Pulmonary surfactant function and molecular architecture is disrupted in the presence of vaping additives.

Inhalation of harmful vaping additives has led to a series of lung illnesses. Some of the selected additives such as vitamin E acetate, and related molecules like vitamin E and cannabidiol, may interfere with the function of the lung surfactant. Proper lipid organization in lung surfactant is key to maintaining low surface tensions, which provides alveolar stability and effective gas exchange throughout respiration. Physiological surfactants, such as bovine lipid extract surfactant used to treat neonatal respiratory distress syndrome, serve as a good model for examining the potential effects of vape additives on proper function. We have found that all additives impede the surfactants' ability to efficiently reach high surface pressures as these systems displayed numerous shoulders throughout compression with accompanying defects to lipid organization. Moreover, the formation of lipid bilayer stacks in the film are hindered by the additives, most notably with vitamin e acetate. Loss of these stacks leave the film prone to buckling and collapse under high compression that occurs at the end of expiration. The data suggest that the additives may interfere with both proper lipid organization and the surfactant protein function.

Caveolin-initiated macropinocytosis is required for efficient silica nanoparticles' transcytosis across the alveolar epithelial barrier.

Removal of particulate materials that would otherwise cumulate within the airspace and hinder the gas exchange is one of the central processes of maintaining lung homeostasis. While the importance of the particle uptake by alveolar macrophages and their expulsion via the airways mucociliary escalator is well established, very little is known about the alternative route for removing the particles via direct crossing the lung epithelium for transfer into the pulmonary lymph and bloodstream. This study dissected sequential mechanisms involved in nanoparticle transcytosis through the alveolar epithelial cell layer. By a combination of live cell, super resolution, and electron microscopy and RNA interference study, we have dissected temporal steps of nanoparticle transcytosis through alveolar epithelium. Our study revealed that caveolin is essential for the firm adhesion of the silica nanoparticle agglomerates to the apical membrane and their subsequent rapid internalization with the help of macropinocytic elements C-terminal-binding protein1 and Rabankyrin-5 but not dynamin. Actin, but not microtubules, played a major role in nanoparticle uptake and subsequent transportation. The compartments with nanoparticles were tethered to trans-Golgi network to be jointly transported along actin stress fibers across the cytoplasm, employing a myosin-dependent mechanism. The trans-Golgi nanoparticle transport machinery was positive to Rab6A, a marker linked to vesicle exocytosis. Exocytosis was primarily occurring at the basolateral plane of the alveolar epithelial cells. The high-proficiency novel caveolin and Rabankyrin-5 associated uptake and transcellular transport of nanoparticles across the AEC barrier supports its importance in clearance of amorphous silica and other types of non-inflammatory nanoparticles that are rapidly removed from the lungs following their inhalation.

Patrolling Alveolar Macrophages Conceal Bacteria from the Immune System to Maintain Homeostasis.

During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

Beta3-Tubulin is Critical for Microtubule Dynamics, Cell Cycle Regulation, and Spontaneous Release of Microvesicles in Human Malignant Melanoma Cells (A375).

Microtubules (MTs), microfilaments, and intermediate filaments, the main constituents of the cytoskeleton, undergo continuous structural changes (metamorphosis), which are central to cellular growth, division, and release of microvesicles (MVs). Altered MTs dynamics, uncontrolled proliferation, and increased production of MVs are hallmarks of carcinogenesis. Class III beta-tubulin (ß3-tubulin), one of seven ß-tubulin isotypes, is a primary component of MT, which correlates with enhanced neoplastic cell survival, metastasis and resistance to chemotherapy. We studied the effects of ß3-tubulin gene silencing on MTs dynamics, cell cycle, and MVs release in human malignant melanoma cells (A375). The knockdown of ß3-tubulin induced G2/M cell cycle arrest, impaired MTs dynamics, and reduced spontaneous MVs release. Additional studies are therefore required to elucidate the pathophysiologic and therapeutic role of ß3-tubulin in melanoma.

Corynebacterium tuberculostearicum, a human skin colonizer, induces the canonical nuclear factor-κB inflammatory signaling pathway in human skin cells.

INTRODUCTION: Corynebacterium tuberculostearicum (C. t.) is a ubiquitous bacterium that colonizes human skin. In contrast to other members of the genus Corynebacterium, such as toxigenic Corynebacterium diphtheriae or the opportunistic pathogen Corynebacterium jeikeium, several studies suggest that C. t. may play a role in skin health and disease. However, the mechanisms underlying these effects remain poorly understood. METHODS: To investigate whether C. t. induces inflammatory pathways in primary human epidermal keratinocytes (HEKs) and human cutaneous squamous carcinoma cells (SCCs), cell culture, reverse transcription-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, immunofluorescence microscopy, Western blot, chromatin immunoprecipitation-PCR, small interfering RNA knockdown and luciferase reporter expression system were used. RESULTS: Herein, we demonstrate that C. t. upregulates the messenger RNA (mRNA) and protein levels of inflammatory mediators in two human skin cell lines, HEKs and SCCs. We further show activation of the canonical nuclear factor-κB (NF-κB) pathway in response to C. t. infection, including phosphorylation of the inhibitor of κB (IκB), the nuclear translocation of NF-κB subunit (NF-κB-P65 ) and the recruitment of NF-κB-P65 and RNA polymerase to the NF-κB response elements at the promoter region of the inflammatory genes. Lastly, the data confirm that C. t.-induced tumor necrosis factor mRNA expression in HEKs is toll-like receptor 2 (TLR2 ) dependent. CONCLUSION: Our results offer a mechanistic model for C. t.-induced inflammation in human keratinocytes via TLR2 and activation of IκB kinase and downstream signaling through the canonical NF-κB pathway. Relevance to chronic inflammatory diseases of the skin and cutaneous oncology is discussed.

In vivo clearance of nanoparticles by transcytosis across alveolar epithelial cells.

Nanoparticles in polluted air or aerosolized drug nanoparticles predominantly settle in the alveolar lung. Here, we describe a novel, highly effective pathway for the particles to cross the alveolar epithelium and reach the lymph and bloodstream. Amorphous silica nanoparticles, suspended in perfluorocarbon, were instilled into the lungs of mice for intravital microscopy. Particles formed agglomerates that settled on the alveolar wall, half of which were removed from the lung within 30 minutes. TEM histology showed agglomerates in stages of crossing the alveolar epithelium, in large compartments inside the epithelial cells and crossing the basal membrane into the interstitium. This pathway is consistent with published kinetic studies in rats and mice, using a host of (negatively) charged and polar nanoparticles.

Pseudomonas aeruginosa quorum-sensing metabolite induces host immune cell death through cell surface lipid domain dissolution.

Bacterial quorum-sensing autoinducers are small chemicals released to control microbial community behaviours. N-(3-oxo-dodecanoyl) homoserine lactone, the autoinducer of the Pseudomonas aeruginosa LasI-LasR circuitry, triggers significant cell death in lymphocytes. We found that this molecule is incorporated into the mammalian plasma membrane and induces dissolution of eukaryotic lipid domains. This event expels tumour necrosis factor receptor 1 into the disordered lipid phase for its spontaneous trimerization without its ligand and drives caspase 3-caspase 8-mediated apoptosis. In vivo, P. aeruginosa releases N-(3-oxo-dodecanoyl) homoserine lactone to suppress host immunity for its own better survival; conversely, blockage of caspases strongly reduces the severity of the infection. This work reveals an unknown communication method between microorganisms and the mammalian host and suggests interventions of bacterial infections by intercepting quorum-sensing signalling.

Cholesterol Crystal-Mediated Inflammation Is Driven by Plasma Membrane Destabilization.

Atherosclerosis is driven by an inflammatory milieu in the walls of artery vessels. Initiated early in life, it progresses to plaque formation and form cell accumulation. A culprit in this cascade is the deposition of cholesterol crystals (CC). The involvement of smaller crystals in the early stage of atherosclerotic changes may be critical to the long-term pathological development. How these small crystals initiate the pro-inflammatory events is under study. We report here an unexpected mechanism that microscopic CC interact with cellular membrane in a phagocytosis-independent manner. The binding of these crystals extracts cholesterol from the cell surface. This process causes a sudden catastrophic rupture of plasma membrane and necrosis of the bound cells independent of any known cell death-inducing pathways, releasing inflammatory agents associated with the necrotic cell death. Our results, therefore, reveal a biophysical aspect of CC in potentially mediating the inflammatory progress in atherosclerosis.

Surfactant Dysfunction in ARDS and Bronchiolitis is Repaired with Cyclodextrins.

Objectives: Acute respiratory distress syndrome (ARDS) is caused by many factors including inhalation of toxicants, acute barotrauma, acid aspiration, and burns. Surfactant function is impaired in ARDS and acute airway injury resulting in high surface tension with alveolar and small airway collapse, edema, hypoxemia, and death. In this study, we explore the mechanisms whereby surfactant becomes dysfunctional in ARDS and bronchiolitis and its repair with a cyclodextrin drug that sequesters cholesterol. Methods: We used in vitro model systems, a mouse model of ARDS, and samples from patients with acute bronchiolitis. Surface tension was measured by captive bubble surfactometry. Results: Patient samples showed severe surfactant inhibition even in the absence of elevated cholesterol levels. Surfactant was also impaired in ARDS mice where the cholesterol to phospholipid ratio (W/W%) was increased. Methyl-ß-cyclodextrin (MßCD) restored surfactant function to normal in both human and animal samples. Model studies showed that the inhibition of surfactant was due to both elevated cholesterol and an interaction between cholesterol and oxidized phospholipids. MßCD was also shown to have anti-inflammatory effects. Conclusions: Inhaled cyclodextrins have potential for the treatment of ARDS. They could be delivered in a portable device carried in combat and used following exposure to toxic gases and fumes or shock secondary to hemorrhage and burns.

Dynamic and Irregular Distribution of RyR2 Clusters in the Periphery of Live Ventricular Myocytes.

Cardiac ryanodine receptors (RyR2s) are Ca2+ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca2+ release. The distribution of these Ca2+ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca2+ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca2+ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca2+- and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca2+ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.

Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida.

Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans and Candida albicans. However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that ß-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that ß-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, ß-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble ß-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating ß-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.

Dysfunction of pulmonary surfactant mediated by phospholipid oxidation is cholesterol-dependent.

Pulmonary surfactant forms a cohesive film at the alveolar air-lung interface, lowering surface tension, and thus reducing the work of breathing and preventing atelectasis. Surfactant function becomes impaired during inflammation due to degradation of the surfactant lipids and proteins by free radicals. In this study, we examine the role of reactive nitrogen (RNS) and oxygen (ROS) species on surfactant function with and without physiological cholesterol levels (5-10%). Surface activity was assessed in vitro in a captive bubble surfactometer (CBS). Surfactant chemistry, monolayer fluidity and thermodynamic behavior were also recorded before and after oxidation. We report that physiologic amounts of cholesterol combined with oxidation results in severe impairment of surfactant function. We also show that surfactant polyunsaturated phospholipids are the most susceptible to oxidative alteration. Membrane thermodynamic experiments showed significant surfactant film stiffening after free radical exposure in the presence of cholesterol. These results point to a previously unappreciated role for cholesterol in amplifying defects in surface activity caused by oxidation of pulmonary surfactant, a finding that may have implications for treating several lung diseases.

Vibrational Profiling of Brain Tumors and Cells.

This study reports vibration profiles of neuronal cells and tissues as well as brain tumor and neocortical specimens. A contact-free method and analysis protocol was designed to convert an atomic force microscope into an ultra-sensitive microphone with capacity to record and listen to live biological samples. A frequency of 3.4 Hz was observed for both cultured rat hippocampal neurons and tissues and vibration could be modulated pharmacologically. Malignant astrocytoma tissue samples obtained from operating room, transported in artificial cerebrospinal fluid, and tested within an hour, vibrated with a much different frequency profile and amplitude, compared to meningioma or lateral temporal cortex providing a quantifiable measurement to accurately distinguish the three tissues in real-time. Vibration signals were converted to audible sound waves by frequency modulation, thus demonstrating, acoustic patterns unique to meningioma, malignant astrocytoma and neocortex.

Pulmonary surfactant dysfunction in pediatric cystic fibrosis: Mechanisms and reversal with a lipid-sequestering drug.

BACKGROUND: Airway surfactant is impaired in cystic fibrosis (CF) and associated with declines in pulmonary function. We hypothesized that surfactant dysfunction in CF is due to an excess of cholesterol with an interaction with oxidation. METHODS: Surfactant was extracted from bronchial lavage fluid from children with CF and surface tension, and lipid content, inflammatory cells and microbial flora were determined. Dysfunctional surfactant samples were re-tested with a lipid-sequestering agent, methyl-ß-cyclodextrin (MßCD). RESULTS: CF surfactant samples were unable to sustain a normal low surface tension. MßCD restored surfactant function in a majority of samples.Mechanistic studies showed that the dysfunction was due to a combination of elevated cholesterol and an interaction with oxidized phospholipids and their pro-inflammatory hydrolysis products. CONCLUSION: We confirm that CF patients have impaired airway surfactant function which could be restored with MßCD. These findings have implications for improving lung function and mitigating inflammation in patients with CF.

The role of multilayers in preventing the premature buckling of the pulmonary surfactant.

The pulmonary surfactant is a protein-lipid mixture that spreads into a film at the air-lung interface. The highly-compacted molecules of the film keep the interface from shrinking under the influence of otherwise high surface tension and thus prevent atelectasis. We have previously shown that for the film to withstand a high film pressure without collapsing it needs to assume a specific architecture of a molecular monolayer with islands of stacks of molecular multilayers scattered over the area. Surface activity was assessed in a captive bubble surfactometer (CBS) and the role of cholesterol and oxidation on surfactant function examined. The surfactant film was conceptualized as a plate under pressure. Finite element analysis was used to evaluate the role of the multilayer stacks in preventing buckling of the plate during compression. The model of film topography was constructed from atomic force microscope (AFM) scans of surfactant films and known physical properties of dipalmitoylphosphatidylcholine (DPPC), a major constituent of surfactant, using ANSYS structural-analysis software. We report that multilayer structures increase film stability. In simulation studies, the critical load required to induce surfactant film buckling increased about two-fold in the presence of multilayers. Our in vitro surfactant studies showed that surface topography varied between functional and dysfunctional films. However, the critical factor for film stability was the anchoring of the multilayers. Furthermore, the anchoring of multilayers and mechanical stability of the film was dependent on the presence of hydrophobic surfactant protein-C. The current study expands our understanding of the mechanism of surfactant inactivation in disease.

Peptide-MHC-based nanomedicines for autoimmunity function as T-cell receptor microclustering devices.

We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner.

Identification and treatment of the Staphylococcus aureus reservoir in vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is reaching epidemic proportions causing morbidity, mortality, and chronic disease due to relapses, suggesting an intracellular reservoir. Using spinning-disk confocal intravital microscopy to track MRSA-GFP in vivo, we identified that within minutes after intravenous infection MRSA is primarily sequestered and killed by intravascular Kupffer cells (KCs) in the liver. However, a minority of the Staphylococci overcome the KC's antimicrobial defenses. These bacteria survive and proliferate for many days within this intracellular niche, where they remain undetected by recruited neutrophils. Over time, the KCs lyse, releasing bacteria into the circulation, enabling dissemination to other organs such as the kidneys. Vancomycin, the antibiotic of choice to treat MRSA bacteremia, could not penetrate the KCs to eradicate intracellular MRSA. However, based on the intravascular location of these specific macrophages, we designed a liposomal formulation of vancomycin that is efficiently taken up by KCs and diminished the intracellular MRSA. Targeting the source of the reservoir dramatically protected the liver but also dissemination to other organs, and prevented mortality. This vancomycin formulation strategy could help treat patients with Staphylococcal bacteremia without a need for novel antibiotics by targeting the previously inaccessible intracellular reservoir in KCs.

Campylobacter jejuni increases flagellar expression and adhesion of noninvasive Escherichia coli: effects on enterocytic Toll-like receptor 4 and CXCL-8 expression.

Campylobacter jejuni is the most common cause of bacterium-induced gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with postinfectious intestinal disorders, including flares in patients with inflammatory bowel disease and postinfectious irritable bowel syndrome (PI-IBS), via mechanisms that remain obscure. Based on the hypothesis that acute campylobacteriosis may cause pathogenic microbiota dysbiosis, we investigated whether C. jejuni may activate dormant virulence genes in noninvasive Escherichia coli and examined the epithelial pathophysiological consequences of these alterations. Microarray and quantitative real-time PCR analyses revealed that E. coli adhesin, flagellum, and hemolysin gene expression were increased when E. coli was exposed to C. jejuni-conditioned medium. Increased development of bacterial flagella upon exposure to live C. jejuni or C. jejuni-conditioned medium was observed under transmission electron microscopy. Atomic force microscopy demonstrated that the forces of bacterial adhesion to colonic T84 enterocytes, and the work required to rupture this adhesion, were significantly increased in E. coli exposed to C. jejuni-conditioned media. Finally, C. jejuni-modified E. coli disrupted TLR4 gene expression and induced proinflammatory CXCL-8 gene expression in colonic enterocytes. Together, these data suggest that exposure to live C. jejuni, and/or to its secretory-excretory products, may activate latent virulence genes in noninvasive E. coli and that these alterations may directly trigger proinflammatory signaling in intestinal epithelia. These observations shed new light on mechanisms that may contribute, at least in part, to postcampylobacteriosis inflammatory disorders.