Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections. We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modelling and network correlation analyses identified 118 differentially expressed proteins, significant through three complementary analytical pipelines. Candidate biomarkers were subsequently analysed in two validation cohorts of differing ethnicity using antibody-based proximity extension assays. TB-specific host biomarkers were confirmed. A six-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14 and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts. This biomarker panel exceeds the World Health Organisation Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.
Hannah F. Schiff, Naomi F. Walker, Cesar Ugarte-Gil, Marc Tebruegge, Antigoni Manousopoulou, Spiros D. Garbis, Salah Mansour, Pak Ho Wong, Gabrielle Rockett, Paolo Piazza, Mahesan Niranjan, Andres F. Vallejo, Christopher H. Woelk, Robert J. Wilkinson, Liku B. Tezera, Diana Garay-Baquero, Paul Elkington
IL-33 is a cytokine central to type 2 immune pathology in chronic airway disease. This cytokine is abundantly expressed in the respiratory epithelium and increased in disease, but how expression is regulated is undefined. Here we show that increased IL33 expression occurs from multiple noncanonical promoters in human chronic obstructive pulmonary disease (COPD), and it facilitates production of alternatively spliced isoforms in airway cells. We found that phorbol 12-myristate 13-acetate (PMA) can activate IL33 promoters through protein kinase C in primary airway cells and lines. Transcription factor (TF) binding arrays combined with RNA interference identified activator protein (AP) TFs as regulators of baseline and induced IL33 promoter activity. ATAC-Seq and ChIP-PCR identified chromatin accessibility and differential TF binding as additional control points for transcription from noncanonical promoters. In support of a role for these TFs in COPD pathogenesis, we found that AP-2 (TFAP2A, TFAP2C) and AP-1 (FOS and JUN) family members are upregulated in human COPD specimens. This study implicates integrative and pioneer TFs in regulating IL33 promoters and alternative splicing in human airway basal cells. Our work reveals a potentially novel approach for targeting IL-33 in development of therapeutics for COPD.
Heather E. Raphael, Ghandi F. Hassan, Omar A. Osorio, Lucy S. Cohen, Morgan D. Payne, Ella Katz-Kiriakos, Ishana Tata, Jamie Hicks, Derek E. Byers, Bo Zhang, Jen Alexander-Brett
Hypercapnia, elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions, and that elevated CO2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3), a mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila, is expressed in mouse and human macrophages. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury and mortality in hypercapnic mice infected with influenza A virus. Our results establish Zfhx3 as the first known mammalian mediator of CO2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung disease.
S. Marina Casalino-Matsuda, Fei Chen, Francisco J. Gonzalez-Gonzalez, Hiroaki Matsuda, Aisha Nair, Hiam Abdala-Valencia, G.R. Scott Budinger, Jin-Tang Dong, Greg J. Beitel, Peter H.S. Sporn
There are no therapies to prevent emphysema progression. Chymotrypsin-like elastase 1 (CELA1) is a serine protease that binds and cleaves lung elastin in a stretch-dependent manner and is required for emphysema in a murine antisense oligonucleotide model of α-1 antitrypsin (AAT) deficiency. This study tested whether CELA1 is important in strain-mediated lung matrix destruction in non–AAT-deficient emphysema and the efficacy of CELA1 neutralization. Airspace simplification was quantified after administration of tracheal porcine pancreatic elastase (PPE), after 8 months of cigarette smoke (CS) exposure, and in aging. In all 3 models, Cela1–/– mice had less emphysema and preserved lung elastin despite increased lung immune cells. A CELA1-neutralizing antibody was developed (KF4), and it inhibited stretch-inducible lung elastase in ex vivo mouse and human lung and immunoprecipitated CELA1 from human lung. In mice, systemically administered KF4 penetrated lung tissue in a dose-dependent manner and 5 mg/kg weekly prevented emphysema in the PPE model with both pre- and postinjury initiation and in the CS model. KF4 did not increase lung immune cells. CELA1-mediated lung matrix remodeling in response to strain is an important contributor to postnatal airspace simplification, and we believe that KF4 could be developed as a lung matrix–stabilizing therapy in emphysema.
Mohit Ojha, Noah J. Smith, Andrew J. Devine, Rashika Joshi, Emily M. Goodman, Qiang Fan, Richard Schuman, Aleksey Porollo, J. Michael Wells, Ekta Tiwary, Matthew R. Batie, Jerilyn Gray, Hitesh Deshmukh, Michael T. Borchers, Samuel A. Ammerman, Brian M. Varisco
BACKGROUND. Information about the size, airway location, and longitudinal behavior of mucus plugs in asthma is needed to understand their role in mechanisms of airflow obstruction and to rationally design muco-active treatments. METHODS. Computed tomography (CT) lung scans from 57 asthma patients were analyzed to quantify mucus plug size and airway location, and paired CT scans obtained 3 years apart were analyzed to determine plug behavior over time. Radiologist annotations of mucus plugs were incorporated in an image-processing pipeline to generate size and location information that was related to measures of airflow. RESULTS. The length distribution of 778 annotated mucus plugs was multimodal and a 12 mm length defined short (“stubby”, ≤12 mm) and long (“stringy”, >12 mm) plug phenotypes. High mucus plug burden was disproportionately attributable to stringy mucus plugs. Mucus plugs localized predominantly to airway generations 6 to 9, and 47% of plugs in baseline scans, persisted in the same airway for three years, and fluctuated in length and volume. Mucus plugs in larger proximal generations had greater effects on spirometry measures than plugs in smaller distal generations, and a model of airflow that estimates the increased airway resistance attributable to plugs predicted higher impact for proximal and more numerous mucus plugs. CONCLUSIONS. Persistent mucus plugs in proximal airway generations occur in asthma and demonstrate a stochastic process of formation and resolution over time. Proximal airway mucus plugs are consequential for airflow and are in locations amenable to treatment by inhaled muco-active drugs or bronchoscopy. TRIAL REGISTRATION. Clinicaltrials.gov NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01759186, NCT01716494, and NCT01760915 FUNDING. NIH Grants: R01 HL080414, UG1 HL139106, P01 HL107202, U01 HL146002, U10 HL109172, U10 HL109168, U10 HL109152, U10 HL109257, U10 HL109146, U10 HL109250, U10 HL109164, U10 109086, and T32 HL007185, F32 HL162422. The following companies provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up: AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi–Genzyme– Regeneron, and TEVA. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative.
Brendan K. Huang, Brett M. Elicker, Travis S. Henry, Kimberly G. Kallianos, Lewis D. Hahn, Monica Tang, Franklin Heng, Charles E. McCulloch, Nirav R. Bhakta, Sharmila Majumdar, Jiwoong Choi, Loren C. Denlinger, Sean B. Fain, Annette T. Hastie, Eric A. Hoffman, Elliot Israel, Nizar N. Jarjour, Bruce D. Levy, David T. Mauger, Kaharu Sumino, Sally E. Wenzel, Mario Castro, Prescott G. Woodruff, John V. Fahy
Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation and airways hyperresponsiveness which is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase (DCLK)-1+ tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57Bl/6 mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ positive tuft cells in the large airways. Compared to wild-type mice, RV-infected Pou2f3–/– mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early life viral infections could potentiate type 2 inflammatory responses to future infections.
Yiran Li, Mingyuan Han, Shilpi Singh, Haley A. Breckenridge, Jordan E. Kreger, Claudia C. Stroupe, Daniel A. Sawicky, Shiuhyang Kuo, Adam M. Goldsmith, Fang Ke, Anukul T. Shenoy, J. Kelley Bentley, Ichiro Matsumoto, Marc B. Hershenson
Cigarette smoking is associated with a higher risk of ICU admissions among flu patients. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice pre-exposed to CS but not room air for four weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS and flu exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest for the first time that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS induced severe flu.
Tomasz W. Kaminski, Tomasz Brzoska, Xiuying Li, Ravi Vats, Omika Katoch, Rikesh K. Dubey, Kamal Bagale, Simon C. Watkins, Bryan J. McVerry, Tirthadipa Pradhan-Sundd, Lianghui Zhang, Keven M. Robinson, Toru Nyunoya, Prithu Sundd
Pulmonary fibrosis is a chronic and often fatal disease. The pathogenesis is characterized by aberrant repair of lung parenchyma resulting in loss of physiological homeostasis, respiratory failure and death. The immune response in pulmonary fibrosis is dysregulated. The gut microbiome is a key regulator of immunity. The role of the gut microbiome in regulating the pulmonary immunity in lung fibrosis is poorly understood. Here, we determine the impact of gut microbiota on pulmonary fibrosis in C57BL/6 mice derived from different vendors (C57BL/6J and C57BL/6NCrl). We use germ free models, fecal microbiota transplantation and cohousing to transmit gut microbiota. Metagenomic studies of feces establish keystone species between sub-strains. Pulmonary fibrosis is microbiota dependent in C57BL/6 mice. Gut microbiota are distinct by β diversity (PERMANOVA P<0.001) and α diversity (P<0.0001). Mortality and lung fibrosis are attenuated in C57BL/6NCrl mice. Elevated CD4+ IL-10+ T cells and lower IL-6 occur in C57BL/6NCrl mice. Horizontal transmission of microbiota by cohousing attenuates mortality in C57BL/6J mice and promotes a transcriptionally altered pulmonary immunity. Temporal changes in lung and gut microbiota demonstrates that gut microbiota contribute largely to immunological phenotype. Key regulatory gut microbiota contribute to lung fibrosis generating rationale for human studies.
Stephen J. Gurczynski, Jay H. Lipinski, Joshua Y. Strauss, Shafiul Alam, Gary B. Huffnagle, Piyush Ranjan, Lucy H. Kennedy, Bethany B. Moore, David N. O'Dwyer
The lymphatic vasculature is the natural pathway for the resolution of inflammation, while the role of pulmonary lymphatic drainage function in sepsis-induced acute respiratory distress syndrome (ARDS) remains poorly characterized. In this study, Indocyanine green (ICG)-Near Infrared (NIR) lymphatic living imaging was performed to examine pulmonary lymphatic drainage function in septic mice models. We found that the pulmonary lymphatic drainage was impaired owing to the damaged lymphatic structure in sepsis-induced ARDS. Moreover, prior lymphatic defects by blocking vascular endothelial growth factor receptor-3 (VEGFR3), worsened sepsis-induced lymphatic dysfunction and inflammation. The post-treatment of vascular endothelial growth factor-C (Cys156Ser) (VEGF-C156S), a ligand of VEGFR3, ameliorated lymphatic drainage through rejuvenating lymphatics to reduce the pulmonary edema and promote pulmonary macrophages and neutrophils to drain to pretracheal lymph nodes (pLNs). Meanwhile, VEGF-C156S post-treatment reversed sepsis-inhibited C-C motif chemokine ligand 21 (CCL21), which co-localizes with the pulmonary lymphatic vessels. Furthermore, the advantages of VEGF-C156S on the drainage of inflammatory cells and edema fluid were abolished by blocking VEGFR3 or CCL21. These results suggest that efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS. Our findings offer a novel therapeutic approach to sepsis-induced ARDS by promoting lymphatic drainage function.
Pu-hong Zhang, Wen-wu Zhang, Shun-shun Wang, Cheng-hua Wu, Yang-dong Ding, Xin-yi Wu, Fang Gao Smith, Yu Hao, Sheng-wei Jin
Chronic lung allograft dysfunction (CLAD) is a major complication after lung transplantation that results from a complex interplay of innate inflammatory and alloimmune factors, culminating in parenchymal and/or obliterative airway fibrosis. Excessive IL-17A signaling and chronic inflammation have been recognized as key factors in these pathological processes. Herein, we developed a model of repeated airway inflammation in mouse minor alloantigen-mismatched single-lung transplantation. Repeated intratracheal LPS instillations augmented pulmonary IL-17A expression. LPS also increased acute rejection, airway epithelial damage, and obliterative airway fibrosis, similar to human explanted lung allografts with antecedent episodes of airway infection. We then investigated the role of donor and recipient IL-17 receptor A (IL-17RA) in this context. Donor IL-17RA deficiency significantly attenuated acute rejection and CLAD features, whereas recipient IL-17RA deficiency only slightly reduced airway obliteration in LPS allografts. IL-17RA immunofluorescence positive staining was greater in human CLAD lungs compared with control human lung specimens, with localization to fibroblasts and myofibroblasts, which was also seen in mouse LPS allografts. Taken together, repeated airway inflammation after lung transplantation caused local airway epithelial damage, with persistent elevation of IL-17A and IL-17RA expression and particular involvement of IL-17RA on donor structural cells in development of fibrosis.
Tatsuaki Watanabe, Stephen C. Juvet, Gregory Berra, Jan Havlin, Wenshan Zhong, Kristen Boonstra, Tina Daigneault, Miho Horie, Chihiro Konoeda, Grace Teskey, Zehong Guan, David M. Hwang, Mingyao Liu, Shaf Keshavjee, Tereza Martinu
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