Biology subjects

Tuvim, M. J.

Publications and source records attributed to Tuvim, M. J..

9 recordsLinked to original sources

bioRxiv2025

Airway Secretory Cells Contain Both a Perinuclear Golgi Ribbon and Dispersed Golgi Satellites

RationaleFinely tuned production and secretion of the polymeric mucins MUC5AC and MUCB are required for lung health, but knowledge of many details between their translation and their packaging into secretory granules is lacking. ObjectivesTo analyze the structure and function of the Golgi apparatus, a key site of mucin glycosylation, folding, polymerization and packaging, in airway epithelial secretory cells. MethodsLung tissue was obtained from mice stimulated or not with IL-13 to upregulate mucin production, and from normal human lungs. Golgi elements in mouse and human tissue were imaged by high-resolution immunofluorescence microscopy and electron microscopy. Tissue from mice with deletion of both polymeric mucins was also examined. Measurements and Main ResultsBy immunofluorescence microscopy, both mouse and human airway secretory cells contained [~]100 dispersed puncta labeled by markers of medial and trans Golgi cisternae and the trans-Golgi network (TGN), but only a few perinuclear puncta were labeled by markers of cis-Golgi cisternae. By electron microscopy, secretory cells contained both a perinuclear Golgi ribbon and numerous dispersed Golgi stacks, termed satellites. In mucous metaplastic cells, satellites were concentrated among immature mucin granules. Increasing mucin production by cytokine stimulation did not increase the number of TGN puncta, nor did preventing polymeric mucin production by gene deletion reduce TGN puncta. ConclusionsMucin-producing airway secretory cells express an unusual Golgi structure consisting of a conventional perinuclear Golgi ribbon as well as dispersed satellites. While the Golgi satellites are likely an adaptation for mucin production and packaging, their presence is specified developmentally, independent of mucin production.

cell biology↗

bioRxiv2023

Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs intrinsic host defenses by therapeutic delivery of a unique dyad of pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential ({Delta}{Psi}m), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during periods of peak vulnerability without reliance on currently available antibiotics. Author SummaryPneumonia is a major cause of death worldwide. Increasing antibiotic resistance and expanding immunocompromised populations continue to enhance the clinical urgency to find new strategies to prevent and treat pneumonia. We have identified a novel inhaled therapeutic that stimulates lung epithelial defenses to protect mice against pneumonia in a manner that depends on production of reactive oxygen species (ROS). Here, we report that the induction of protective ROS from lung epithelial mitochondria occurs following the interaction of one component of the treatment, an oligodeoxynucleotide, with the mitochondrial voltage-dependent anion channel 1. This interaction alters energy transfer between the mitochondria and the cytosol, resulting in metabolic reprogramming that drives more electrons into the electron transport chain, then causes electrons to leak from the electron transport chain to form protective ROS. While antioxidant therapies are endorsed in many other disease states, we present here an example of therapeutic induction of ROS that is associated with broad protection against pneumonia without reliance on administration of antibiotics.

immunology↗

bioRxiv2021

Epithelial Immunomodulation by Aerosolized Toll-like Receptor Agonists Attenuates Allergic Responsiveness in Mice

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness (AHR), and airway remodeling. Epidemiologic data has revealed that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via myriad expression of Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs). We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 ("Pam2", TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 ("ODN", TLR9 ligand) when delivered together by aerosol ("Pam2ODN"), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of airway epithelial cells. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy. One Sentence SummaryA synergistic combination of Toll-like Receptor agonists, delivered directly into the lung mucosa, can attenuate allergic responsiveness of airway epithelial cells and prevent host sensitization to aeroallergens. What is already knownO_LIAllergic sensitization has increased in the 20th century due to reduced contact with microbial organisms in industrialized society (ie. hygiene hypothesis) C_LIO_LIWe have previously identified a pharmacological means to stimulate innate immunity of lung epithelial cells. C_LI What this study addsO_LIActivation of innate immunity in lung epithelial cells attenuates the allergic responsiveness of mice. C_LIO_LISynergistic cooperation of pattern recognition receptors induces stronger immunomodulatory responses C_LI What is the clinical significanceO_LIAerosolized Toll-like Receptor agonists have been demonstrated as safe in human clinical trials C_LIO_LIThis study provides proof-of-principle that aerosolized toll-like receptor agonists could have clinical efficacy in the setting of the allergen immunotherapy C_LI

immunology↗

bioRxiv2021

Intermediary role of lung alveolar type 1 cells in epithelial repair upon Sendai virus infection

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how >90% of this barrier - made of alveolar type 1 (AT1) cells - responds to injury, in contrast to our accumulating knowledge of epithelial progenitor and stem cells whose importance lies in their ability to restore the barrier. Using Sendai virus to model natural infection in mice, we combine 3D imaging, lineage-tracing, and single-cell genomics to show that AT1 cells have an intermediary role by persisting in areas depleted of alveolar type 2 (AT2) cells, mounting an interferon response, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2+ clusters without differentiating into AT1 or AT2 cells, as shown in influenza models. Intriguingly, large AT2-cell-depleted areas remain covered by AT1 cells, which we name "AT2-less regions", and are replaced by SOX2+ clusters spreading both basally and luminally around AT1 cell extensions. AT2 cell proliferation and differentiation are largely confined to topologically distal regions - the end of airspace that could be in the periphery or middle of the lung - and form de novo alveolar surface, with limited contribution to in situ repair of AT2-less regions. Time course single-cell RNA-seq and AT1-cell interactome analyses suggest enhanced recognition of AT1 cells by immune cells and altered growth signals. Our comprehensive spatiotemporal and genome-wide study highlights the hitherto unappreciated role of AT1 cells during Sendai virus infection and possibly other injury-repair processes.

developmental biology↗

bioRxiv2020

Immune modulation to improve survival of respiratory virus infections in mice

Viral pneumonia remains a global health threat requiring novel treatment strategies, as strikingly exemplified in the SARS-CoV-2 pandemic of 2019-2020. We have reported that mice treated with a combination of inhaled Toll-like receptor (TLR) 2/6 and TLR 9 agonists (Pam2-ODN) to stimulate innate immunity are broadly protected against respiratory pathogens, but the mechanisms underlying this protection remain incompletely elucidated. Here, we show in a lethal paramyxovirus model that Pam2-ODN-enhanced survival is associated with robust virus inactivation by reactive oxygen species (ROS), which occurs prior to internalization by lung epithelial cells. However, we also found that mortality in sham-treated mice temporally corresponded with CD8+ T cell-enriched lung inflammation that peaks on days 11-12 after viral challenge, when the viral burden has waned to a scarcely detectable level. Pam2-ODN treatment blocked this injurious inflammation by reducing the viral burden, and alternatively, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. These findings reveal opportunities for targeted immunomodulation to protect susceptible individuals against the morbidity and mortality of respiratory viral infections.

immunology↗

bioRxiv2020

Potentiating TMEM16A channel function has no effect on airway goblet cells or bronchial and pulmonary vascular smooth muscle function

The calcium-activated chloride channel TMEM16A enables chloride secretion across several transporting epithelia, including in the airway where it represents a therapeutic target for the treatment of cystic fibrosis. Additional roles for TMEM16A have also been proposed, including enhancing goblet cell exocytosis, increasing goblet cell numbers and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, both potentiation and inhibition, could affect any of these proposed biological roles. In vitro, a recently described potent and selective TMEM16A potentiator (ETX001) failed to stimulate mucin release from primary human bronchial epithelial (HBE) cells over a 24h exposure period using both biochemical and imaging endpoints. In addition, treatment of HBE cells with ETX001 or a potent and selective TMEM16A inhibitor (Ani9) for 4 days did not influence mucin release or goblet cell formation. In vivo, a TMEM16A potentiator was without effect on goblet cell emptying in an IL-13 driven goblet cell metaplasia model. Using freshly isolated human bronchi and pulmonary arteries, neither ETX001 or Ani9 had any effect on the contractile or relaxant responses of the tissues. In vivo, ETX001 also failed to influence either lung or cardiovascular function when delivered directly into the airways of telemetered rats. Together, these studies do not support a role for TMEM16A in the regulation of goblet cell numbers or mucin release, or on the regulation of airway or pulmonary artery smooth muscle contraction.

physiology↗

bioRxiv2020

Inducible epithelial resistance improves survival of Sendai virus pneumonia in mice by both inactivating virus and preventing CD8+ T cell-mediated immunopathology

Viral pneumonias remain a global health threat necessitating novel strategies to prevent and treat these lower respiratory tract infections. We have reported that mice treated with a combination of inhaled Toll-like receptor (TLR) 2/6 and TLR 9 agonists (Pam2-ODN) are broadly protected against respiratory pathogens. Although a single inhalation of Pam-ODN prevents acute morbidity and chronic complications associated with viral pneumonias, the mechanisms underlying this protection remain incompletely elucidated. Here, we show in a lethal paramyxovirus model that Pam2-ODN-enhanced survival is associated with robust virus inactivation that occurs prior to internalization by lung epithelial cells. However, it was also noted that viral mortality in sham-treated mice temporally corresponded with CD8+ T cell-enriched lung inflammation that peaks after the viral burden wanes. Pam2-ODN treatment also blocked this injurious inflammation, but the attenuation of lymphocytic inflammation and the reduction in virus burden were both lost when inducible reactive oxygen species generation was inhibited. Depleting CD8+ T cells before or after viral challenge underscored the balanced roles of CD8+ T cells in antiviral immunity and fatal immunopathology, but did not obviate the Pam2-ODN antiviral protection. These findings identify multifunctional inducible antiviral mechanisms and may reveal means to protect susceptible individuals against respiratory infections.

immunology↗

bioRxiv2018

Different Munc18 Proteins Mediate Baseline and Stimulated Airway Mucin Secretion

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens, but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all three Munc18 isoforms. Using conditional airway epithelial deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane, and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.

cell biology↗

bioRxiv2017

Control of Mast Cell Regulated Exocytosis by Munc13 Proteins

Mast cells (MCs) are involved in pathogen defense and inflammatory reactions. Upon stimulation, they release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 proteins play essential roles in regulated exocytosis. We found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knockout (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and histological evidence of defective MC degranulation, and not with changes in MC development, distribution, numbers or morphology. In vitro assays revealed that the defective MC response in the absence of Munc13-4 was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants with different expression levels of Munc13-4 in their MCs showed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and the total amount of exocytosis follows. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and electron microscopy studies showed that the number of multigranular compound events (granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that while Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full IgE-mediated anaphylactic response.

cell biology↗