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Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2, that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network, highly upregulated by type 2 (T2) inflammation through the action of interleukin-13, and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally, we define airway responses to common coronavirus infections in children, finding that these infections generate host responses similar to other viral species, including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

The high prevalence of Johne&rsquo, s disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

Air pollution particulate matter 100 genes), which included transcriptional drivers of mucus metaplasia (SPDEF and FOXA3). Exposure to a higher OE dose modified the expression of 1,240 genes and further exacerbated expression responses observed at the moderate dose, including the mucus secretory program. Moreover, the higher OE dose significantly increased the MUC5AC/MUC5B gel-forming mucin expression ratio and strongly downregulated ciliated cell expression programs, including key ciliating cell transcription factors (e.g., FOXJ1 and MCIDAS). Chronic OE stimulation induced mucus metaplasia–like remodeling characterized by increases in MUC5AC(+) secretory cells and MUC5AC mucus secretions. This epithelial remodeling may underlie poor respiratory outcomes associated with high PM(2.5) exposure.

SUMMARY The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function., In Brief Using airway epithelial cell cultures, Jackson et al. show that IL-13, a driver of type 2-high asthma, induces emergent mucus secretory expression states for each cell type. This program universally diminishes innate airway defense, produces a pathologic mucus secretome that arrests mucociliary movement, and is recapitulated in type 2 inflamed children., Graphical Abstract

Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and non-smokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium., Chronic lung diseases are characterized by molecular and cellular composition changes. Here the authors use single-cell RNA sequencing to map cell type-specific changes in human tracheal epithelium related to smoking, and to provide evidence for a tuft-like progenitor for pulmonary neuroendocrine cells and ionocytes.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2, that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network, highly upregulated by type 2 (T2) inflammation through the action of interleukin-13, and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally, we define airway responses to common coronavirus infections in children, finding that these infections generate host responses similar to other viral species, including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes., ACE2 and TMPRSS2 have received recent attention as entry factors for SARS-CoV-2. Here the authors analyze nasal airway transcriptome data from 695 children determining ACE2 and TMPRSS2 expression is induced by viral and type2 inflammation, respectively, and both exhibit eQTLs that vary across world populations.

Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and nonsmokers, we generated a comprehensive atlas of epithelial cell types and states, connected these into lineages, and defined cell-specific responses to smoking. Our analysis inferred multi-state lineages that develop into surface mucus secretory and ciliated cells and contrasted these to the unique lineage and specialization of submucosal gland (SMG) cells. Our analysis also suggests a lineage relationship between tuft, pulmonary neuroendocrine, and the newly discovered CFTR-rich ionocyte cells. Our smoking analysis found that all cell types, including protected stem and SMG populations, are affected by smoking, through both pan-epithelial smoking response networks and hundreds of cell type-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, chronic and ultimately fatal enteritis that affects ruminant populations worldwide. One mode of MAP transmission is oral when young animals ingest bacteria from the collostrum and milk of infected dams. The exposure to raw milk has a dramatic impact on MAP, resulting in a more invasive and virulent phenotype. The MAP1203 gene is upregulated over 28-fold after exposure of the bacterium to milk. In this study, the role of MAP1203 in binding and invasion of the bovine epithelial cells was investigated. By over-expressing the native MAP1203 gene and two clones of deletion mutant in the signal sequence and of missense mutations changing the integrin domain from RGD into RDE, we demonstrate that MAP1203 plays a role in increasing binding in more than 50% and invasion in 35% of bovine MDBK epithelial cells during early phase of infection. Furthermore, results obtained suggest that MAP1203 is a surface-exposed protein in MAP and the signal sequence is required for processing and expression of functional protein on the surface of the bacterium. Using the protein pull-down assay and far-Western blot, we also demonstrate that MAP1203 interacts with the host dihydropyrimidinase-related protein 2 and glyceraldehyde 3-phosphate dehydrogenase proteins, located on the membrane of epithelial cell and involved in the remodeling of the cytoskeleton. Our data suggests that MAP1203 plays a significant role in the initiation of MAP infection of the bovine epithelium.

Mycobacterium avium subsp. hominissuis (M. avium) is a member of the non-tuberculous mycobacteria (NTM), and is a common cause of lung infection in patients with chronic NTM lung conditions. M. avium is an environmental bacterium believed to be transmitted from environmental sources. In this work we used a recently developed model in Caenorhabditis elegans to ask whether M. avium can be transmitted from host-to-host, and the bacterial genes associated with host colonization. Infection of C. elegans was carried out by placing the nematode in cultured with M. avium. Bacteria eliminated from the intestines of infected C. elegans were used to infect naïve nematodes. In parallel experiments, to identify colonization associated genes, a transposon library of M. avium was screened for the ability to bind to HEp-2 mucosal cells. Thirty clones were identified and five selected clones with impaired adherence to HEp-2 epithelial cells were used to infect C. elegans to determine the degree of colonization. It was determined that M. avium eliminated from infected C. elegans were able to colonize a naïve C. elegans with high efficiency. Thirty of the most adherence-deficient M. avium clones obtained from the HEp-2 cell screening were sequenced to identify the location of the transposon. Many of the genes associated with the bacterial cell wall synthesis were shown to be inactivated in the selected mutants. Five out of the 30 bacterial clones were then used to infect C. elegans. All five mutants had impaired ability to colonize C. elegans compared with the wild type bacteria (decrease of 1.5–2.0 logs, p < 0.05). The limitation of this work is that the model can be used for initial screening, but other more complex systems should be used to confirm the findings. C. elegans can be used as a model to test for M. avium adherence/colonization-associated virulence determinants. All the tested adherence-deficient clones that were examined had impaired ability to colonize the host C. elegans, and some can be potentially used to prevent colonization.

Respiratory illness caused by viral infection is associated with the development and exacerbation of childhood asthma. Little is known about the effects of respiratory viral infections in the absence of illness. Using quantitative PCR (qPCR) for common respiratory viruses and for two genes known to be highly upregulated in viral infections (CCL8/CXCL11), we screened 92 asthmatic and 69 healthy children without illness for respiratory virus infections.We found 21 viral qPCR-positive and 2 suspected virus-infected subjects with high expression of CCL8/CXCL11. We applied a dual RNA-seq workflow to these subjects, together with 25 viral qPCR-negative subjects, to compare qPCR with sequencing-based virus detection and to generate the airway transcriptome for analysis. RNA-seq virus detection achieved 86% sensitivity when compared to qPCR-based screening. We detected additional respiratory viruses in the two CCL8/CXCL11-high subjects and in two of the qPCR-negative subjects. Viral read counts varied widely and were used to stratify subjects into Virus-High and Virus-Low groups. Examination of the host airway transcriptome found that the Virus-High group was characterized by immune cell airway infiltration, downregulation of cilia genes, and dampening of type 2 inflammation. Even the Virus-Low group was differentiated from the No-Virus group by 100 genes, some involved in eIF2 signaling.Respiratory virus infection without illness is not innocuous but may determine the airway function of these subjects by driving immune cell airway infiltration, cellular remodeling, and alteration of asthmogenic gene expression.

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFβ. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.

The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.

Background Respiratory illness caused by viral infection is associated with the development and exacerbation of childhood asthma. Little is known about the effects of respiratory viral infections in the absence of illness. Using quantitative PCR (qPCR) for common respiratory viruses and for two genes known to be highly upregulated in viral infections (CCL8/CXCL11), we screened 92 asthmatic and 69 healthy children without illness for respiratory virus infections. Results We found 21 viral qPCR-positive and 2 suspected virus-infected subjects with high expression of CCL8/CXCL11. We applied a dual RNA-seq workflow to these subjects, together with 25 viral qPCR-negative subjects, to compare qPCR with sequencing-based virus detection and to generate the airway transcriptome for analysis. RNA-seq virus detection achieved 86% sensitivity when compared to qPCR-based screening. We detected additional respiratory viruses in the two CCL8/CXCL11-high subjects and in two of the qPCR-negative subjects. Viral read counts varied widely and were used to stratify subjects into Virus-High and Virus-Low groups. Examination of the host airway transcriptome found that the Virus-High group was characterized by immune cell airway infiltration, downregulation of cilia genes, and dampening of type 2 inflammation. Even the Virus-Low group was differentiated from the No-Virus group by 100 genes, some involved in eIF2 signaling. Conclusions Respiratory virus infection without illness is not innocuous but may determine the airway function of these subjects by driving immune cell airway infiltration, cellular remodeling, and alteration of asthmogenic gene expression. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1140-8) contains supplementary material, which is available to authorized users.

Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 - rs1420101 and rs11685480 - are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33-driven type 2 inflammation.

Alveolar macrophages are the main line of innate immune response against M. tuberculosis (Mtb) infection. However, these cells serve as the major intracellular niche for Mtb enhancing its survival, replication and, later on, cell-to-cell spread. Mtb-associated cytotoxicity of macrophages has been well documented, but limited information exists about mechanisms by which the pathogen induces cell necrosis. To identify virulence factors involved in the induction of necrosis, we screened 5,000 transposon mutants of Mtb for clones that failed to promote the host cell necrosis in a similar manner as the wild-type bacterium. Five Mtb mutants were identified as potential candidates inducing significantly lower levels of THP-1 cell damage in contrast to the H37Rv wild-type infection. Reduced levels of the cell damage by necrosis deficient mutants (NDMs) were also associated with delayed damage of mitochondrial membrane permeability when compared with the wild-type infection over time. Two knockout mutants of the Rv3873 gene, encoding a cell wall PPE68 protein of RD1 region, were identified out of 5 NDMs. Further investigation lead to the observation that PPE68 protein interacts and exports several unknown or known surface/secreted proteins, among them Rv2626c is associated with the host cell necrosis. When the Rv2626c gene is deleted from the genome of Mtb, the bacterium displays significantly less necrosis in THP-1 cells and, conversely, the overexpression of Rv2626c promotes the host cell necrosis at early time points of infections in contrast to the wild-type strain.

The nematode Caenorhabditis elegans has become a model system for studying the disease interaction between pathogens and the host. To determine whether the transparent nematode could serve as a useful model for Mycobacterium avium subspecies hominissuis (MAH) infection of the intestinal tract, worms were fed MAH and assayed for the effects of the bacterial infection on the worm. It was observed during feeding that viable MAH increases in the intestinal lumen in a time dependent manner. Ingestion of MAH was deemed non-toxic to worms as MAH-fed populations have similar survival curves to those fed E. coli strain OP50. Pulse-chase analysis using E. coli strain OP50 revealed that MAH colonize the intestinal tract, as viable MAH remain within the intestine after the assay. Visualization of intestinal MAH using histology and transmission electron microscopy demonstrates that MAH localizes to the intestinal lumen, as well as establishes direct contact with intestinal epithelium. Bacterial colonization appears to have a detrimental effect on the microvilli of the intestinal epithelial cells. The MAH ΔGPL/4B2 strain with a mutation in glycopeptidolipid production is deficient in binding to human epithelial cells (HEp-2), as well as deficient in its ability to bind to and colonize the intestinal tract of C. elegans as efficiently as wild-type MAH. These data indicate the C. elegans may serve as a useful model system for MAH pathogenesis and in determining the mechanisms used by MAH during infection and colonization of the intestinal epithelium.

Johne’s disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne’s disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne’s disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne’s-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection.

Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins. IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte., IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte.

Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne’s disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321 and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.