gene expression in response to CS was most significantly changed at 4 h after exposure. Early transcriptomic changes (1 h, 4 h post CS exposure) were related to oxidative stress, xenobiotic metabolism, higher expression of immediate early genes and pro-inflammatory pathways (i.e., Nrf2, AP-1, AhR). At 24 h, ferroptosis-associated genes were significantly increased, whereas PRKN, involved in removing dysfunctional mitochondria, was downregulated. Importantly, the transcriptome dynamics of the current study mirrored in-vivo human studies of acute CS exposure, chronic smokers, and inversely mirrored smoking cessation. CONCLUSION: These findings show that early after CS exposure xenobiotic metabolism and pro-inflammatory pathways were activated, followed by activation of the ferroptosis-related cell death pathway. Moreover, significant overlap between these transcriptomic responses in the in-vitro model and human in-vivo studies was found, with an early response of ciliated cells. These results provide validation for the use of ALI-PBEC cultures to study the human lung epithelial response to inhaled toxicants.
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Fumar Cigarrillos , Xenobióticos , Bronquios/metabolismo , Fumar Cigarrillos/efectos adversos , Fumar Cigarrillos/genética , Células Epiteliales/metabolismo , Humanos , Membrana Mucosa , Nicotiana , Xenobióticos/metabolismo , Xenobióticos/farmacologíaAsunto(s)
Enfisema , Enfisema Pulmonar , Humanos , Células Epiteliales Alveolares , Células Endoteliales , Pulmón , Alveolos PulmonaresRESUMEN
Cationic host defence peptides (CHDPs), also known as antimicrobial peptides, exhibit a wide range of activities contributing to immune responses and resolution of infections. CHDPs are expressed across diverse species, are generally amphipathic with less than 50 amino acids in length, and differ significantly in sequence and structure. This chapter focuses on the role of these peptides in immunity. CHDPs are known to function in both innate and adaptive immune responses. These peptides exert both pro- and anti-inflammatory properties, which are likely context dependent based on cell and tissue type, concentration of the peptides, and its interaction with other factors in the microenvironment. Furthermore, the crosstalk between CHDPs and the microbiome and how this may influence mucosal immunity is a rapidly emerging field of research. Overall, the immunomodulatory functions of CHDPs play an important role in the control of infections, regulation of inflammation, and maintaining immune homeostasis. It is thus not surprising that dysregulation of expression of CHDPs is implicated in the susceptibility, pathology, and progression of various diseases. In this chapter, we summarize the immunomodulatory functions of CHDPs, its clinical relevance, and the translational opportunities that these peptides provide for the development of new therapies.
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Inmunidad Adaptativa , Péptidos Catiónicos Antimicrobianos/inmunología , Inmunidad Innata , Inflamación/inmunología , HumanosRESUMEN
BACKGROUND: Exacerbation in asthma is associated with decreased expression of specific host defence peptides (HDPs) in the lungs. We examined the effects of a synthetic derivative of HDP, innate defence regulator (IDR) peptide IDR-1002, in house dust mite (HDM)-challenged murine model of asthma, in interleukin (IL)-33-challenged mice and in human primary bronchial epithelial cells (PBECs). METHODS: IDR-1002 (6 mg/kg per mouse) was administered (subcutaneously) in HDM-challenged and/or IL-33-challenged BALB/c mice. Lung function analysis was performed with increasing dose of methacholine by flexiVent small animal ventilator, cell differentials in bronchoalveolar lavage performed by modified Wright-Giemsa staining, and cytokines monitored by MesoScale Discovery assay and ELISA. PBECs stimulated with tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), with or without IDR-1002, were analysed by western blots. RESULTS: IDR-1002 blunted HDM challenge-induced airway hyper-responsiveness (AHR), and lung leucocyte accumulation including that of eosinophils and neutrophils, in HDM-challenged mice. Concomitantly, IDR-1002 suppressed HDM-induced IL-33 in the lungs. IFN-γ/TNF-α-induced IL-33 production was abrogated by IDR-1002 in PBECs. Administration of IL-33 in HDM-challenged mice, or challenge with IL-33 alone, mitigated the ability of IDR-1002 to control leucocyte accumulation in the lungs, suggesting that the suppression of IL-33 is essential for the anti-inflammatory activity of IDR-1002. In contrast, the peptide significantly reduced either HDM, IL-33 or HDM+IL-33 co-challenge-induced AHR in vivo. CONCLUSION: This study demonstrates that an immunomodulatory IDR peptide controls the pathophysiology of asthma in a murine model. As IL-33 is implicated in steroid-refractory severe asthma, our findings on the effects of IDR-1002 may contribute to the development of novel therapies for steroid-refractory severe asthma.
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Péptidos Catiónicos Antimicrobianos/farmacología , Asma/tratamiento farmacológico , Citocinas/metabolismo , Inmunomodulación/efectos de los fármacos , Hipersensibilidad Respiratoria/tratamiento farmacológico , Animales , Asma/inmunología , Asma/metabolismo , Western Blotting , Líquido del Lavado Bronquioalveolar/citología , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Pyroglyphidae/inmunología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismoRESUMEN
It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3â weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections.
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Bronquios/citología , Diferenciación Celular/efectos de los fármacos , Células Epiteliales/citología , Humo , Productos de Tabaco/toxicidad , Bronquios/inmunología , Células Cultivadas , Células Epiteliales/inmunología , Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina A/inmunología , Microscopía ConfocalRESUMEN
Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells to proinflammatory cytokines alters their vitamin D metabolism, antibacterial activity, and expression of hCAP18/LL-37. To investigate this, primary bronchial epithelial cells were differentiated at the air-liquid interface for 14 days in the presence of the proinflammatory cytokines, TNF-α and IL-1ß (TNF-α/IL-1ß), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor, and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using quantitative PCR, Western blot, and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using nontypeable Haemophilus influenzae. We found that TNF-α/IL-1ß treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of nontypeable H. influenzae. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1ß, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and vitamin D receptor expression remained unaffected. Furthermore, we have demonstrated that the TNF-α/IL-1ß-mediated induction of CYP24A1 was, at least in part, mediated by the transcription factor specific protein 1, and the epidermal growth factor receptor-mitogen-activated protein kinase pathway. These findings indicate that TNF-α/IL-1ß decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1 and suggest that chronic inflammation impairs protective responses induced by vitamin D.
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Bronquios/citología , Citocinas/metabolismo , Células Epiteliales/inmunología , Mediadores de Inflamación/metabolismo , Vitamina D/farmacología , Lesión Pulmonar Aguda/patología , Péptidos Catiónicos Antimicrobianos , Calcifediol/farmacología , Catelicidinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Haemophilus influenzae/efectos de los fármacos , Humanos , Interleucina-17/farmacología , Interleucina-1beta/farmacología , Viabilidad Microbiana/efectos de los fármacos , Mucinas/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacología , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
OBJECTIVE: Loss of endothelial barrier function in arterial blood vessels is characteristic of vascular pathologies, including atherosclerosis. Here, we present a near-infrared fluorescence (NIRF) imaging methodology for quantifying endothelial permeability and macromolecular uptake in large arteries in the mouse and evaluate its applicability for studying mechanisms of vascular inflammation. APPROACH AND RESULTS: To validate the NIRF methodology, macrovascular inflammation was induced in C57bl/6 mice by local tumor necrosis factor-α stimulation of the carotid artery or in apolipoprotein E-deficient mice by Western diet for 4 weeks. Evans blue dye, serving as plasma protein marker and fluorescent in the near-infrared spectrum, was given intravenously at different doses. Carotids and aorta were excised, and Evans blue dye fluorescence was assessed through whole vessel scan in an infrared imaging system. NIRF correlated to extraction-absorbance methodology for Evans blue dye quantification and was superior at discriminating plasma protein accumulation in tumor necrosis factor-α-stimulated carotids. NIRF allowed for focal quantification of increased arterial wall Evans blue dye uptake in (apolipoprotein E-deficient) mice. Importantly, NIRF left vessels intact for subsequent histological analysis or quantification of leukocyte subpopulations by flow cytometry. CONCLUSIONS: The described NIRF methodology provides a sensitive and rapid tool to locate and quantify macromolecular uptake in the wall of arterial blood vessels in vascular pathologies in mice.
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Aorta/metabolismo , Aterosclerosis/metabolismo , Permeabilidad Capilar , Arterias Carótidas/metabolismo , Células Endoteliales/metabolismo , Microscopía Confocal , Imagen Óptica/métodos , Espectroscopía Infrarroja Corta , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Colorantes/administración & dosificación , Colorantes/metabolismo , Modelos Animales de Enfermedad , Azul de Evans/administración & dosificación , Azul de Evans/metabolismo , Femenino , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Inyecciones Intravenosas , Ratones Endogámicos C57BL , Ratones Noqueados , Reproducibilidad de los Resultados , Factor de Necrosis Tumoral alfaRESUMEN
In humans, LL-37 and eicosanoids are important mediators of inflammation and immune responses. Here we report that LL-37 promotes leukotriene B4 (LTB4) and thromboxane A2 (TXA2) generation by human monocyte-derived macrophages (HMDMs). LL-37 evokes calcium mobilization apparently via the P2X7 receptor (P2X7R), activation of ERK1/2 and p38 MAPKs, as well as cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase in HMDMs, leading to an early (1 h) release of LTB4. Similarly, TXA2 production at an early time involved the same signaling sequence along an LL-37-P2X7R-cPLA2-cyclooxygenase-1 (COX-1) axis. However, at later (6-8 h) time points, internalized LL-37 up-regulates COX-2 expression, promoting TXA2 production. Furthermore, intraperitoneal injection of mice with murine cathelicidin-related antimicrobial peptide (mCRAMP) induces significantly higher levels of LTB4 and TXA2 in mouse ascites rich in macrophages. Conversely, cathelicidin-deficient (Cnlp(-/-)) mice produce much less LTB4 and TXB2 in vivo in response to TNF-α compared with control mice. We conclude that LL-37 elicits a biphasic release of eicosanoids in macrophages with early, Ca(2+)-dependent formation of LTB4 and TXA2 followed by a late peak of TXA2, generated via induction of COX-2 by internalized LL-37, thus allowing eicosanoid production in a temporally controlled manner. Moreover, our findings provide evidence that LL-37 is an endogenous regulator of eicosanoid-dependent inflammatory responses in vivo.
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Péptidos Catiónicos Antimicrobianos/fisiología , Eicosanoides/biosíntesis , Leucotrieno B4/metabolismo , Macrófagos/efectos de los fármacos , Peritonitis/metabolismo , Tromboxano A2/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/deficiencia , Araquidonato 5-Lipooxigenasa/metabolismo , Señalización del Calcio , Catelicidinas/deficiencia , Catelicidinas/fisiología , Catelicidinas/toxicidad , Células Cultivadas , Humanos , Inflamación/fisiopatología , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Peritonitis/inducido químicamente , Peritonitis/patología , Fosfolipasas A2 Citosólicas/metabolismo , Fosforilación , Prostaglandina-Endoperóxido Sintasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional , Receptores Purinérgicos P2X7/fisiología , Proteínas Recombinantes/toxicidad , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
RATIONALE: The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood. OBJECTIVE: To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation. METHODS AND RESULTS: Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of nonclassical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium, where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates formyl-peptide receptor 2 on classical monocytes, resulting in monocytic ß1- and ß2-integrin conformational change toward an extended, active conformation that allows for adhesion to their respective ligands, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. CONCLUSIONS: These data elucidate a novel mechanism of neutrophil-mediated monocyte recruitment, which could be targeted in conditions where recruitment of classical monocytes plays an unfavorable role.
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Péptidos Catiónicos Antimicrobianos/metabolismo , Comunicación Celular/fisiología , Inflamación/patología , Monocitos/metabolismo , Monocitos/patología , Neutrófilos/metabolismo , Neutrófilos/patología , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/genética , Catelicidinas/genética , Catelicidinas/metabolismo , Adhesión Celular/fisiología , Endotelio Vascular/metabolismo , Humanos , Inflamación/metabolismo , Integrinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismoRESUMEN
Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte-macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.
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Factores Inmunológicos/fisiología , Lactoferrina/fisiología , Peroxidasa/antagonistas & inhibidores , Peroxidasa/metabolismo , Antibacterianos , Células Cultivadas , Humanos , Monocitos/enzimología , Monocitos/inmunologíaRESUMEN
INTRODUCTION: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). METHODS: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. RESULTS: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by â¼10-fold, and MMP13 was increased by â¼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. CONCLUSIONS: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.
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Remodelación de las Vías Aéreas (Respiratorias) , Péptidos Catiónicos Antimicrobianos , Asma , Bronquios , Catelicidinas , Células Epiteliales , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 9 de la Matriz , Factor de Necrosis Tumoral alfa , Humanos , Péptidos Catiónicos Antimicrobianos/metabolismo , Asma/inmunología , Asma/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteómica , Mucosa Respiratoria/inmunología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of a class of highly pathogenic coronaviruses. The large family of coronaviruses, however, also includes members that cause only mild symptoms, like human coronavirus-229E (HCoV-229E) or OC43 (HCoV-OC43). Unravelling how molecular (and cellular) pathophysiology differs between highly and low pathogenic coronaviruses is important for the development of therapeutic strategies. Methods: Here, we analysed the transcriptome of primary human bronchial epithelial cells (PBEC), differentiated at the air-liquid interface (ALI) after infection with SARS-CoV-2, SARS-CoV, Middle East Respiratory Syndrome (MERS)-CoV and HCoV-229E using bulk RNA sequencing. Results: ALI-PBEC were efficiently infected by all viruses, and SARS-CoV, MERS-CoV and HCoV-229E infection resulted in a largely similar transcriptional response. The response to SARS-CoV-2 infection differed markedly as it uniquely lacked the increase in expression of immediate early genes, including FOS, FOSB and NR4A1 that was observed with all other coronaviruses. This finding was further confirmed in publicly available experimental and clinical datasets. Interfering with NR4A1 signalling in Calu-3 lung epithelial cells resulted in a 100-fold reduction in extracellular RNA copies of SARS-CoV-2 and MERS-CoV, suggesting an involvement in virus replication. Furthermore, a lack in induction of interferon-related gene expression characterised the main difference between the highly pathogenic coronaviruses and low pathogenic viruses HCoV-229E and HCoV-OC43. Conclusion: Our results demonstrate a previously unknown suppression of a host response gene set by SARS-CoV-2 and confirm a difference in interferon-related gene expression between highly pathogenic and low pathogenic coronaviruses.
RESUMEN
Our study focuses on the intricate connection between tissue-level organization and ciliated organ function in humans, particularly in understanding the morphological organization of airways and their role in mucociliary clearance. Mucociliary clearance is a key mechanical defense mechanism of human airways, and clearance failure is associated with many respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma. While single-cell transcriptomics have unveiled the cellular complexity of the human airway epithelium, our understanding of the mechanics that link epithelial structure to clearance function mainly stem from animal models. This reliance on animal data limits crucial insights into human airway barrier function and hampers the human-relevant in vitro modeling of airway diseases. This study, for the first time, maps the distribution of ciliated and secretory cell types along the airway tree in both rats and humans, noting species-specific differences in ciliary function and elucidates structural parameters of airway epithelia that predict clearance function in both native and in vitro tissues alike. By uncovering how tissue organization influences ciliary function, we can better understand disruptions in mucociliary clearance, which could have implications for various ciliated organs beyond the airways.
RESUMEN
Mucociliary clearance is a key mechanical defense mechanism of human airways, and clearance failure is linked to major respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. While single-cell transcriptomics have unveiled the cellular complexity of the human airway epithelium, our understanding of the mechanics that link epithelial structure to clearance function mainly stem from animal models. This reliance on animal data limits crucial insights into human airway barrier function and hampers the human-relevant in vitro modeling of airway diseases. Our study fills this crucial knowledge gap and for the first time (1) maps the distribution of ciliated and secretory cell types on the mucosal surface along the proximo-distal axis of the rat and human airway tree, (2) identifies species-specific differences in ciliary beat and clearance function, and (3) elucidates structural parameters of airway epithelia that predict clearance function in both native and in vitro tissues alike. Our broad range of experimental approaches and physics-based modeling translate into generalizable parameters to quantitatively benchmark the human-relevancy of mucociliary clearance in experimental models, and to characterize distinct disease states.
RESUMEN
Early career members of Assembly 3 (Basic and Translational Sciences) of the European Respiratory Society (ERS) summarise the key messages discussed during six selected sessions that took place at the ERS International Congress 2023 in Milan, Italy. Aligned with the theme of the congress, the first session covered is "Micro- and macro-environments and respiratory health", which is followed by a summary of the "Scientific year in review" session. Next, recent advances in experimental methodologies and new technologies are discussed from the "Tissue modelling and remodelling" session and a summary provided of the translational science session, "What did you always want to know about omics analyses for clinical practice?", which was organised as part of the ERS Translational Science initiative's aims. The "Lost in translation: new insights into cell-to-cell crosstalk in lung disease" session highlighted how next-generation sequencing can be integrated with laboratory methods, and a final summary of studies is presented from the "From the transcriptome landscape to innovative preclinical models in lung diseases" session, which links the transcriptome landscape with innovative preclinical models. The wide range of topics covered in the selected sessions and the high quality of the research discussed demonstrate the strength of the basic and translational science being presented at the international respiratory conference organised by the ERS.