Gain-of-function variants in SMAD4 compromise respiratory epithelial function.

BACKGROUND: Myhre syndrome is an exceedingly rare yet increasingly diagnosed genetic disorder arising from germline variants in the SMAD4 gene. Its core manifestation is the progression of stiffness and fibrosis across multiple organs. Individuals with Myhre syndrome exhibit a propensity for upper respiratory tract remodeling and infections. The molecular and cellular mechanisms underlying this phenotype remain unclear. OBJECTIVE: We sought to investigate how SMAD4 pathogenic variants associated with Myhre syndrome affect SMAD4 protein levels, activation, and physiological functions in patient-derived nasal epithelial cells. METHODS: Clinical observations were conducted on a cohort of 47 patients recruited at Massachusetts General Hospital from 2016 to 2023. Nasal epithelial basal cells were isolated and cultured from inferior turbinate brushings of healthy subjects (n = 8) and patients with Myhre syndrome (n = 3; SMAD4-Ile500Val, Arg496Cys, and Ile500Thr). Transcriptomic analysis and functional assays were performed to assess SMAD4 levels, transcriptional activity, and epithelial cell host defense functions, including cell proliferation, mucociliary differentiation, and bacterial elimination. RESULTS: Clinical observations revealed a prevalent history of otitis media and sinusitis among most individuals with Myhre syndrome. Analyses of nasal epithelial cells indicated that SMAD4 mutations do not alter SMAD4 protein stability or upstream regulatory SMAD phosphorylation but enhance signaling transcriptional activity, supporting a gain-of-function mechanism, likely attributable to increased protein-protein interaction of the SMAD complex. Consequently, Myhre syndrome nasal basal cells exhibit reduced potential in cell proliferation and mucociliary differentiation. Furthermore, Myhre syndrome nasal epithelia are impaired in bacterial killing. CONCLUSIONS: Compromised innate immunity originating from epithelial cells in Myhre syndrome may contribute to increased susceptibility to upper respiratory tract infections.

Airway Basal Stem Cells in COVID-19 Exhibit a Proinflammatory Signature and Impaired Mucocililary Differentiation.

Airway basal stem cells (BSCs) play a critical role in epithelial regeneration. Whether coronavirus disease (COVID-19) affects BSC function is unknown. Here, we derived BSC lines from patients with COVID-19 using tracheal aspirates (TAs) to circumvent the biosafety concerns of live-cell derivation. We show that BSCs derived from the TAs of control patients are bona fide bronchial BSCs. TA BSCs from patients with COVID-19 tested negative for severe acute respiratory syndrome coronavirus 2 RNA; however, these so-termed COVID-19-exposed BSCs in vitro resemble a predominant BSC subpopulation uniquely present in patients with COVID-19, manifested by a proinflammatory gene signature and STAT3 hyperactivation. Furthermore, the sustained STAT3 hyperactivation drives goblet cell differentiation of COVID-19-exposed BSCs in an air-liquid interface. Last, these phenotypes of COVID-19-exposed BSCs can be induced in control BSCs by cytokine cocktail pretreatment. Taken together, acute inflammation in COVID-19 exerts a long-term impact on mucociliary differentiation of BSCs.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

RATIONALE: Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. OBJECTIVES: To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. METHODS: In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. RESULTS: GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. CONCLUSIONS: GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

Emergence of the natural history of Myhre syndrome: 47 patients evaluated in the Massachusetts General Hospital Myhre Syndrome Clinic (2016-2023).

Myhre syndrome is an increasingly diagnosed ultrarare condition caused by recurrent germline autosomal dominant de novo variants in SMAD4. Detailed multispecialty evaluations performed at the Massachusetts General Hospital (MGH) Myhre Syndrome Clinic (2016-2023) and by collaborating specialists have facilitated deep phenotyping, genotyping and natural history analysis. Of 47 patients (four previously reported), most (81%) patients returned to MGH at least once. For patients followed for at least 5 years, symptom progression was observed in all. 55% were female and 9% were older than 18 years at diagnosis. Pathogenic variants in SMAD4 involved protein residues p.Ile500Val (49%), p.Ile500Thr (11%), p.Ile500Leu (2%), and p.Arg496Cys (38%). Individuals with the SMAD4 variant p.Arg496Cys were less likely to have hearing loss, growth restriction, and aortic hypoplasia than the other variant groups. Those with the p.Ile500Thr variant had moderate/severe aortic hypoplasia in three patients (60%), however, the small number (n = 5) prevented statistical comparison with the other variants. Two deaths reported in this cohort involved complex cardiovascular disease and airway stenosis, respectively. We provide a foundation for ongoing natural history studies and emphasize the need for evidence-based guidelines in anticipation of disease-specific therapies.

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.

STAT3 mutation-associated airway epithelial defects in Job syndrome.

BACKGROUND: Job syndrome is a disease of autosomal dominant hyper-IgE syndrome (AD-HIES). Patients harboring STAT3 mutation are particularly prone to airway remodeling and airway infections. OBJECTIVES: Airway epithelial cells play a central role as the first line of defense against pathogenic infection and express high levels of STAT3. This study thus interrogates how AD-HIES STAT3 mutations impact the physiological functions of airway epithelial cells. METHODS: This study created human airway basal cells expressing 4 common AD-HIES STAT3 mutants (R382W, V463del, V637M, and Y657S). In addition, primary airway epithelial cells were isolated from a patient with Job syndrome who was harboring a STAT3-S560del mutation and from mice harboring a STAT3-V463del mutation. Cell proliferation, differentiation, barrier function, bacterial elimination, and innate immune responses to pathogenic infection were quantitatively analyzed. RESULTS: STAT3 mutations reduce STAT3 protein phosphorylation, nuclear translocation, transcription activity, and protein stability in airway basal cells. As a consequence, STAT3-mutated airway basal cells give rise to airway epithelial cells with abnormal cellular composition and loss of coordinated mucociliary clearance. Notably, AD-HIES STAT3 airway epithelial cells are defective in bacterial killing and fail to initiate vigorous proinflammatory responses and neutrophil transepithelial migration in response to an experimental model of Pseudomonas aeruginosa infection. CONCLUSIONS: AD-HIES STAT3 mutations confer numerous abnormalities to airway epithelial cells in cell differentiation and host innate immunity, emphasizing their involvement in the pathogenesis of lung complications in Job syndrome. Therefore, therapies must address the epithelial defects as well as the previously noted immune cell defects to alleviate chronic infections in patients with Job syndrome.

Basal Cell-derived WNT7A Promotes Fibrogenesis at the Fibrotic Niche in Idiopathic Pulmonary Fibrosis.

Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function in vitro and in a repetitive bleomycin model in vivo. We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.

Human Airway Basal Cells Undergo Reversible Squamous Differentiation and Reshape Innate Immunity.

Histological and lineage immunofluorescence examination revealed that healthy conducting airways of humans and animals harbor sporadic poorly differentiated epithelial patches mostly in the dorsal noncartilage regions that remarkably manifest squamous differentiation. In vitro analysis demonstrated that this squamous phenotype is not due to intrinsic functional change in underlying airway basal cells. Rather, it is a reversible physiological response to persistent Wnt signaling stimulation during de novo differentiation. Squamous epithelial cells have elevated gene signatures of glucose uptake and cellular glycolysis. Inhibition of glycolysis or a decrease in glucose availability suppresses Wnt-induced squamous epithelial differentiation. Compared with pseudostratified airway epithelial cells, a cascade of mucosal protective functions is impaired in squamous epithelial cells, featuring increased epithelial permeability, spontaneous epithelial unjamming, and enhanced inflammatory responses. Our study raises the possibility that the squamous differentiation naturally occurring in healthy airways identified herein may represent "vulnerable spots" within the airway mucosa that are sensitive to damage and inflammation when confronted by infection or injury. Squamous metaplasia and hyperplasia are hallmarks of many airway diseases, thereby expanding these areas of vulnerability with potential pathological consequences. Thus, investigation of physiological and reversible squamous differentiation from healthy airway basal cells may provide critical knowledge to understand pathogenic squamous remodeling, which is often nonreversible, progressive, and hyperinflammatory.

Rho/SMAD/mTOR triple inhibition enables long-term expansion of human neonatal tracheal aspirate-derived airway basal cell-like cells.

BACKGROUND: Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture. METHODS: We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates. RESULTS: Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium. CONCLUSIONS: Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease. IMPACT: Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.

Genome-Wide Association Study: Functional Variant rs2076295 Regulates Desmoplakin Expression in Airway Epithelial Cells.

Rationale: Genetic association studies have identified rs2076295 in association with idiopathic pulmonary fibrosis (IPF). We hypothesized that rs2076295 is the functional variant regulating DSP (desmoplakin) expression in human bronchial epithelial cells, and DSP regulates extracellular matrix-related gene expression and cell migration, which is relevant to IPF development.Objectives: To determine whether rs2076295 regulates DSP expression and the function of DSP in airway epithelial cells.Methods: Using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 editing (including regional deletion, indel, CRISPR interference, and single-base editing), we modified rs2076295 and measured DSP expression in edited 16HBE14o- and primary airway epithelial cells. Cellular integrity, migration, and genome-wide gene expression changes were examined in 16HBE14o- single colonies with DSP knockout. The expression of DSP and its relevant matrix genes was measured by quantitative PCR and also analyzed in single-cell RNA-sequencing data from control and IPF lungs.Measurements and Main Results:DSP is expressed predominantly in bronchial and alveolar epithelial cells, with reduced expression in alveolar epithelial cells in IPF lungs. The deletion of the DNA region-spanning rs2076295 led to reduced expression of DSP, and the edited rs2076295GG 16HBE14o- line has lower expression of DSP than the rs2076295TT lines. Knockout of DSP in 16HBE14o- cells decreased transepithelial resistance but increased cell migration, with increased expression of extracellular matrix-related genes, including MMP7 and MMP9. Silencing of MMP7 and MMP9 abolished increased migration in DSP-knockout cells.Conclusions: rs2076295 regulates DSP expression in human airway epithelial cells. The loss of DSP enhances extracellular matrix-related gene expression and promotes cell migration, which may contribute to the pathogenesis of IPF.

Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.

SMAD Signaling Restricts Mucous Cell Differentiation in Human Airway Epithelium.

Mucin-secreting goblet cell metaplasia and hyperplasia (GCMH) is a common pathological phenotype in many human respiratory diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, primary ciliary dyskinesia, and infections. A better understanding of how goblet cell quantities or proportions in the airway epithelium are regulated may provide novel therapeutic targets to mitigate GCMH in these devastating diseases. We identify canonical SMAD signaling as the principal pathway restricting goblet cell differentiation in human airway epithelium. Differentiated goblet cells express low levels of phosphorylated SMAD. Accordingly, inhibition of SMAD signaling markedly amplifies GCMH induced by mucous mediators. In contrast, SMAD signaling activation impedes goblet cell generation and accelerates the resolution of preexisting GCMH. SMAD signaling inhibition can override the suppressive effects imposed by a GABAergic receptor inhibitor, suggesting the GABAergic pathway likely operates through inhibition of SMAD signaling in regulating mucous differentiation. Collectively, our data demonstrate that SMAD signaling plays a determining role in mucous cell differentiation, and thus raise the possibility that dysregulation of this pathway contributes to respiratory pathophysiology during airway inflammation and pulmonary diseases. In addition, our study also highlights the potential for SMAD modulation as a therapeutic target in mitigating GCMH.

Neutrophil-Derived Cytosolic PLA2α Contributes to Bacterial-Induced Neutrophil Transepithelial Migration.

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography, a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.

Dedifferentiation of committed epithelial cells into stem cells in vivo.

Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer.

Airway Serous Cells: A Comparative Study of Spatial Distribution and Abundance among Species.

The conducting airways of the respiratory system play a crucial role in filtering, humidifying, and directing air into the lungs. Among the specialized cell types within these airways, airway serous cells are notable for their secretion of watery, protein-rich fluids and enzymes, which contribute to maintaining airway surface liquid homeostasis and defending against pathogens. However, the distribution and abundance of serous cells across different species in the conducting airways remain poorly understood. In this study, we addressed this gap by investigating the spatial distribution of the airway serous cell-specific marker BPI fold containing family A member 1 (BPIFA1) in humans, pigs, and mice. Our findings demonstrate significant variations in the distribution and abundance of serous cells among these species, potentially reflecting their different respiratory anatomy and evolutionary adaptations to diverse environmental challenges and respiratory demands. In humans and pigs, airway serous cells are predominantly found in the submucosal glands of the trachea and segmental bronchi, frequently overlapping with lysozyme-positive secretory cells. In contrast, rodents like mice exhibit a distinct pattern where serous cells are scarce in submucosal glands. Instead, rodent serous cells are primarily located at the epithelial surface from the trachea to the main bronchi, where many co-express the Club cell-specific protein SCGB1A1. The abundance of serous cells diminishes progressively in the intrapulmonary airways. Given that rodent models are widely utilized in respiratory research, understanding anatomical and cellular differences in airway serous cells is critical for interpreting experimental outcomes and translating findings to human respiratory diseases and therapeutic strategies. This comparative analysis enhances our understanding of airway biology across species and informs the selection and interpretation of animal models in respiratory studies.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant threefold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin, triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased the release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier-disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.IMPORTANCEStreptococcus pneumoniae (Sp), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp. Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp-infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin (PLY), triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface (ALI) cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant fMLP did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.

XBP1-mediated transcriptional regulation of SLC5A1 in human epithelial cells in disease conditions.

BACKGROUND: Sodium-Glucose cotransporter 1 and 2 (SGLT1/2) belong to the family of glucose transporters, encoded by SLC5A1 and SLC5A2, respectively. SGLT2 is almost exclusively expressed in the renal proximal convoluted tubule cells. SGLT1 is expressed in the kidneys but also in other organs throughout the body. Many SGLT inhibitor drugs have been developed based on the mechanism of blocking glucose (re)absorption mediated by SGLT1/2, and several have gained major regulatory agencies' approval for treating diabetes. Intriguingly these drugs are also effective in treating diseases beyond diabetes, for example heart failure and chronic kidney disease. We recently discovered that SGLT1 is upregulated in the airway epithelial cells derived from patients of cystic fibrosis (CF), a devastating genetic disease affecting greater than 70,000 worldwide. RESULTS: In the present work, we show that the SGLT1 upregulation is coupled with elevated endoplasmic reticulum (ER) stress response, indicated by activation of the primary ER stress senor inositol-requiring protein 1α (IRE1α) and the ER stress-induced transcription factor X-box binding protein 1 (XBP1), in CF epithelial cells, and in epithelial cells of other stress conditions. Through biochemistry experiments, we demonstrated that the spliced form of XBP1 (XBP1s) acts as a transcription factor for SLC5A1 by directly binding to its promoter region. Targeting this ER stress → SLC5A1 axis by either the ER stress inhibitor Rapamycin or the SGLT1 inhibitor Sotagliflozin was effective in attenuating the ER stress response and reducing the SGLT1 level in these cellular model systems. CONCLUSIONS: The present work establishes a causal relationship between ER stress and SGLT1 upregulation and provides a mechanistic explanation why SGLT inhibitor drugs benefit diseases beyond diabetes.

Activation of STAT3-mediated ciliated cell survival protects against severe infection by respiratory syncytial virus.

Respiratory syncytial virus (RSV) selectively targets ciliated cells in human bronchial epithelium and can cause bronchiolitis and pneumonia, mostly in infants. To identify molecular targets of intervention during RSV infection in infants, we investigated how age regulates RSV interaction with the bronchial epithelium barrier. Employing precision-cut lung slices and air-liquid interface cultures generated from infant and adult human donors, we found robust RSV virus spread and extensive apoptotic cell death only in infant bronchial epithelium. In contrast, adult bronchial epithelium showed no barrier damage and limited RSV infection. Single nuclear RNA-Seq revealed age-related insufficiency of an antiapoptotic STAT3 activation response to RSV infection in infant ciliated cells, which was exploited to facilitate virus spread via the extruded apoptotic ciliated cells carrying RSV. Activation of STAT3 and blockade of apoptosis rendered protection against severe RSV infection in infant bronchial epithelium. Lastly, apoptotic inhibitor treatment of a neonatal mouse model of RSV infection mitigated infection and inflammation in the lung. Taken together, our findings identify a STAT3-mediated antiapoptosis pathway as a target to battle severe RSV disease in infants.