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Epithelial barriers are programmed for defense and repair but are also the site of long-term structural remodeling and disease. In general, this paradigm features epithelial stem cell (ESCs) that are called on to regenerate damaged tissues but can also be reprogrammed for detrimental remodeling. Here we identified a Wfdc21-dependent monocyte-derived dendritic cell (moDC) population that functioned as an early sentinel niche for basal-ESC reprogramming in mouse models of epithelial injury after respiratory viral infection. Niche function depended on moDC delivery of ligand GPNMB to basal-ESC receptor CD44 so that properly timed antibody blockade of ligand or receptor provided long-lasting correction of reprogramming and broad disease phenotypes. These same control points worked directly in mouse and human basal-ESC organoids. Together, the findings identify a mechanism to explain and modify what is otherwise a stereotyped but sometimes detrimental response to epithelial injury.
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All living organisms are charged with repair after injury particularly at epithelial barrier sites, but in some cases this response leads instead to structural remodeling and long-term disease. Identifying the molecular and cellular control of this divergence is key to disease modification. In that regard, stress kinase control of epithelial stem cells is a rational entry point for study. Here we examine the potential for mitogen-activated protein kinase 13 (MAPK13) regulation of epithelial stem cells using models of respiratory viral injury and post-viral lung disease. We show that Mapk13 gene-knockout mice handle acute infectious illness as expected but are protected against structural remodeling manifest as basal-epithelial stem cell (basal-ESC) hyperplasia-metaplasia, immune activation, and mucinous differentiation. In corresponding cell models, Mapk13-deficiency directly attenuates basal-ESC growth and organoid formation. Extension to human studies shows marked induction/activation of basal-cell MAPK13 in clinical samples of comparable remodeling found in asthma and COPD. Here again, MAPK13 gene-knockdown inhibits human basal-ESC growth in culture. Together, the data identify MAPK13 as a control for structural remodeling and disease after epithelial injury and as a suitable target for down-regulation as a disease-modifying strategy.
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Importance: Adding fulvestrant to anastrozole (A+F) improved survival in postmenopausal women with advanced estrogen receptor (ER)-positive/ERBB2 (formerly HER2)-negative breast cancer. However, the combination has not been tested in early-stage disease. Objective: To determine whether neoadjuvant fulvestrant or A+F increases the rate of pathologic complete response or ypT1-2N0/N1mic/Ki67 2.7% or less residual disease (referred to as endocrine-sensitive disease) over anastrozole alone. Design, Setting, and Participants: A phase 3 randomized clinical trial assessing differences in clinical and correlative outcomes between each of the fulvestrant-containing arms and the anastrozole arm. Postmenopausal women with clinical stage II to III, ER-rich (Allred score 6-8 or >66%)/ERBB2-negative breast cancer were included. All analyses were based on data frozen on March 2, 2023. Interventions: Patients received anastrozole, fulvestrant, or a combination for 6 months preoperatively. Tumor Ki67 was assessed at week 4 and optionally at week 12, and if greater than 10% at either time point, the patient switched to neoadjuvant chemotherapy or immediate surgery. Main Outcomes and Measures: The primary outcome was the endocrine-sensitive disease rate (ESDR). A secondary outcome was the percentage change in Ki67 after 4 weeks of neoadjuvant endocrine therapy (NET) (week 4 Ki67 suppression). Results: Between February 2014 and November 2018, 1362 female patients (mean [SD] age, 65.0 [8.2] years) were enrolled. Among the 1298 evaluable patients, ESDRs were 18.7% (95% CI, 15.1%-22.7%), 22.8% (95% CI, 18.9%-27.1%), and 20.5% (95% CI, 16.8%-24.6%) with anastrozole, fulvestrant, and A+F, respectively. Compared to anastrozole, neither fulvestrant-containing regimen significantly improved ESDR or week 4 Ki67 suppression. The rate of week 4 or week 12 Ki67 greater than 10% was 25.1%, 24.2%, and 15.7% with anastrozole, fulvestrant, and A+F, respectively. Pathologic complete response/residual cancer burden class I occurred in 8 of 167 patients and 17 of 167 patients, respectively (15.0%; 95% CI, 9.9%-21.3%), after switching to neoadjuvant chemotherapy due to week 4 or week 12 Ki67 greater than 10%. PAM50 subtyping derived from RNA sequencing of baseline biopsies available for 753 patients (58%) identified 394 luminal A, 304 luminal B, and 55 nonluminal tumors. A+F led to a greater week 4 Ki67 suppression than anastrozole alone in luminal B tumors (median [IQR], -90.4% [-95.2 to -81.9%] vs -76.7% [-89.0 to -55.6%]; P < .001), but not luminal A tumors. Thirty-six nonluminal tumors (65.5%) had a week 4 or week 12 Ki67 greater than 10%. Conclusions and Relevance: In this randomized clinical trial, neither fulvestrant nor A+F significantly improved the 6-month ESDR over anastrozole in ER-rich/ERBB2-negative breast cancer. Aromatase inhibition remains the standard-of-care NET. Differential NET response by PAM50 subtype in exploratory analyses warrants further investigation. Trial Registration: ClinicalTrials.gov Identifier: NCT01953588.
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Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Anciano , Femenino , Humanos , Anastrozol/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/patología , Fulvestrant , Antígeno Ki-67 , Terapia Neoadyuvante , Nitrilos/efectos adversos , Posmenopausia , Receptor ErbB-2 , Receptores de Estrógenos , Triazoles/efectos adversos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Persona de Mediana EdadRESUMEN
Respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can trigger chronic lung disease that persists and even progresses after expected clearance of infectious virus. To gain an understanding of this process, the current study examined a series of consecutive fatal cases of coronavirus disease 2019 (COVID-19) that came to autopsy at 27 to 51 days after hospital admission. In each patient, a stereotyped bronchiolar-alveolar pattern of lung remodeling was identified with basal epithelial cell hyperplasia, immune activation, and mucinous differentiation. Remodeling regions featured macrophage infiltration and apoptosis and a marked depletion of alveolar type 1 and 2 epithelial cells. This pattern closely resembled findings from an experimental model of post-viral lung disease that requires basal-epithelial stem cell growth, immune activation, and differentiation. Together, these results provide evidence of basal epithelial cell reprogramming in long-term COVID-19 and thereby yield a pathway for explaining and correcting lung dysfunction in this type of disease.
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COVID-19 , Humanos , Reprogramación Celular , SARS-CoV-2 , Pulmón , Células EpitelialesRESUMEN
Respiratory viruses, including SARS-CoV-2, can trigger chronic lung disease that persists and even progresses after expected clearance of infectious virus. To gain an understanding of this process, we examined a series of consecutive fatal cases of Covid-19 that came to autopsy at 27-51 d after hospital admission. In each patient, we identify a stereotyped bronchiolar-alveolar pattern of lung remodeling with basal epithelial cell hyperplasia and mucinous differentiation. Remodeling regions also feature macrophage infiltration and apoptosis and a marked depletion of alveolar type 1 and 2 epithelial cells. This entire pattern closely resembles findings from an experimental model of post-viral lung disease that requires basal-epithelial stem cell growth, immune activation, and differentiation. The present results thereby provide evidence of possible basal epithelial cell reprogramming in long-term Covid-19 as well and thereby a pathway for explaining and correcting lung dysfunction in this type of disease.
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Mannose-binding lectins effectively inhibit most seasonal strains of influenza A virus and contribute to the innate host defense vs. these viruses. In contrast, pandemic IAV strains are largely resistant to these lectins, likely contributing to increased spread and worse outcomes. In this paper, we evaluated the inhibition of IAV by mannose-binding lectins of human, bacterial, and fungal origin to understand and possibly increase activity vs. the pandemic IAV. A modified version of the human surfactant protein D (SP-D) neck and carbohydrate recognition domain (NCRD) with combinatorial substitutions at the 325 and 343 positions, previously shown to inhibit pandemic H3N2 IAV in vitro and in vivo, and to inhibit pandemic H1N1 in vitro, failed to protect mice from pandemic H1N1 in vivo in the current study. We attempted a variety of maneuvers to improve the activity of the mutant NCRDs vs. the 2009 pandemic H1N1, including the formation of full-length SP-D molecules containing the mutant NCRD, cross-linking of NCRDs through the use of antibodies, combining SP-D or NCRDs with alpha-2-macroglobulin, and introducing an additional mutation to the double mutant NCRD. None of these substantially increased the antiviral activity for the pandemic H1N1. We also tested the activity of bacterial and algal mannose-binding lectins, cyanovirin, and griffithsin, against IAV. These had strong activity against seasonal IAV, which was largely retained against pandemic H1N1. We propose mechanisms to account for differences in activity of SP-D constructs against pandemic H3N2 and H1N1, and for differences in activity of cyanovirin vs. SP-D constructs.
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Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.
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Alarminas/fisiología , Células Epiteliales/fisiología , Interleucina-33/fisiología , Enfermedades Pulmonares/fisiopatología , Infecciones por Respirovirus/complicaciones , Virus Sendai , Células Madre/fisiología , Animales , Diferenciación Celular , Interleucina-33/genética , Ratones , Análisis de la Célula Individual , Células Madre/citologíaRESUMEN
Since the largest 2014â»2016 Ebola virus disease outbreak in West Africa, understanding of Ebola virus infection has improved, notably the involvement of innate immune mediators. Amongst them, collectins are important players in the antiviral innate immune defense. A screening of Ebola glycoprotein (GP)-collectins interactions revealed the specific interaction of human surfactant protein D (hSP-D), a lectin expressed in lung and liver, two compartments where Ebola was found in vivo. Further analyses have demonstrated an involvement of hSP-D in the enhancement of virus infection in several in vitro models. Similar effects were observed for porcine SP-D (pSP-D). In addition, both hSP-D and pSP-D interacted with Reston virus (RESTV) GP and enhanced pseudoviral infection in pulmonary cells. Thus, our study reveals a novel partner of Ebola GP that may participate to enhance viral spread.
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Ebolavirus/química , Glicoproteínas/química , Fiebre Hemorrágica Ebola/inmunología , Proteína D Asociada a Surfactante Pulmonar/química , Animales , Chlorocebus aethiops , Colectinas/química , Ebolavirus/efectos de los fármacos , Células HEK293 , Interacciones Microbiota-Huesped , Humanos , Inmunidad Innata , Unión Proteica , Proteína D Asociada a Surfactante Pulmonar/genética , Porcinos , Células Vero , Proteínas Virales/químicaRESUMEN
PURPOSE: The recent publication of the ACOSOG Z1031 trial results demonstrated that Ki-67 proliferation marker-based neoadjuvant endocrine therapy response monitoring could be used for tailoring the use of adjuvant chemotherapy in ER+HER2-negative breast cancer patients. In this paper, we describe the development of the Ki-67 clinical trial assay used for this study. METHODS: Ki-67 assay assessment focused on reproducing a 2.7% Ki-67 cut-point (CP) required for calculating the Preoperative Endocrine Prognostic Index and a 10% CP for poor endocrine therapy response identification within the first month of neoadjuvant endocrine treatment. Image analysis was assessed to increase the efficiency of the scoring process. Clinical outcome concordance for two independent Ki-67 scores was the primary performance metric. RESULTS: Discordant scores led to a triage approach where cases with complex histological features that software algorithms could not resolve were flagged for visual point counting (17%). The final Ki-67 scoring approach was run on T1/2 N0 cases from the P024 and POL trials (N = 58). The percent positive agreement for the 2.7% CP was 87.5% (95% CI 61.7-98.5%); percent negative agreement 88.9% (95% CI: 65.3-98.6%). Minor discordance did not affect the ability to predict similar relapse-free outcomes (Log-Rank P = 0.044 and P = 0.055). The data for the 10% early triage CP in the POL trial were similar (N = 66), the percentage positive agreement was 100%, and percent negative agreement 93.55% (95% CI: 78.58-99.21%). The independent survival predictions were concordant (Log-rank P = 0.0001 and P = 0.01). CONCLUSIONS: We have developed an efficient and reproducible Ki-67 scoring system that was approved by the Clinical Trials Evaluation Program for NCI-supported neoadjuvant endocrine therapy trials. Using the methodology described here, investigators are able to identify a subgroup of patients with ER+HER2-negative breast cancer that can be safely managed without the need of adjuvant chemotherapy.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Antígeno Ki-67/metabolismo , Antineoplásicos Hormonales/administración & dosificación , Antineoplásicos Hormonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/mortalidad , Quimioterapia Adyuvante , Toma de Decisiones Clínicas , Femenino , Humanos , Estimación de Kaplan-Meier , Terapia Neoadyuvante , Pronóstico , Curva ROC , Reproducibilidad de los Resultados , Resultado del TratamientoRESUMEN
Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) -positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inhibidores de la Aromatasa/uso terapéutico , Neoplasias de la Mama/química , Neoplasias de la Mama/tratamiento farmacológico , Antígeno Ki-67/análisis , Recurrencia Local de Neoplasia , Anciano , Anastrozol , Androstadienos/uso terapéutico , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Toma de Decisiones Clínicas , Femenino , Estudios de Seguimiento , Humanos , Antígeno Ki-67/genética , Letrozol , Persona de Mediana Edad , Índice Mitótico , Terapia Neoadyuvante/métodos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Nitrilos/uso terapéutico , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Receptores de Progesterona/genética , Tasa de Supervivencia , Transcriptoma , Triazoles/uso terapéuticoRESUMEN
Infiltrating activated monocytes are important mediators of damaging inflammation during influenza A virus (IAV) infection. We show that soluble respiratory proteins [collectins, surfactant proteins D (SP-D) and mannose binding lectin (MBL), H-ficolin and LL-37] inhibit replication of seasonal IAV in human monocytes. The collectins and H-ficolin also increased viral uptake by the cells, while LL-37 did not. H-ficolin was able to inhibit replication of the 2009 pandemic H1N1 strain (Cal09) in monocytes, but SP-D and LL-37 had significantly fewer inhibitory effects on this strain than on seasonal IAV. All of these proteins reduced IAV-induced TNF-α production, even in instances when viral replication was not reduced. We used modified recombinant versions of SP-D, MBL and ficolin to elucidate mechanisms through which these proteins alter monocyte interactions with IAV. We demonstrate the importance of the multimeric structure, and of binding properties of the lectin domain, in mediating antiviral and opsonic activity of the proteins. Hence, soluble inhibitors present in airway lining fluid may aid clearance of IAV by promoting monocyte uptake of the virus, while reducing viral replication and virus-induced TNF-α responses in these cells. However, SP-D and LL-37 have reduced ability to inhibit replication of pandemic IAV in monocytes.
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Péptidos Catiónicos Antimicrobianos/metabolismo , Colectinas/metabolismo , Glicoproteínas/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Lectinas/metabolismo , Mucosa Respiratoria/inmunología , Células Cultivadas , Glicoproteínas/genética , Humanos , Inmunidad Innata , Lectinas/genética , Neutrófilos/inmunología , Neutrófilos/virología , Fagocitosis/genética , Unión Proteica/genética , Ingeniería de Proteínas , Multimerización de Proteína/genética , Proteína D Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Mucosa Respiratoria/virología , Factor de Necrosis Tumoral alfa/metabolismo , Carga Viral , Replicación Viral , CatelicidinasRESUMEN
As a major player of the innate immune system, surfactant protein D (SP-D) recognizes and promotes elimination of various pathogens such as Gram-negative bacteria. SP-D binds to l-glycero-d-manno-heptose (Hep), a constituent of the partially conserved lipopolysaccharide (LPS) inner core of many Gram-negative bacteria. Binding and affinity of trimeric human SP-D to Hep in distinct LPS inner core glycans differing in linkages and adjacent residues was elucidated using glycan array and surface plasmon resonance measurements that were compared to in silico interaction studies. The combination of in vitro assays using defined glycans and molecular docking and dynamic simulation approaches provides insights into the interaction of trimeric SP-D with those glycan ligands. Trimeric SP-D wildtype recognized larger LPS inner core oligosaccharides with slightly enhanced affinity than smaller compounds suggesting the involvement of stabilizing secondary interactions. A trimeric human SP-D mutant D324N+D325N+R343K resembling rat SP-D bound to various LPS inner core structures in a similar pattern as observed for the wildtype but with higher affinity. The selective mutation of SP-D promotes targeting of LPS inner core oligosaccharides on Gram-negative bacteria to develop novel therapeutic agents.
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Lipopolisacáridos/química , Proteína D Asociada a Surfactante Pulmonar/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Humanos , Cinética , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
UNLABELLED: Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills over 600,000 people annually. Here, we report integrated computational and experimental investigations of the role and mechanisms of transcriptional regulation in cryptococcal infection. Major cryptococcal virulence traits include melanin production and the development of a large polysaccharide capsule upon host entry; shed capsule polysaccharides also impair host defenses. We found that both transcription and translation are required for capsule growth and that Usv101 is a master regulator of pathogenesis, regulating melanin production, capsule growth, and capsule shedding. It does this by directly regulating genes encoding glycoactive enzymes and genes encoding three other transcription factors that are essential for capsule growth: GAT201, RIM101, and SP1. Murine infection with cryptococci lacking Usv101 significantly alters the kinetics and pathogenesis of disease, with extended survival and, unexpectedly, death by pneumonia rather than meningitis. Our approaches and findings will inform studies of other pathogenic microbes. IMPORTANCE: Cryptococcus neoformans causes fatal meningitis in immunocompromised individuals, mainly HIV positive, killing over 600,000 each year. A unique feature of this yeast, which makes it particularly virulent, is its polysaccharide capsule; this structure impedes host efforts to combat infection. Capsule size and structure respond to environmental conditions, such as those encountered in an infected host. We have combined computational and experimental tools to elucidate capsule regulation, which we show primarily occurs at the transcriptional level. We also demonstrate that loss of a novel transcription factor alters virulence factor expression and host cell interactions, changing the lethal condition from meningitis to pneumonia with an exacerbated host response. We further demonstrate the relevant targets of regulation and kinetically map key regulatory and host interactions. Our work elucidates mechanisms of capsule regulation, provides methods and resources to the research community, and demonstrates an altered pathogenic outcome that resembles some human conditions.
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Criptococosis/microbiología , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Factores de Transcripción/metabolismo , Animales , Biología Computacional , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Femenino , Proteínas Fúngicas/genética , Redes Reguladoras de Genes , Humanos , Melaninas/metabolismo , Ratones , Factores de Transcripción/genética , VirulenciaRESUMEN
Mutations in the autophagy gene EPG5 are linked to the multisystem human disease Vici syndrome, which is characterized in part by pulmonary abnormalities, including recurrent infections. We found that Epg5-deficient mice exhibited elevated baseline innate immune cellular and cytokine-based lung inflammation and were resistant to lethal influenza virus infection. Lung transcriptomics, bone marrow transplantation experiments, and analysis of cellular cytokine expression indicated that Epg5 plays a role in lung physiology through its function in macrophages. Deletion of other autophagy genes including Atg14, Fip200, Atg5, and Atg7 in myeloid cells also led to elevated basal lung inflammation and influenza resistance. This suggests that Epg5 and other Atg genes function in macrophages to limit innate immune inflammation in the lung. Disruption of this normal homeostatic dampening of lung inflammation results in increased resistance to influenza, suggesting that normal homeostatic mechanisms that limit basal tissue inflammation support some infectious diseases.
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Inmunidad Innata , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Neumonía/inmunología , Proteínas/inmunología , Animales , Proteína 7 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Homeostasis , Humanos , Gripe Humana/genética , Gripe Humana/virología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/inmunología , Neumonía/genética , Neumonía/virología , Proteínas/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/inmunologíaRESUMEN
Surfactant protein D (SP-D) is critical for maintenance of lung homeostasis and provides a first line of defense to pathogens at mucosal surfaces. Polymorphisms in the SP-D-encoding gene SFTPD have been associated with chronic obstructive pulmonary disease and ulcerative colitis. Identification of the immunoreceptors that bind SP-D is essential for understanding its contribution to lung homeostasis and mucosal defense. We located a putative binding motif for the osteoclast-associated receptor (OSCAR) within the SP-D collagenous domain. An OSCAR-Fc fusion protein specifically bound to the collagenous region of recombinant SP-D and captured native SP-D from human bronchoalveolar lavage. OSCAR localized in an intracellular compartment of alveolar macrophages together with SP-D. Moreover, we found OSCAR on the surface of interstitial lung and blood CCR2(+) inflammatory monocytes, which secreted TNF-α when exposed to SP-D in an OSCAR-dependent fashion. OSCAR and SP-D did not exclusively colocalize in lung, as they were also highly expressed in atherosclerotic plaques of human aorta, supporting a role for this interaction in atherosclerosis. Our results identify the OSCAR:SP-D interaction as a potential therapeutic target in chronic inflammatory diseases of the lung as well as other diseases involving tissue accumulation of SP-D, infiltration of inflammatory monocytes, and release of TNF-α.
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Inflamación/metabolismo , Monocitos/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Receptores CCR2/metabolismo , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Línea Celular , Membrana Celular/metabolismo , Biología Computacional , Células Espumosas/inmunología , Células Espumosas/metabolismo , Células Espumosas/patología , Expresión Génica , Humanos , Inflamación/inmunología , Inflamación/patología , Espacio Intracelular/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Monocitos/inmunología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.
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Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Proteína D Asociada a Surfactante Pulmonar/genética , Animales , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Resistencia a la Enfermedad , Perros , Femenino , Interacciones Huésped-Patógeno , Humanos , Gripe Humana/inmunología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos DBA , Mutación Missense , Pandemias , Unión Proteica , Proteína D Asociada a Surfactante Pulmonar/química , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Carga ViralRESUMEN
Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. Interactions of SP-D with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with that of full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have determined crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). On the basis of the D325A+R343V-Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding caused by additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V because of alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D-HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Modelos Moleculares , Proteína D Asociada a Surfactante Pulmonar/química , Adhesividad , Sustitución de Aminoácidos , Sitios de Unión , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Subtipo H3N2 del Virus de la Influenza A/química , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Viabilidad Microbiana , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Conformación Proteica , Proteína D Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27.
Asunto(s)
Proteínas Portadoras/metabolismo , Pulmón/inmunología , Proteína D Asociada a Surfactante Pulmonar/inmunología , Virus Vaccinia/metabolismo , Vaccinia/inmunología , Proteínas Virales de Fusión/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/virología , Masculino , Proteínas de la Membrana , Ratones , Ratones Noqueados , Unión Proteica , Proteína D Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Vaccinia/genética , Vaccinia/metabolismo , Vaccinia/virología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
LL-37, the only human cathelicidin, is a cationic antimicrobial peptide with antibacterial and antifungal activity. LL-37 is released from neutrophil granules and produced by epithelial cells. It has been implicated in host defence against influenza A virus (IAV) in recent studies. We now demonstrate dose-related neutralizing activity of LL-37 against several seasonal and mouse-adapted IAV strains. The ability of LL-37 to inhibit these IAV strains resulted mainly from direct effects on the virus, since pre-incubation of virus with LL-37 was needed for optimal inhibition. LL-37 bound high-density lipoprotein (HDL), and pre-incubation of LL-37 with human serum or HDL reduced its antiviral activity. LL-37 did not inhibit viral association with epithelial cells as assessed by quantitative RT-PCR or confocal microscopy. This finding contrasted with results obtained with surfactant protein D (SP-D). Unlike collectins or human neutrophil defensins (HNPs), LL-37 did not induce viral aggregation under electron microscopy. In the electron microscopy studies, LL-37 appeared to cause disruption of viral membranes. LL-37 had additive antiviral activity when combined with other innate inhibitors like SP-D, surfactant protein A and HNPs. Unlike HNPs, LL-37 did not bind SP-D significantly. These findings indicate that LL-37 contributes to host defence against IAV through a mechanism distinct from that of SP-D and HNPs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Defensinas/metabolismo , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Virus de la Influenza A/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Péptidos Catiónicos Antimicrobianos/inmunología , Células CHO , Colectinas/inmunología , Colectinas/metabolismo , Cricetinae , Cricetulus , Defensinas/inmunología , Perros , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Subtipo H3N2 del Virus de la Influenza A/inmunología , Virus de la Influenza A/inmunología , Lipoproteínas HDL/inmunología , Lipoproteínas HDL/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/virología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Proteína A Asociada a Surfactante Pulmonar/inmunología , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Proteína D Asociada a Surfactante Pulmonar/inmunología , CatelicidinasRESUMEN
Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.