EPITHELIAL REMODELING AND MICROBIAL DYSBIOSIS IN THE LOWER RESPIRATORY TRACT OF VITAMIN A-DEFICIENT MOUSE LUNGS.

The World Health Organization identified vitamin A deficiency (VAD) as a major public health issue in low-income communities and developing countries, while additional studies have shown dietary VAD leads to various lung pathologies. Once believed to be sterile, research now shows that transient microbial communities exist within healthy lungs and are often dysregulated in patients suffering from malnourishment, respiratory infections, and disease. The inability to parse vitamin A-mediated mechanisms from other metabolic mechanisms in humans with pathogenic endotypes, as well as the lack of data investigating how VAD affects the lung microbiome, remains a significant gap in the field. To address this unmet need, we compared molecular, metatranscriptomic, and morphometric data to identify how dietary VAD affects the lung as well as the lung microbiome. Our research shows structural and functional alterations in host-microbe-diet interactions in VAD lungs compared to vitamin A-sufficient (VAS) lungs; these changes are associated with epithelial remodeling, a breakdown in mucociliary clearance, microbial imbalance, and altered microbial colonization patterns after 8 weeks of vitamin A deficient diet. These findings confirm vitamin A is critical for lung homeostasis and provide mechanistic insights that could be valuable for the prevention of respiratory infections and disease.

Multicenter evaluation of the Selux Next-Generation Phenotyping antimicrobial susceptibility testing system.

The Selux Next-Generation Phenotyping (NGP) system (Charlestown, MA) is a new antimicrobial susceptibility testing system that utilizes two sequential assays performed on all wells of doubling dilution series to determine MICs. A multicenter evaluation of the performance of the Selux NGP system compared with reference broth microdilution was conducted following FDA recommendations and using FDA-defined breakpoints. A total of 2,488 clinical and challenge isolates were included; gram-negative isolates were tested against 24 antimicrobials, and gram-positive isolates were tested against 15 antimicrobials. Data is provided for all organism-antimicrobial combinations evaluated, including those that did and did not meet FDA performance requirements. Overall very major error and major error rates were less than 1% (31/3,805 and 107/15,606, respectively), essential agreement and categorical agreement were >95%, reproducibility was ≥95%, and the average time-to-result (from time of assay start to time of MIC result) was 5.65 hours.

Sequestration of gut pathobionts in intraluminal casts, a mechanism to avoid dysregulated T cell activation by pathobionts.

T cells that express the transcription factor RORγ, regulatory (Treg), or conventional (Th17) are strongly influenced by intestinal symbionts. In a genetic approach to identify mechanisms underlying this influence, we performed a screen for microbial genes implicated, in germfree mice monocolonized with Escherichia coli Nissle. The loss of capsule-synthesis genes impaired clonal expansion and differentiation of intestinal RORγ+ T cells. Mechanistic exploration revealed that the capsule-less mutants remained able to induce species-specific immunoglobulin A (IgA) and were highly IgA-coated. They could still trigger myeloid cells, and more effectively damaged epithelial cells in vitro. Unlike wild-type microbes, capsule-less mutants were mostly engulfed in intraluminal casts, large agglomerates composed of myeloid cells extravasated into the gut lumen. We speculate that sequestration in luminal casts of potentially harmful microbes, favored by IgA binding, reduces the immune system's actual exposure, preserving host-microbe equilibrium. The variable immunostimulation by microbes that has been charted in recent years may not solely be conditioned by triggering molecules or metabolites but also by physical limits to immune system exposure.

A Disease Identification Algorithm for Medical Crowdfunding Campaigns: Validation Study.

BACKGROUND: Web-based crowdfunding has become a popular method to raise money for medical expenses, and there is growing research interest in this topic. However, crowdfunding data are largely composed of unstructured text, thereby posing many challenges for researchers hoping to answer questions about specific medical conditions. Previous studies have used methods that either failed to address major challenges or were poorly scalable to large sample sizes. To enable further research on this emerging funding mechanism in health care, better methods are needed. OBJECTIVE: We sought to validate an algorithm for identifying 11 disease categories in web-based medical crowdfunding campaigns. We hypothesized that a disease identification algorithm combining a named entity recognition (NER) model and word search approach could identify disease categories with high precision and accuracy. Such an algorithm would facilitate further research using these data. METHODS: Web scraping was used to collect data on medical crowdfunding campaigns from GoFundMe (GoFundMe Inc). Using pretrained NER and entity resolution models from Spark NLP for Healthcare in combination with targeted keyword searches, we constructed an algorithm to identify conditions in the campaign descriptions, translate conditions to International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) codes, and predict the presence or absence of 11 disease categories in the campaigns. The classification performance of the algorithm was evaluated against 400 manually labeled campaigns. RESULTS: We collected data on 89,645 crowdfunding campaigns through web scraping. The interrater reliability for detecting the presence of broad disease categories in the campaign descriptions was high (Cohen κ: range 0.69-0.96). The NER and entity resolution models identified 6594 unique (276,020 total) ICD-10-CM codes among all of the crowdfunding campaigns in our sample. Through our word search, we identified 3261 additional campaigns for which a medical condition was not otherwise detected with the NER model. When averaged across all disease categories and weighted by the number of campaigns that mentioned each disease category, the algorithm demonstrated an overall precision of 0.83 (range 0.48-0.97), a recall of 0.77 (range 0.42-0.98), an F1 score of 0.78 (range 0.56-0.96), and an accuracy of 95% (range 90%-98%). CONCLUSIONS: A disease identification algorithm combining pretrained natural language processing models and ICD-10-CM code-based disease categorization was able to detect 11 disease categories in medical crowdfunding campaigns with high precision and accuracy.

A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune-epithelial interface.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

Profound Treg perturbations correlate with COVID-19 severity.

The hallmark of severe COVID-19 is an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We hypothesized that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, correlating with poor outcomes. These Tregs showed a distinct transcriptional signature, with overexpression of several suppressive effectors, but also proinflammatory molecules like interleukin (IL)-32, and a striking similarity to tumor-infiltrating Tregs that suppress antitumor responses. Most marked during acute severe disease, these traits persisted somewhat in convalescent patients. A screen for candidate agents revealed that IL-6 and IL-18 may individually contribute different facets of these COVID-19-linked perturbations. These results suggest that Tregs may play nefarious roles in COVID-19, by suppressing antiviral T cell responses during the severe phase of the disease, and by a direct proinflammatory role.

Retinoic Acid Signaling and Development of the Respiratory System.

Retinoic acid (RA), the bioactive metabolite of vitamin A (VA), has long been recognized as a critical regulator of the development of the respiratory system. During embryogenesis, RA signaling is involved in the development of the trachea, airways, lung, and diaphragm. During postnatal life, RA continues to impact respiratory health. Disruption of RA activity during embryonic development produces dramatic phenotypes in animal models and human diseases, including tracheoesophageal fistula, tracheomalacia, congenital diaphragmatic hernia (CDH), and lung agenesis or hypoplasia. Several experimental methods have been used to target RA pathways during the formation of the embryonic lung. These have been performed in different animal models using gain- and loss-of-function strategies and dietary, pharmacologic, and genetic approaches that deplete retinoid stores or disrupt retinoid signaling. Experiments utilizing these methods have led to a deeper understanding of RA's role as an important signaling molecule that influences all stages of lung development. Current research is uncovering RA cross talk interactions with other embryonic signaling factors, such as fibroblast growth factors, WNT, and transforming growth factor-beta.

Transcriptional landscape of pulmonary lymphatic endothelial cells during fetal gestation.

The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to collect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3+/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, E18.5). Gene expression profiling was performed using whole-genome microarrays, and 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregulated at E18.5 were significantly enriched in genes with functional annotations corresponding to innate immune response, positive regulation of angiogenesis, complement & coagulation cascade, ECM/cell-adhesion, and lipid metabolism. Gene Set Enrichment Analysis identified several pathways coordinately upregulated during late gestation, the strongest of which was the type-I IFN-α/ß signaling pathway. Upregulation of canonical interferon target genes was confirmed by qRT-PCR and in situ hybridization in E18.5 PLECs. We also identified transcriptional events consistent with a prenatal PLEC maturation program. This PLEC-specific program included individual genes (Ch25h, Itpkc, Pcdhac2 and S1pr3) as well as a set of chemokines and genes containing an NF-κB binding site in their promoter. Overall, this work reveals transcriptional insights into the genes, signaling pathways and biological processes associated with pulmonary lymphatic maturation in the fetal lung.

Microplate-based surface area assay for rapid phenotypic antibiotic susceptibility testing.

Rapid delivery of proper antibiotic therapies to infectious disease patients is essential for improving patient outcomes, decreasing hospital lengths-of-stay, and combating the antibiotic resistance epidemic. Antibiotic stewardship programs are designed to address these issues by coordinating hospital efforts to rapidly deliver the most effective antibiotics for each patient, which requires bacterial identification and antimicrobial susceptibility testing (AST). Despite the clinical need for fast susceptibility testing over a wide range of antibiotics, conventional phenotypic AST requires overnight incubations, and new rapid phenotypic AST platforms restrict the number of antibiotics tested for each patient. Here, we introduce a novel approach to AST based on signal amplification of bacterial surfaces that enables phenotypic AST within 5 hours for non-fastidious bacteria. By binding bacterial surfaces, this novel method allows more accurate measurements of bacterial replication in instances where organisms filament or swell in response to antibiotic exposure. Further, as an endpoint assay performed on standard microplates, this method should enable parallel testing of more antibiotics than is currently possible with available automated systems. This technology has the potential to revolutionize clinical practice by providing rapid and accurate phenotypic AST data for virtually all available antibiotics in a single test.

Retinoic acid signaling is essential for airway smooth muscle homeostasis.

Airway smooth muscle (ASM) is a dynamic and complex tissue involved in regulation of bronchomotor tone, but the molecular events essential for the maintenance of ASM homeostasis are not well understood. Observational and genome-wide association studies in humans have linked airway function to the nutritional status of vitamin A and its bioactive metabolite retinoic acid (RA). Here, we provide evidence that ongoing RA signaling is critical for the regulation of adult ASM phenotype. By using dietary, pharmacologic, and genetic models in mice and humans, we show that (a) RA signaling is active in adult ASM in the normal lung, (b) RA-deficient ASM cells are hypertrophic, hypercontractile, profibrotic, but not hyperproliferative, (c) TGF-ß signaling, known to cause ASM hypertrophy and airway fibrosis in human obstructive lung diseases, is hyperactivated in RA-deficient ASM, (d) pharmacologic and genetic inhibition of the TGF-ß activity in ASM prevents the development of the aberrant phenotype induced by RA deficiency, and (e) the consequences of transient RA deficiency in ASM are long-lasting. These results indicate that RA signaling actively maintains adult ASM homeostasis, and disruption of RA signaling leads to aberrant ASM phenotypes similar to those seen in human chronic airway diseases such as asthma.

Expression of Piwi protein MIWI2 defines a distinct population of multiciliated cells.

P-element-induced wimpy testes (Piwi) proteins are known for suppressing retrotransposon activation in the mammalian germline. However, whether Piwi protein or Piwi-dependent functions occur in the mammalian soma is unclear. Contrary to germline-restricted expression, we observed that Piwi-like Miwi2 mRNA is indeed expressed in epithelial cells of the lung in adult mice and that it is induced during pneumonia. Further investigation revealed that MIWI2 protein localized to the cytoplasm of a discrete population of multiciliated airway epithelial cells. Isolation and next-generation sequencing of MIWI2-positive multiciliated cells revealed that they are phenotypically distinct from neighboring MIWI2-negative multiciliated cells. Mice lacking MIWI2 exhibited an altered balance of airway epithelial cells, demonstrating fewer multiciliated cells and an increase in club cells. During pneumococcal pneumonia, Miwi2-deficient mice exhibited increased expression of inflammatory mediators and increased immune cell recruitment, leading to enhanced bacterial clearance. Taken together, our data delineate MIWI2-dependent functions outside of the germline and demonstrate the presence of distinct subsets of airway multiciliated cells that can be discriminated by MIWI2 expression. By demonstrating roles for MIWI2 in airway cell identity and pulmonary innate immunity, these studies elucidate unanticipated physiological functions for Piwi proteins in somatic tissues.

Stem Cells in Lung Injury and Repair.

In this review, we summarize the recent literature on the biology of endogenous stem cells in adult lung injury repair. We focus on in vivo studies in mice with an emphasis on data generated using cell-specific Cre-dependent lineage-tracing systems. These studies provide new information on the identification of lung stem cells, their hierarchical relationships, the plasticity of their behavior in different types of injury, and the molecular signals that control their fates. Although most of this work has been on epithelial hierarchies, we expect that further development of robust genetic tools will foster meaningful investigations into how nonepithelial cell populations are controlled during lung injury repair in adults. The ultimate challenge will be to translate these findings to the pathogenesis and treatment of human lung diseases.

Culture of mouse embryonic foregut explants.

The ability to culture embryonic organ rudiments and follow their development ex vivo has helped to understand how tissues are constructed and what cellular and biological events are important in this process. Here we outline a technique for isolation and ex vivo growth of foregut explants from E8.5 mouse embryos. This technique serves as a reliable tool for the analysis of the morphogenetic processes and signaling networks during early development of foregut derivatives, such as the lungs.

Prenatal retinoid deficiency leads to airway hyperresponsiveness in adult mice.

There is increasing evidence that vitamin A deficiency in utero correlates with abnormal airway smooth muscle (SM) function in postnatal life. The bioactive vitamin A metabolite retinoic acid (RA) is essential for formation of the lung primordium; however, little is known about the impact of early fetal RA deficiency on postnatal lung structure and function. Here, we provide evidence that during murine lung development, endogenous RA has a key role in restricting the airway SM differentiation program during airway formation. Using murine models of pharmacological, genetic, and dietary vitamin A/RA deficiency, we found that disruption of RA signaling during embryonic development consistently resulted in an altered airway SM phenotype with markedly increased expression of SM markers. The aberrant phenotype persisted postnatally regardless of the adult vitamin A status and manifested as structural changes in the bronchial SM and hyperresponsiveness of the airway without evidence of inflammation. Our data reveal a role for endogenous RA signaling in restricting SM differentiation and preventing precocious and excessive SM differentiation when airways are forming.

Cutting edge: Crohn's disease-associated Nod2 mutation limits production of proinflammatory cytokines to protect the host from Enterococcus faecalis-induced lethality.

Nucleotide-binding oligomerization domain 2 (Nod2) mutations including L1007fsinsC are associated with the development of Crohn's disease (CD). These CD-associated Nod2 mutations are common in healthy white populations, suggesting that they may confer some protective function, but experimental evidence is lacking. Using a mouse strain that expresses Nod2(2939iCstop), the equivalent of the L1007fsinsC mutation, we found that macrophages homozygous for Nod2(2939iCstop) are impaired in the recognition of muramyl dipeptide and Enterococcus faecalis, a commensal bacterium that is a common cause of sepsis-associated lethality in humans. Notably, Nod2 deficiency and homozygocity for Nod2(2939iCstop) were associated with reduced production of TNF-α and IL-6 and lethality after systemic infection with E. faecalis despite normal bacteria loads. Consistently, inhibition of TNF-α signaling protected wild-type mice from E. faecalis-induced lethality. These results suggest that the same Nod2 mutation can increase the susceptibility to CD, but also protect the host from systemic infection by a common enteric bacterium.

A retinoic acid-dependent network in the foregut controls formation of the mouse lung primordium.

The developmental abnormalities associated with disruption of signaling by retinoic acid (RA), the biologically active form of vitamin A, have been known for decades from studies in animal models and humans. These include defects in the respiratory system, such as lung hypoplasia and agenesis. However, the molecular events controlled by RA that lead to formation of the lung primordium from the primitive foregut remain unclear. Here, we present evidence that endogenous RA acts as a major regulatory signal integrating Wnt and Tgfbeta pathways in the control of Fgf10 expression during induction of the mouse primordial lung. We demonstrated that activation of Wnt signaling required for lung formation was dependent on local repression of its antagonist, Dickkopf homolog 1 (Dkk1), by endogenous RA. Moreover, we showed that simultaneously activating Wnt and repressing Tgfbeta allowed induction of both lung buds in RA-deficient foreguts. The data in this study suggest that disruption of Wnt/Tgfbeta/Fgf10 interactions represents the molecular basis for the classically reported failure to form lung buds in vitamin A deficiency.

Mechanisms of lung development: contribution to adult lung disease and relevance to chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) results in major remodeling of the distal airspaces and changes in the differentiation profile of the airway epithelium. The cellular and molecular mechanisms involved in initiation and progression of this disease are little understood. Although environmental factors, including cigarette smoke, have been directly implicated in the pathogenesis of COPD, genetic risk factors also appear to play a fundamental role in the individual's susceptibility to this disease. Lung development depends on precise coordination of signals, such as fibroblast growth factors (Fgf), Sonic Hedgehog (Shh), retinoic acid, Notch, and Tgf beta. Dramatic changes in the pattern of branching and differentiation of the lung epithelium results from disruption of these signals in genetically altered mice. Recent studies, including whole-genome expression and genome-wide association analyses, suggest that some molecular regulators originally described in developmental processes may be altered in patients with COPD. Whether disturbances in the molecular and cellular events mediated by these genes during development participate in the initiation or exacerbation of COPD, needs further investigation. The role of selected pathways, including Sonic hedgehog, Notch, retinoid, and Tgf beta in the developing lung and the potential association with COPD are discussed.

Gamma-secretase activation of notch signaling regulates the balance of proximal and distal fates in progenitor cells of the developing lung.

Little is known about the mechanisms by which the lung epithelial progenitors are initially patterned and how proximal-distal boundaries are established and maintained when the lung primordium forms and starts to branch. Here we identified a number of Notch pathway components in respiratory progenitors of the early lung, and we investigated the role of Notch in lung pattern formation. By preventing gamma-secretase cleavage of Notch receptors, we have disrupted global Notch signaling in the foregut and in the lung during the initial stages of murine lung morphogenesis. We demonstrate that Notch signaling is not necessary for lung bud initiation; however, Notch is required to maintain a balance of proximal-distal cell fates at these early stages. Disruption of Notch signaling dramatically expands the population of distal progenitors, altering morphogenetic boundaries and preventing formation of proximal structures. Our data suggest a novel mechanism in which Notch and fibroblast growth factor signaling interact to control the proximal-distal pattern of forming airways in the mammalian lung.

Inhibition of Tgf beta signaling by endogenous retinoic acid is essential for primary lung bud induction.

Disruption of retinoic acid (RA) signaling during early development results in severe respiratory tract abnormalities, including lung agenesis. Previous studies suggest that this might result from failure to selectively induce fibroblast growth factor 10 (Fgf10) in the prospective lung region of the foregut. Little is known about the RA-dependent pathways present in the foregut that may be crucial for lung formation. By performing global gene expression analysis of RA-deficient foreguts from a genetic [retinaldehyde dehydrogenase 2 (Raldh2)-null] and a pharmacological (BMS493-treated) mouse model, we found upregulation of a large number of Tgfbeta targets. Increased Smad2 phosphorylation further suggested that Tgfbeta signaling was hyperactive in these foreguts when lung agenesis was observed. RA rescue of the lung phenotype was associated with low levels of Smad2 phosphorylation and downregulation of Tgfbeta targets in Raldh2-null foreguts. Interestingly, the lung defect that resulted from RA-deficiency could be reproduced in RA-sufficient foreguts by hyperactivating Tgfbeta signaling with exogenous TGF beta 1. Preventing activation of endogenous Tgfbeta signaling with a pan-specific TGFbeta-blocking antibody allowed bud formation and gene expression in the lung field of both Raldh2-null and BMS493-treated foreguts. Our data support a novel mechanism of RA-Tgfbeta-Fgf10 interactions in the developing foregut, in which endogenous RA controls Tgfbeta activity in the prospective lung field to allow local expression of Fgf10 and induction of lung buds.

Distinct roles for retinoic acid receptors alpha and beta in early lung morphogenesis.

Retinoic acid (RA) signaling is required for normal development of multiple organs. However, little is known about how RA influences the initial stages of lung development. Here, we used a combination of genetic, pharmacological and explant culture approaches to address this issue, and to investigate how signaling by different RA receptors (RAR) mediates the RA effects. We analyzed initiation of lung development in retinaldehyde dehydrogenase-2 (Raldh2) null mice, a model in which RA signaling is absent from the foregut from its earliest developmental stages. We provide evidence that RA is dispensable for specification of lung cell fate in the endoderm. By using synthetic retinoids to selectively activate RAR alpha or beta signaling in this model, we demonstrate novel and unique functions of these receptors in the early lung. We show that activation of RAR beta, but not alpha, induces expression of the fibroblast growth factor Fgf10 and bud morphogenesis in the lung field. Similar analysis of wild type foregut shows that endogenous RAR alpha activity is required to maintain overall RA signaling, and to refine the RAR beta effects in the lung field. Our data support the idea that balanced activation of RAR alpha and beta is critical for proper lung bud initiation and endodermal differentiation.