RESUMEN
Aside from centrally induced trained immunity in the bone marrow (BM) and peripheral blood by parenteral vaccination or infection, evidence indicates that mucosal-resident innate immune memory can develop via a local inflammatory pathway following mucosal exposure. However, whether mucosal-resident innate memory results from integrating distally generated immunological signals following parenteral vaccination/infection is unclear. Here we show that subcutaneous Bacillus Calmette-Guérin (BCG) vaccination can induce memory alveolar macrophages (AMs) and trained immunity in the lung. Although parenteral BCG vaccination trains BM progenitors and circulating monocytes, induction of memory AMs is independent of circulating monocytes. Rather, parenteral BCG vaccination, via mycobacterial dissemination, causes a time-dependent alteration in the intestinal microbiome, barrier function and microbial metabolites, and subsequent changes in circulating and lung metabolites, leading to the induction of memory macrophages and trained immunity in the lung. These data identify an intestinal microbiota-mediated pathway for innate immune memory development at distal mucosal tissues and have implications for the development of next-generation vaccine strategies against respiratory pathogens.
Asunto(s)
Vacuna BCG , Macrófagos Alveolares , Inmunidad Entrenada , Pulmón , Vacunación , Inmunidad InnataRESUMEN
Innate immune memory is an emerging area of research. However, innate immune memory at major mucosal sites remains poorly understood. Here, we show that respiratory viral infection induces long-lasting memory alveolar macrophages (AMs). Memory AMs are programed to express high MHC II, a defense-ready gene signature, and increased glycolytic metabolism, and produce, upon re-stimulation, neutrophil chemokines. Using a multitude of approaches, we reveal that the priming, but not maintenance, of memory AMs requires the help from effector CD8 T cells. T cells jump-start this process via IFN-γ production. We further find that formation and maintenance of memory AMs are independent of monocytes or bone marrow progenitors. Finally, we demonstrate that memory AMs are poised for robust trained immunity against bacterial infection in the lung via rapid induction of chemokines and neutrophilia. Our study thus establishes a new paradigm of immunological memory formation whereby adaptive T-lymphocytes render innate memory of mucosal-associated macrophages.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Innata , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos T CD8-positivos/citología , Memoria Inmunológica , Pulmón/citología , Macrófagos Alveolares/citología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Monocitos/citología , Monocitos/inmunología , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
RATIONALE: In asthma, sputum group 2 innate lymphoid cells (ILC2) are activated within 7h after allergen challenge. Neuroimmune interactions mediate rapid host responses at mucosal interfaces. In murine models of asthma, lung ILC2 co-localize to sensory neuronal termini expressing the neuropeptide, neuromedin U (NMU) and NMU stimulates type 2 cytokines secretion by ILC2 with additive effects to alarmins, in vitro. OBJECTIVES: Investigate effect of NMU/NMUR1 axis on early activation of ILC2 in asthma. METHODS: M ild asthmatics (n=8) were enrolled in a diluent-controlled, allergen-inhalation challenge study. Sputum ILC2 expression of NMU receptor 1 (NMUR1) and T2 cytokines were enumerated by flow cytometry and airway NMU levels were assessed by ELISA. This was compared to samples from moderate-severe asthmatics (n=9). Flow sort-purified and ex-vivo expanded ILC2 were used for functional assays and transcriptomic analyses. RESULTS: Significant increases in sputum ILC2 expressing NMUR1 were detected 7h post- allergen versus diluent challenge where the majority of NMUR1+ILC2 expressed IL-5/IL-13. Sputum NMUR1+ILC2 were significantly greater in mild versus moderate-severe asthmatics and NMUR1+ILC2 correlated inversely with the dose of inhaled corticosteroid in the latter group. Co-culturing with alarmins upregulated NMUR1 in ILC2, which was attenuated by dexamethasone. NMU stimulated T2 cytokine expression by ILC2, maximal at 6h was abrogated by dexamethasone or specific signaling inhibitors for mitogen-activated protein kinase ½, phospho-inositol 3 kinase but not IL-33 signaling moiety MyD88, in vitro. CONCLUSIONS: The NMU/NMUR1 axis stimulates rapid effects on ILC2, and maybe an important early activator of these cells in eosinophilic inflammatory responses in asthma.
RESUMEN
Rationale: Localized autoimmune responses have been reported in patients with severe eosinophilic asthma, characterized by eosinophil degranulation and airway infections. Objective: To determine the presence of autoantibodies against macrophage scavenger receptors within the airways and their effects on macrophage function and susceptibility to infection. Methods: Anti-EPX (eosinophil peroxidase), anti-MARCO (macrophage receptor with collagenous structure) IgG titers, and T1 and T2 (type 1/2) cytokines were measured in 221 sputa from 143 well-characterized patients with severe asthma. Peripheral monocytes and MDMs (monocyte-derived macrophages) isolated from healthy control subjects were treated with immunoprecipitated immunoglobulins from sputa with high anti-MARCO titers or nonspecific IgG to assess uptake of Streptococcus pneumoniae or response to the bacterial product LPS. Measurements and Main Results: Anti-MARCO IgG was detected in 36% of patients, with significantly higher titers (up to 1:16) in patients with mixed granulocytic sputa, indicative of airway infections. Multivariate regression analysis confirmed increased frequency of degranulation (free eosinophil granules), increased blood eosinophils (indicative of high T2 burden), increased sputum total cell count, peripheral blood leukocytes (indicative of infection), and lymphopenia were associated with increased anti-MARCO IgG titers; IL-15 (odds ratio [OR], 1.79; confidence interval [CI], 1.19-2.70), IL-13 (OR, 1.06; CI, 1.02-1.12), and IL-12p70 (OR, 3.34; CI, 1.32-8.40) were the associated cytokines. Patients with anti-MARCO antibodies had higher chances of subsequent infective versus eosinophilic exacerbations (P = 0.01). MDMs treated with immunoprecipitated immunoglobulins (anti-MARCO+ sputa) had reduced bacterial uptake by 39% ± 15% and significantly reduced release of IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05) in response to an LPS stimulus. Conclusions: Autoantibodies against macrophage scavenger receptors in eosinophilic asthma airways may impede effective host defenses and lead to recurrent infective bronchitis.
Asunto(s)
Asma , Bronquitis , Eosinofilia Pulmonar , Humanos , Autoanticuerpos , Lipopolisacáridos , Eosinófilos , Citocinas , Macrófagos , Inmunoglobulina GRESUMEN
Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive lung scarring. IPF-related pulmonary vascular remodeling and pulmonary hypertension (PH) result in a particularly poor prognosis. Objectives: To study the pathogenesis of vascular remodeling in fibrotic lungs and its contribution to progression of fibrosis. Methods: We used an experimental model of lung fibrosis associated with PH by transient overexpression of active TGF-ß1 (transforming growth factor-ß1). Samples from patients with fibrotic lung diseases were analyzed in depth using immunostaining, gene expression, and gene mutations. Measurements and Main Results: We found a reduction in endothelial cells (ECs) and activation of vascular smooth muscle cells (VSMCs) in fibrotic lungs. Coculturing fibroblasts with VSMCs or ECs from fibrotic lungs induced fibrotic phenotypes in fibroblasts. IPF fibroblasts induced EC death and activation of VSMCs in coculture systems. Decreased concentrations of BMPR2 (bone morphogenic protein receptor 2) and its signaling were observed in ECs and VSMCs from fibrotic lungs in both rats and humans. On fibroblasts treated with media from VSMCs, BMPR2 suppression in VSMCs led to fibrogenic effects. Tacrolimus activated BMPR2 signaling and attenuated fibrosis and PH in rodent lungs. Whole-exome sequencing revealed rare mutations in PH-related genes, including BMPR2, in patients with IPF undergoing transplantation. A unique missense BMPR2 mutation (p.Q721R) was discovered to have dysfunctional effects on BMPR2 signaling. Conclusions: Endothelial dysfunction and vascular remodeling in PH secondary to pulmonary fibrosis enhance fibrogenesis through impaired BMPR2 signaling. Tacrolimus may have value as a treatment of advanced IPF and concomitant PH. Genetic abnormalities may determine the development of PH in advanced IPF.
Asunto(s)
Hipertensión Pulmonar , Fibrosis Pulmonar Idiopática , Humanos , Ratas , Animales , Remodelación Vascular , Células Endoteliales/metabolismo , Tacrolimus , Pulmón/patología , Fibrosis Pulmonar Idiopática/patología , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Fibroblastos/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genéticaRESUMEN
BACKGROUND: Kidney development is regulated by cellular interactions between the ureteric epithelium, mesenchyme, and stroma. Previous studies demonstrate essential roles for stromal ß-catenin in kidney development. However, how stromal ß-catenin regulates kidney development is not known. We hypothesize that stromal ß-catenin modulates pathways and genes that facilitate communications with neighboring cell populations to regulate kidney development. RESULTS: We isolated purified stromal cells with wild type, deficient, and overexpressed ß-catenin by fluorescence-activated cell sorting and conducted RNA Sequencing. A Gene Ontology network analysis demonstrated that stromal ß-catenin modulates key kidney developmental processes, including branching morphogenesis, nephrogenesis and vascular formation. Specific stromal ß-catenin candidate target genes that may mediate these effects included secreted, cell-surface and transcriptional factors that regulate branching morphogenesis and nephrogenesis (Wnts, Bmp, Fgfr, Tcf/Lef) and secreted vascular guidance cues (Angpt1, VEGF, Sema3a). We validated established ß-catenin targets including Lef1 and novel candidate ß-catenin targets including Sema3e which have unknown roles in kidney development. CONCLUSIONS: These studies advance our understanding of gene and biological pathway dysregulation in the context of stromal ß-catenin misexpression during kidney development. Our findings suggest that during normal kidney development, stromal ß-catenin may regulate secreted and cell-surface proteins to communicate with adjacent cell populations.
Asunto(s)
Uréter , beta Catenina , beta Catenina/genética , beta Catenina/metabolismo , Riñón/metabolismo , Factores de Transcripción/metabolismo , Uréter/metabolismo , Transducción de SeñalRESUMEN
Type 1 alveolar epithelial cells (AT1s) and type 2 alveolar epithelial cells (AT2s) regulate the structural integrity and function of alveoli. AT1s mediate gas exchange, whereas AT2s serve multiple functions, including surfactant secretion and alveolar repair through proliferation and differentiation into AT1s as progenitors. However, mechanisms regulating AT2 proliferation and differentiation remain unclear. Here we demonstrate that Gremlin, an intrinsic inhibitor of bone morphogenetic protein (BMP), induces AT2 proliferation and differentiation. Transient overexpression of Gremlin in rat lungs by adenovirus vector delivery suppressed BMP signaling, induced proliferation of AT2s and the production of Bmp2, which in turn led to the recovery of BMP signaling and induced AT2 differentiation into AT1s. Bleomycin-induced lung injury upregulated Gremlin and showed a similar time course of biomarker expression comparable to the adenovirus model. TGF-ß and IL-1ß induced Gremlin expression in fibroblasts. Taken together, our findings implicate that Gremlin expression during lung injury leads to precisely timed inhibition of BMP signaling and activates AT2s, leading to alveolar repair.
Asunto(s)
Células Epiteliales Alveolares , Lesión Pulmonar , Ratas , Animales , Células Epiteliales Alveolares/metabolismo , Lesión Pulmonar/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/fisiología , Proliferación CelularRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14+ circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodeling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in the lung of a patient with IPF and in CD14+ circulating monocytes obtained from blood of a patient with IPF. After bleomycin administration, the myeloid-specific deletion of Atf6α altered the pulmonary macrophage composition, expanding CD11b+ subpopulations with dual polarized CD38+ CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. A further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b+ macrophages which had altered function during lung injury and fibrosis.
Asunto(s)
Fibrosis Pulmonar Idiopática , Lesión Pulmonar , Ratones , Animales , Lesión Pulmonar/metabolismo , Factor de Transcripción Activador 6/metabolismo , Ratones Endogámicos C57BL , Macrófagos/metabolismo , Pulmón/patología , Fibrosis Pulmonar Idiopática/patología , Fibrosis , Bleomicina/efectos adversos , Bleomicina/metabolismoRESUMEN
BACKGROUND: Autoimmunity has been reported in patients with severe coronavirus disease 2019 (COVID-19). We investigated whether anti-nuclear/extractable-nuclear antibodies (ANAs/ENAs) were present up to a year after infection, and if they were associated with the development of clinically relevant post-acute sequalae of COVID-19 (PASC) symptoms. METHODS: A rapid-assessment line immunoassay was used to measure circulating levels of ANAs/ENAs in 106 convalescent COVID-19 patients with varying acute phase severities at 3, 6 and 12â months post-recovery. Patient-reported fatigue, cough and dyspnoea were recorded at each time point. Multivariable logistic regression model and receiver operating curves were used to test the association of autoantibodies with patient-reported outcomes and pro-inflammatory cytokines. RESULTS: Compared to age- and sex-matched healthy controls (n=22) and those who had other respiratory infections (n=34), patients with COVID-19 had higher detectable ANAs at 3â months post-recovery (p<0.001). The mean number of ANA autoreactivities per individual decreased between 3 and 12â months (from 3.99 to 1.55) with persistent positive titres associated with fatigue, dyspnoea and cough severity. Antibodies to U1-snRNP and anti-SS-B/La were both positively associated with persistent symptoms of fatigue (p<0.028, area under the curve (AUC) 0.86) and dyspnoea (p<0.003, AUC=0.81). Pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α and C-reactive protein predicted the elevated ANAs at 12â months. TNF-α, D-dimer and interleukin-1ß had the strongest association with symptoms at 12â months. Regression analysis showed that TNF-α predicted fatigue (ß=4.65, p=0.004) and general symptomaticity (ß=2.40, p=0.03) at 12â months. INTERPRETATION: Persistently positive ANAs at 12â months post-COVID are associated with persisting symptoms and inflammation (TNF-α) in a subset of COVID-19 survivors. This finding indicates the need for further investigation into the role of autoimmunity in PASC.
Asunto(s)
Autoanticuerpos , COVID-19 , Humanos , Síndrome Post Agudo de COVID-19 , Factor de Necrosis Tumoral alfa , Tos , Anticuerpos Antinucleares , Citocinas , FatigaRESUMEN
BACKGROUND: Local airway autoimmune responses may contribute to steroid dependence and persistent eosinophilia in severe asthma. Auto-IgG antibodies directed against granule proteins such as eosinophil peroxidase (EPX), macrophage scavenger receptor with collagenous structure (MARCO) and nuclear/extranuclear antigens (antinuclear antibodies (ANAs)) have been reported. Our objective was to describe the prevalence and clinical characteristics of asthmatic patients with airway autoreactivity, and to assess if this could be predicted from clinical history of autoreactivity. METHODS: We analysed anti-EPX, anti-MARCO and ANAs in 218 sputum samples collected prospectively from 148 asthmatic patients, and evaluated their association with lung function parameters, blood/airway inflammation, severity indices and exacerbations. Additionally, 107 of these patients consented to fill out an autoimmune checklist to determine personal/family history of systemic autoimmune disease and symptoms. RESULTS: Out of the 148 patients, 59 (40%) were anti-EPX IgG+, 53 (36%) were anti-MARCO IgG+ and 64 out of 129 (50%) had ≥2 nuclear/extranuclear autoreactivities. A composite airway autoreactivity score (CAAS) demonstrated that 82 patients (55%) had ≥2 airway autoreactivities (considered as CAAS+). Increased airway eosinophil degranulation (OR 15.1, 95% CI 1.1-199.4), increased blood leukocytes (OR 3.5, 95% CI 1.3-10.1) and reduced blood lymphocytes (OR 0.19, 95% CI 0.04-0.84) predicted CAAS+. A third of CAAS+ patients reported an exacerbation, associated with increased anti-EPX and/or anti-MARCO IgG (p<0.05). While no association was found between family history or personal diagnosis of autoimmune disease, 30% of CAAS+ asthmatic patients reported sicca symptoms (p=0.02). Current anti-inflammatory (inhaled/oral corticosteroids and/or adjunct anti-interleukin-5 biologics) treatment does not attenuate airway autoantibodies, irrespective of eosinophil suppression. CONCLUSION: We report 55% of moderate-severe asthmatic patients to have airway autoreactivity that persists despite anti-inflammatory treatment and is associated with exacerbations.
Asunto(s)
Asma , Enfermedades Autoinmunes , Humanos , Autoanticuerpos , Esputo/química , Eosinófilos , Antiinflamatorios/uso terapéutico , Inmunoglobulina GRESUMEN
BACKGROUND: Cigarette smokers are at increased risk of acquiring influenza, developing severe disease and requiring hospitalisation/intensive care unit admission following infection. However, immune mechanisms underlying this predisposition are incompletely understood, and therapeutic strategies for influenza are limited. METHODS: We used a mouse model of concurrent cigarette smoke exposure and H1N1 influenza infection, colony-stimulating factor (CSF)3 supplementation/receptor (CSF3R) blockade and single-cell RNA sequencing (scRNAseq) to investigate this relationship. RESULTS: Cigarette smoke exposure exacerbated features of viral pneumonia such as oedema, hypoxaemia and pulmonary neutrophilia. Smoke-exposed infected mice demonstrated an increase in viral (v)RNA, but not replication-competent viral particles, relative to infection-only controls. Interstitial rather than airspace neutrophilia positively predicted morbidity in smoke-exposed infected mice. Screening of pulmonary cytokines using a novel dysregulation score identified an exacerbated expression of CSF3 and interleukin-6 in the context of smoke exposure and influenza. Recombinant (r)CSF3 supplementation during influenza aggravated morbidity, hypothermia and oedema, while anti-CSF3R treatment of smoke-exposed infected mice improved alveolar-capillary barrier function. scRNAseq delineated a shift in the distribution of Csf3 + cells towards neutrophils in the context of cigarette smoke and influenza. However, although smoke-exposed lungs were enriched for infected, highly activated neutrophils, gene signatures of these cells largely reflected an exacerbated form of typical influenza with select unique regulatory features. CONCLUSION: This work provides novel insight into the mechanisms by which cigarette smoke exacerbates influenza infection, unveiling potential therapeutic targets (e.g. excess vRNA accumulation, oedematous CSF3R signalling) for use in this context, and potential limitations for clinical rCSF3 therapy during viral infectious disease.
Asunto(s)
Fumar Cigarrillos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Animales , Fumar Cigarrillos/efectos adversos , Humanos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Neutrófilos , NicotianaRESUMEN
BACKGROUND: Progressive fibrosing interstitial lung disease (PF-ILD) is characterised by progressive physiological, symptomatic and/or radiographic worsening. The real-world prevalence and characteristics of PF-ILD remain uncertain. METHODS: Patients were enrolled from the Canadian Registry for Pulmonary Fibrosis between 2015 and 2020. PF-ILD was defined as a relative forced vital capacity (FVC) decline ≥10%, death, lung transplantation or any two of: relative FVC decline ≥5% and <10%, worsening respiratory symptoms or worsening fibrosis on computed tomography of the chest, all within 24â months of diagnosis. Time-to-event analysis compared progression between key diagnostic subgroups. Characteristics associated with progression were determined by multivariable regression. RESULTS: Of 2746 patients with fibrotic ILD (mean±sd age 65±12â years; 51% female), 1376 (50%) met PF-ILD criteria in the first 24â months of follow-up. PF-ILD occurred in 427 (59%) patients with idiopathic pulmonary fibrosis (IPF), 125 (58%) with fibrotic hypersensitivity pneumonitis (HP), 281 (51%) with unclassifiable ILD (U-ILD) and 402 (45%) with connective tissue disease-associated ILD (CTD-ILD). Compared with IPF, time to progression was similar in patients with HP (hazard ratio (HR) 0.96, 95% CI 0.79-1.17), but was delayed in patients with U-ILD (HR 0.82, 95% CI 0.71-0.96) and CTD-ILD (HR 0.65, 95% CI 0.56-0.74). Background treatment varied across diagnostic subtypes, with 66% of IPF patients receiving antifibrotic therapy, while immunomodulatory therapy was utilised in 49%, 61% and 37% of patients with CHP, CTD-ILD and U-ILD, respectively. Increasing age, male sex, gastro-oesophageal reflux disease and lower baseline pulmonary function were independently associated with progression. CONCLUSIONS: Progression is common in patients with fibrotic ILD, and is similarly prevalent in HP and IPF. Routinely collected variables help identify patients at risk for progression and may guide therapeutic strategies.
Asunto(s)
Alveolitis Alérgica Extrínseca , Fibrosis Pulmonar Idiopática , Enfermedades Pulmonares Intersticiales , Anciano , Alveolitis Alérgica Extrínseca/complicaciones , Alveolitis Alérgica Extrínseca/epidemiología , Canadá/epidemiología , Progresión de la Enfermedad , Femenino , Humanos , Fibrosis Pulmonar Idiopática/complicaciones , Fibrosis Pulmonar Idiopática/diagnóstico , Fibrosis Pulmonar Idiopática/epidemiología , Enfermedades Pulmonares Intersticiales/complicaciones , Enfermedades Pulmonares Intersticiales/diagnóstico , Enfermedades Pulmonares Intersticiales/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Sistema de RegistrosRESUMEN
Discovery of the microbiota-gut-brain axis has led to proposed microbe-based therapeutic strategies in mental health, including the use of mood-altering bacterial species, termed psychobiotics. However, we still have limited understanding of the key signaling pathways engaged by specific organisms in modulating brain function, and evidence suggests that bacteria with broadly similar neuroactive and immunomodulatory actions can drive different behavioral outcomes. We sought to identify pathways distinguishing two psychoactive bacterial strains that seemingly engage similar gut-brain signaling pathways but have distinct effects on behaviour. We used RNAseq to identify mRNAs differentially expressed in the blood and hippocampus of mice following Lacticaseibacillus rhamnosus JB-1, and Limosilactobacillus reuteri 6475 treatment and performed Gene Set Enrichment Analysis (GSEA) to identify enrichment in pathway activity. L. rhamnosus, but not L. reuteri treatment altered several pathways in the blood and hippocampus, and the rhamnosus could be clearly distinguished based on mRNA profile. In particular, L. rhamnosus treatment modulated the activity of interferon signaling, JAK/STAT, and TNF-alpha via NF-KB pathways. Our results highlight that psychobiotics can induce complex changes in host gene expression, andin understanding these changes, we may help fine-tune selection of psychobiotics for treating mood disorders.
Asunto(s)
Lacticaseibacillus rhamnosus , Probióticos , Afecto , Animales , Encéfalo/metabolismo , Hipocampo , Masculino , Ratones , Probióticos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and excessive extracellular matrix deposition. We sought to investigate the role of FKBP13 (13-kD FK506-binding protein), an endoplasmic reticulum-resident molecular chaperone, in various forms of pulmonary fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy specimens from 24 patients with idiopathic pulmonary fibrosis and 17 control subjects. FKBP13 expression was found to be elevated in the fibrotic regions of idiopathic pulmonary fibrosis lung tissues and correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also increased in lung biopsy specimens of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using FKBP13-/- mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 21), and resolution (Day 50) phases. FKBP13-/- mice showed increased infiltration of inflammatory cells and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of fibrosis at Day 50. These changes were associated with an increased number of cells that stained positive for TUNEL and cleaved caspase 3 in the FKBP13-/- lungs, indicating a heightened cellular sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity of interstitial lung diseases and that it has a biologically relevant role in protecting mice against bleomycin-induced injury, inflammation, and fibrosis.
Asunto(s)
Enfermedades Pulmonares Intersticiales/metabolismo , Enfermedades Pulmonares Intersticiales/patología , Proteínas de Unión a Tacrolimus/metabolismo , Regulación hacia Arriba/fisiología , Animales , Biomarcadores/metabolismo , Biopsia/métodos , Bleomicina/efectos adversos , Citocinas/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Inflamación/metabolismo , Inflamación/patología , Pulmón , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Chronic obstructive pulmonary disease (COPD) is a complex and progressive respiratory disease. Autoimmune processes have been hypothesized to contribute to disease progression; however, the presence of autoantibodies in the serum has been variable. Given that COPD is a lung disease, we sought to investigate whether autoantibodies in sputum supernatant would better define pulmonary autoimmune processes. Matched sputum and serum samples were obtained from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study and at the Guangzhou Institute of Respiratory Health (GIRH). Samples were collected from patients with varying severity of COPD, asymptomatic smokers, and healthy control subjects. IgG and IgM autoantibodies were detected in sputum and serum of all subjects in both cohorts using a broad-spectrum autoantigen array. No differences were observed in sputum autoantibodies between COPD and asymptomatic smokers in either cohort. In contrast, 16% of detectable sputum IgG autoantibodies were decreased in subjects with COPD compared to healthy controls in the ADEPT cohort. Compared to asymptomatic smokers, approximately 13% of detectable serum IgG and 40% of detectable serum IgM autoantibodies were differentially expressed in GIRH COPD subjects. Of the differentially expressed specificities, anti-nuclear autoantibodies were predominately decreased. A weak correlation between increased serum IgM anti-tissue autoantibodies and a measure of airspace enlargement was observed. The differential expression of specificities varied between the cohorts. In closing, using a comprehensive autoantibody array, we demonstrate that autoantibodies are present in subjects with COPD, asymptomatic smokers, and healthy controls. Cohorts displayed high levels of heterogeneity, precluding the utilization of autoantibodies for diagnostic purposes.
Asunto(s)
Autoanticuerpos/inmunología , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Esputo/metabolismo , Estudios de Casos y Controles , Progresión de la Enfermedad , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Pulmón/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Fumadores , Fumar/metabolismoRESUMEN
Since the discovery of the myofibroblast over 50 years ago, much has been learned about its role in wound healing and fibrosis. Its origin, however, remains controversial, with a number of progenitor cells being proposed. Macrophage-myofibroblast transition (MMT) is a recent term coined in 2014 that describes the mechanism through which macrophages, derived from circulating monocytes originating in the bone marrow, transformed into myofibroblasts and contributed to kidney fibrosis. Over the past years, several studies have confirmed the existence of MMT in various systems, suggesting that MMT could potentially occur in all fibrotic conditions and constitute a reasonable therapeutic target to prevent progressive fibrotic disease. In this perspective, we examined recent evidence supporting the notion of MMT in both human disease and experimental models across organ systems. Mechanistic insight from these studies and information from in vitro studies is provided. The findings substantiating plausible MMT showcased the co-expression of macrophage and myofibroblast markers, including CD68 or F4/80 (macrophage) and α-SMA (myofibroblast), in fibroblast-like cells. Furthermore, fate-mapping experiments in murine models exhibiting myeloid-derived myofibroblasts in the tissue further provide direct evidence for MMT. Additionally, we provide some evidence from single cell RNA sequencing experiments confirmed by fluorescent in situ hybridisation studies, showing monocyte/macrophage and myofibroblast markers co-expressed in lung tissue from patients with fibrotic lung disease. In conclusion, MMT is likely a significant contributor to myofibroblast formation in wound healing and fibrotic disease across organ systems. Circulating precursors including monocytes and the molecular mechanisms governing MMT could constitute valid targets and provide insight for the development of novel antifibrotic therapies; however, further understanding of these processes is warranted.
Asunto(s)
Monocitos , Miofibroblastos , Animales , Diferenciación Celular , Fibrosis , Humanos , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/patología , Cicatrización de HeridasRESUMEN
BACKGROUND: In clinical trials, the two anti-interleukin (IL)-5 monoclonal antibodies (mAbs: mepolizumab and reslizumab) approved to treat severe eosinophilic asthma reduce exacerbations by â¼50-60%. OBJECTIVE: To observe response to anti-IL-5 mAbs in a real-life clinical setting, and to evaluate predictors of suboptimal response. METHODS: In four Canadian academic centres, predefined clinical end-points in 250 carefully characterised moderate-to-severe asthmatic patients were collected prospectively to assess response to the two anti-IL-5 mAbs. Suboptimal response was determined based on failure to reduce maintenance corticosteroid (MCS) or asthma symptoms scores (Asthma Control Questionnaire (ACQ)) or exacerbations, in addition to persistence of sputum/blood eosinophils. Worsening in suboptimal responders was assessed based on reduced lung function by 25% or increase in MCS/ACQ. A representative subset of 39 patients was evaluated for inflammatory mediators, autoantibodies and complement activation in sputum (by ELISA) and for immune-complex deposition by immunostaining formalin-fixed paraffin-embedded sputum plugs. RESULTS: Suboptimal responses were observed in 42.8% (107 out of 250) patients treated with either mepolizumab or reslizumab. Daily prednisone requirement, sinus disease and late-onset asthma diagnoses were the strongest predictors of suboptimal response. Asthma worsened in 13.6% (34 out of 250) of these patients. The majority (79%) of them were prednisone-dependent. Presence of sputum anti-eosinophil peroxidase immunoglobulin (Ig)G was a predictor of suboptimal response to an anti-IL-5 mAb. An increase in sputum C3c (marker of complement activation) and deposition of C1q-bound/IL-5-bound IgG were observed in the sputa of those patients who worsened on therapy, suggesting an underlying autoimmune-mediated pathology. CONCLUSION: A significant number of patients who meet currently approved indications for anti-IL5 mAbs show suboptimal response to them in real-life clinical practice, particularly if they are on high doses of prednisone. Monitoring blood eosinophil count is not helpful to identify these patients. The concern of worsening of symptoms associated with immune-complex mediated complement activation in a small proportion of these patients highlights the relevance of recognising airway autoimmune phenomena and this requires further evaluation.
Asunto(s)
Antiasmáticos , Asma , Antiasmáticos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Asma/tratamiento farmacológico , Canadá , Eosinófilos , Humanos , Interleucina-5RESUMEN
In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.
Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus , Pandemias , Peptidil-Dipeptidasa A , Neumonía Viral , Serina Endopeptidasas , Enzima Convertidora de Angiotensina 2 , COVID-19 , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Chaperón BiP del Retículo Endoplásmico , Expresión Génica , Perfilación de la Expresión Génica/métodos , Humanos , Pulmón/metabolismo , Pulmón/virología , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/metabolismo , Neumonía Viral/virología , Receptores Virales/clasificación , Receptores Virales/genética , Receptores Virales/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , SARS-CoV-2 , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Internalización del VirusRESUMEN
BACKGROUND: Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR). METHODS: We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. RESULTS: We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. CONCLUSIONS: Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.