Healthy Human Lung Research Articles

Integrating N -glycan and CODEX imaging reveal cell-specific protein glycosylation in healthy human lung.

N -linked glycosylation, the major post-translational modification of cellular proteins, is important for proper lung functioning, serving to fold, traffic, and stabilize protein structures and to mediate various cell-cell recognition events. Identifying cell-specific N -glycan structures in human lungs is critical for understanding the chemistry and mechanisms that guide cell-cell and cell-matrix interactions and determining nuanced functions of specific N -glycosylation. Our study, which used matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) combined with co-detection by indexing (CODEX) to reveal the cellular origin of N -glycans, is a significant step in this direction. This innovative technological combination enabled us to detect and differentiate N -glycans located in the vicinity of cells surrounding airways and blood vessels, parenchyma, submucosal glands, cartilage, and smooth muscles. The potential impact of our findings on future research is immense. For instance, our algorithm for grouping N -glycans based on their functional chemical features, combined with identifying group niches, paves the way for targeted studies. We found that fucosylated N -glycans are dominant around immune cells, tetra antennary N -glycans in the cartilage, high-mannose N -glycans surrounding the bronchus originate from associated collagenous structures, complex fucosylated-tetra antennary-polylactosamine N -glycans are spread over smooth muscle structures and in epithelial cells surrounding arteries, and N -glycans with Hex:6 HexNAc:6 compositions, which, according to our algorithm, can be ascribed to either tetra antennary or bisecting N -glycan, are highly abundant in the parenchyma. The findings suggest cell or region-specific functions for these localized glycan structures.

The bronchoalveolar lavage dilution conundrum: an updated view on a long-standing problem.

Bronchoalveolar lavage (BAL) is used by researchers to study molecular interactions within healthy and diseased human lungs. However, the utility of BAL fluid measurements may be limited by difficulties accounting for dilution of the epithelial lining fluid (ELF) sampled and inconsistent collection techniques. The use of endogenous markers to estimate ELF dilution has been proposed as a potential method to normalize acellular molecule measurements in BAL fluid, but these markers are also imperfect and prone to inaccuracy. The focus of this report is to review factors that affect the interpretation of acellular molecule measurements in lung ELF and present original data comparing the performance of several BAL dilution markers during health and in a human endobronchial endotoxin challenge model of acute inflammation. Our findings suggest that incomplete ELF and lavage fluid mixing, flux of markers across the alveolar barrier, and lung inflammation are all possible factors that can affect marker performance. Accounting for these factors, we show that commonly used markers including urea, total protein, albumin, and immunoglobulin M are likely unreliable BAL dilution markers. In contrast, surfactant protein D appears to be less affected by these factors and may be a more accurate and biologically plausible marker to improve the reproducibility of acellular BAL component measurements across individuals during health and inflammatory states.NEW & NOTEWORTHY In this report, mathematical prediction models and real-world measurements are used to compare the performance of molecular markers of dilution in bronchoalveolar lavage fluid samples. Effects of acute inflammation within individual subjects are highlighted. These findings inform recommendations for normalizing measurements across bronchoalveolar lavage samples and highlight the need for additional markers to improve the rigor of translational studies utilizing bronchoalveolar lavage measurements.

Aging-Associated Molecular Changes in Human Alveolar Type I Cells.

Human alveolar type I (AT1) cells are specialized epithelial cells that line the alveoli in the lungs where gas exchange occurs. The primary function of AT1 cells is not only to facilitate efficient gas exchange between the air and the blood in the lungs, but also to contribute to the structural integrity of the alveoli to maintain lung function and homeostasis. Aging has notable effects on the structure, function, and regenerative capacity of human AT1 cells. However, our understanding of the molecular mechanisms driving these age-related changes in AT1 cells remains limited. Leveraging a recent single-cell transcriptomics dataset we generated on healthy human lungs, we identified a series of significant molecular alterations in AT1 cells from aged lungs. Notably, the aged AT1 cells exhibited increased cellular senescence and chemokine gene expression, alongside diminished epithelial features such as decreases in cell junctions, endocytosis, and pulmonary matrisome gene expression. Gene set analyses also indicated that aged AT1 cells were resistant to apoptosis, a crucial mechanism for turnover and renewal of AT1 cells, thereby ensuring alveolar integrity and function. Further research on these alterations is imperative to fully elucidate the impact on AT1 cells and is indispensable for developing effective therapies to preserve lung function and promote healthy aging.

Visceral pleura mechanics: Characterization of human, pig, and rat lung material properties

Pulmonary air leaks are amongst the most common complications in lung surgery. Lung sealants are applied to the organ surface and need to synchronously stretch with the visceral pleura, the layer of tissue which encompasses the lung parenchymal tissue. These adhesives are commonly tested on pig and rat lungs, but applied to human lungs. However, the unknown mechanics of human lung visceral pleura undermines the clinical translatability of such animal-tested sealants and the absence of how pig and rat lung visceral pleura compare to human tissues is necessary to address. Here we quantify the biaxial planar tensile mechanics of visceral pleura from healthy transplant-eligible and smoker human lungs for the first time, and further compare the material behaviors to pig and rat lung visceral pleura. Initial and final stiffness moduli, maximum stress, low-to-high strain transition, and stress relaxation are analyzed and compared between and within groups, further considering regional and directional dependencies. Visceral pleura tissue from all species behave isotropically, and pig and human visceral pleura exhibits regional heterogeneity (i.e. upper versus lower lobe differences). We find that pig visceral pleura exhibits similar initial stiffness moduli and regional trends compared to human visceral pleura, suggesting pig tissue may serve as a viable animal model candidate for lung sealant testing. The outcomes and mechanical characterization of these scarce tissues enables future development of biomimetic lung sealants for improved surgical applications. Statement of significanceSurgical lung sealants must synchronously deform with the underlying tissue and with each breath to minimize post-operative air leaks, which remain the most frequent complications of pulmonary intervention. These adhesives are often tested on pig and rat lungs, but applied to humans; however, the material properties of human lung visceral pleura were previously unexplored. Here, for the first time, the mechanics of human visceral pleura tissue are investigated, further contrasting rarely acquired donated lungs from healthy and smoking individuals, and additionally, comparing biaxial planar material characterizations to animal models often employed for pulmonary sealant development. This fundamental material characterization addresses key hindrances in the advancement of biomimetic sealants and evaluates the translatability of animal model experiments for clinical applications.

Quantification of circadian rhythms in mammalian lung tissue snapshot data

Healthy mammalian cells have a circadian clock, a gene regulatory network that allows them to schedule their physiological processes to optimal times of the day. When healthy cells turn into cancer cells, the circadian clock often becomes cancer specifically disturbed, so there is an interest in the extraction of circadian features from gene expression data of cancer. This is challenging, as clinical gene expression samples of cancer are snapshot-like and the circadian clock is best examined using gene expression time series. In this study, we obtained lists of intersecting circadian genes in public gene expression time series data of lung tissue of mouse and baboon. We base our circadian gene lists on correlations of gene expression levels of circadian genes, which are closely associated to the phase differences between them. Combining circadian gene expression patterns of diurnal and nocturnal species of different ages provides circadian genes that are also important in healthy and cancerous human lung tissue. We tested the quality of the representation of the circadian clock in our gene lists by PCA-based reconstructions of the circadian times of the mouse and baboon samples. Then we assigned potential circadian times to the human lung tissue samples and find an intact circadian clock in the healthy human lung tissue, but an altered, weak clock in the adjacent cancerous lung tissue.

Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity

Studying human fetal lungs can inform how developmental defects and disease states alter the function of the lungs. Here, we sequenced >150,000 single cells from 19 healthy human pseudoglandular fetal lung tissues ranging between gestational weeks 10–19. We capture dynamic developmental trajectories from progenitor cells that express abundant levels of the cystic fibrosis conductance transmembrane regulator (CFTR). These cells give rise to multiple specialized epithelial cell types. Combined with spatial transcriptomics, we show temporal regulation of key signalling pathways that may drive the temporal and spatial emergence of specialized epithelial cells including ciliated and pulmonary neuroendocrine cells. Finally, we show that human pluripotent stem cell-derived fetal lung models contain CFTR-expressing progenitor cells that capture similar lineage developmental trajectories as identified in the native tissue. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development and benchmarks fetal lung cultures from human pluripotent stem cell differentiations to similar developmental window.

TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation.

Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9. A combination of large-scale proteomic analyses, lung intravital microscopy, and adoptive transfer of ILC9 cells revealed that high IL-9 expression distinguishes a multicytokine-producing state-of-activated ILC2s with an increased capacity to initiate IL-5-dependent allergic airway inflammation. Similar to IL-33 and TSLP, TL1A is expressed in airway basal cells in healthy and asthmatic human lungs. Together, these results indicate that TL1A is an epithelium-derived cytokine and an important cofactor of IL-33 in the airways.

Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung.

Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

Quantitative profiling and baseline intervals of trace elements in healthy lung tissues

BackgroundHuman lung tissue, as an interface with the environment, is susceptible to various environmental pollutants, including trace metals. However, quantitative data on trace metals in human lung tissues remain poorly described. MethodsThis study aimed to characterize the elemental composition of histologically healthy, unaffected parts of human lung tissues, associated with non-infective, non-infiltrative, and non-malignant diseases (n = 60) for essential (Cr, Mn, Fe, Co, Cu, Zn, and Se) and toxic trace elements (Sr, Ni, As, Cd, and Pb). Additionally, we investigated the influence of personal factors (sex, age, and smoking habits) on the examined trace element profiles, as well as between the trace elements correlations in the healthy human lungs. ResultsAmong the analyzed trace elements, Fe was the most prevalent, while As was the least prevalent in healthy lung tissues. Stratifying by age revealed significantly higher Cr and Co (less Sr, Ni, and Pb) and lower Se levels in older individuals (above 65 years) compared to their younger counterparts. Sex-based differences were also notable, with Cu and Co 1.2- and 2.3-fold higher levels in females than in males. Exploring the impact of smoking habits revealed a striking 10-fold increase in Cd levels in the lung tissues of smokers compared to non-smokers. Correlation analyses showed significant positive associations between concentrations of certain toxic and essential trace elements in healthy lung tissues. ConclusionsThis study could contribute to the establishment of baseline intervals for essential and toxic trace elements, valuable for toxicological and clinical assessment, in healthy, unaffected human lungs, and indicates the influence of sex, age, and smoking. However, further larger-scale studies are needed to make more stable conclusions.

Synthesis, Characterization, Antioxidant, and Anticancer Activity against Colon Cancer Cells of Some Cinnamaldehyde-Based Chalcone Derivatives.

The purpose of the current investigation was to produce cinammaldehyde-based chalcone derivatives (3a-k) to evaluate their potential effectiveness as antioxidant and inhibitory agents versus human Caco-2 cancer cells. The findings obtained using the DPPH assay showed that compound 3e had the highest effective antioxidant activity with the best IC50 value compared with the other compounds. Moreover, the cytotoxic findings revealed that compound 3e was the best compound for inhibiting Caco-2 development in contrast to all other produced derivatives, with the lowest IC50 concentration (32.19 ± 3.92 µM), and it also had no detrimental effects on healthy human lung cells (wi38 cells). Exposure of Caco-2 cells with this IC50 value of compound 3e resulted in a substantial rise in the number of early and late cells that are apoptotic with a significant comet nucleus when compared with control cells employing the annexin V/PI and comet evaluations, respectively. Furthermore, qRT-PCR and ELISA examinations indicated that compound 3e significantly altered the expression of genes and their relative proteins related to apoptosis in the treated Caco-2 cells, thus significantly inhibiting Caco-2 growth through activating Caspase-3 via an intrinsic apoptotic pathway. As a result, compound 3e could serve as an effective therapy for human colon cancer.

Systematic Prediction of Minor Histocompatibility Antigens to Inform GvHD Outcomes after Allogeneic Stem Cell Transplantation

T cell alloreactivity against minor histocompatibility antigens (mHAgs), polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms (SNPs, indels, frameshifts), is at the core of the therapeutic effect of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite the crucial role of mHAgs in graft-versus-leukemia and graft-versus-host (GvHD) reactions, it has not been possible thus far to consistently link patient-specific mHAg repertoires to clinical outcomes. As a result, only D-R HLA matching and the activity of GvHD prophylaxis strategies are currently available to help clinicians in this challenge. We hypothesized that post-transplant outcomes could be impacted by genome-wide mHAg load, delineated in an organ- and malignancy-specific fashion. We have therefore devised an analytic framework to systematically identify autosomal and Y-encoded mHAgs, based on the integration of polymorphism detection by whole-exome sequencing (WES) of germline DNA from D-R pairs together with organ-specific transcriptional- and proteome-level expression together with HLA class I epitope prediction. A critical component of the pipeline was building a reliable expression atlas for acute or chronic GvHD-targeted tissues. To capture less frequent but biologically relevant cell types consistently underestimated in bulk RNA expression profiles, we evaluated multiple external single-cell datasets of healthy human skin, liver, lung, GI, oral mucosa, and lacrimal gland. For each tissue, we merged the corresponding single-cell data sets and then clustered and annotated tissue-resident cell types, for which specific expressed genes were then identified, totaling 13,512 genes expressed in ≥1 GvHD target tissue. To determine how mHAg load related to GvHD risk, we applied our mHAg pipeline to paired WES (≥85% target base coverage at ≥20x depth) generated from 220 D-R pairs treated with a matched-related donor allo-HSCT for AML/MDS. We considered the impact of diagnosis, prognostic risk score, status at transplant, conditioning regimen, and graft source against median mHAg load on risk for acute and chronic GvHD. Across these patients, only total autosomal mHAg load > median was associated with increased risk of developing grade II-IV aGvHD (HR = 2.54, 95% confidence interval: 1.03 - 6.26, p = 0.043). While the total autosomal mHAg load was not associated with development of cGvHD, subgroup analysis of 55 FàM transplants revealed that a combined score of autosomal and Y-encoded mHAgs > median was associated with development of NIH moderate/severe cGvHD (p = 0.042). We next asked whether organ-specific mHAg load could predict the risk for organ-restricted acute or chronic GvHD. By evaluating organ-specific GvHD occurrence across deciles of mHAg load, we identified 2 distinct patterns of association. In the case of lung cGvHD, all 14 positive cases occurred in patients with a lung-specific autosomal mHAg load > median. By logistic regression modelling, lung mHAg load > median was the sole factor associated with increased risk of lung cGvHD (OR = 17.22, 95% CI: 3.18 - 321.63, p = 0.008). In contrast, for aGvHD of the liver, we observed an opposite pattern, with 12 of 13 affected patients having a liver mHAg load < median. We hypothesized that a limited set of immunodominant liver mHAgs might drive the clinical manifestations in these patients. By analyzing the mHAg landscape of patients affected by liver aGvHD, we observed an enriched representation of 7 polymorphisms in genes with preferential expression in liver. Notably, these SNPs were less frequent in patients developing aGvHD without liver involvement (p = 0.002) and co-occurred at a lower frequency in patients experiencing liver cGvHD (p = 0.0002). For one patient experiencing liver aGvHD, we could trace ex vivo T cells specific for 1 (of the 3 predicted) driver liver mHAgs. Overall, our findings indicate that molecular characterization of D-R pairs to define GvHD risk could facilitate the design of personalized post-HSCT treatments to minimize this highly morbid condition, including incorporation of additional immunosuppressive therapies, introduced early for high-risk patients, or reduced-intensity approaches in low-risk settings. This is particularly valuable for lung GvHD, which is associated with the highest morbidity and mortality.

Wirelessly Actuated Ciliary Airway Stent for Excessive Mucus Transportation

AbstractSmall‐scale cilia‐like devices that can manipulate fluids in narrow spaces have great potential in microfluidics, biomechanics, biomedical engineering, and other applications. However, prior studies mostly focus on artificial cilia for pumping fluids in lab‐on‐a‐chip microfluidic applications. The design and control of artificial cilia for transporting viscous mucus in confined and tubular structures remain challenging and medical devices such as airway stents with ciliary function are still missing. Herein, a method is reported that enables integrating artificial cilia arrays on 3D curved surfaces and an airway ciliary stent is presented for excessive mucus transportation. The method allows encoding bioinspired non‐reciprocal motion and metachronal waves for efficient fluid pumping in tubular structures. The method also introduces a lubricant hydrogel coating layer on artificial cilia inspired by the periciliary layer in airway cilia, which further enhances viscous fluid transportation. It is demonstrated that a novel ciliary airway stent can transport viscous porcine mucus in a lung phantom even faster than the respiratory cilia in a healthy human lung. The methods of designing, integrating, and controlling artificial cilia on 3D curved surfaces thus enable the unprecedented function of removing excessive mucus beyond traditional airway stents for treating various lung diseases in a minimally invasive manner.

Transcriptional changes of the aging lung.

Aging is a natural process associated with declined organ function and higher susceptibility to developing chronic diseases. A systemic single-cell type-based study provides a unique opportunity to understand the mechanisms behind age-related pathologies. Here, we use single-cell gene expression analysis comparing healthy young and aged human lungs from nonsmoker donors to investigate age-related transcriptional changes. Our data suggest that aging has a heterogenous effect on lung cells, as some populations are more transcriptionally dynamic while others remain stable in aged individuals. We found that monocytes and alveolar macrophages were the most transcriptionally affected populations. These changes were related to inflammation and regulation of the immune response. Additionally, we calculated the LungAge score, which reveals the diversity of lung cell types during aging. Changes in DNA damage repair, fatty acid metabolism, and inflammation are essential for age prediction. Finally, we quantified the senescence score in aged lungs and found that the more biased cells toward senescence are immune and progenitor cells. Our study provides a comprehensive and systemic analysis of the molecular signatures of lung aging. Our LungAge signature can be used to predict molecular signatures of physiological aging and to detect common signatures of age-related lung diseases.

Histological and immunohistochemical characterization of mucosa-associated lymphoid tissue and antigen-presenting cells in trachea and lung of cattle.

The presence of bronchus-associated lymphoid tissue (BALT) and its structural components has been described in different healthy animal species and in animals with diseases of the respiratory tract. In contrast to normal mammals, BALT is absent in healthy human adult lungs, but has been found in the lungs of children. The histological characteristics of organized mucosa-associated lymphoid tissue (MALT), its subsets of immune cells and their in situ distribution in the lung of healthy subadult and adult cattle shows close similarities with BALT in humans and other animal species such as sheep, horses and pigs. This study clearly demonstrates that organized MALT also occurs in the tracheal mucosa of cattle. The absence of tracheal MALT and BALT in calves suggest that these structures are not constitutive. In the mucosa of bovine trachea, bronchi and bronchioli, MHC II+ and CD11c+ dendritic cells (DCs) are located in the epithelium and in the lamina propria mucosae. These DCs are already present in calves soon after birth. Examination of tangential epithelial sheets shows that in the bovine tracheal epithelium, like in man and rat, a dense network of MHC II+ and CD11c+ DCs exists and that their number is considerably higher than in conventional transverse sections. In the bovine tracheal and bronchial epithelium, MHC II+ DCs are extending their dendrites towards the lumen indicating that these DCs possibly are involved in sampling of luminal antigens. The presence of significantly higher numbers of MHC II+ DCs in the tracheal and bronchial/bronchiolar mucosa of older cattle in than in calves possibly results from local stimulation with exogenous antigens during postnatal life. Detection of DCs expressing the costimulatory molecules CD80 and CD86 in calves and cattle suggests maturation of DCs, which is most likely induced by stimulation with exogenous antigens.

Synthesis, characterization and biological evaluation of three novel copper(II) clonixinate complexes with methylpyridines: Insights into structure, DNA and BSA binding properties and anticancer activity

Three novel mononuclear copper(II) complexes 1–3 with clonixinate anion (clon) derived from clonixin (Hclon) and α-, β-, or γ-methylpyridine (α-, β-, or γ-pic) were synthesised. The compounds were characterised by IR, UV–Vis and EPR spectroscopy and elemental analysis. The redox properties of the prepared complexes were studied by square wave voltammetry. The structure of the complexes [Cu(clon)2(α-pic)2] (1), [Cu(clon)2(β-pic)2] (2), [Cu(clon)2(γ-pic)2] (3) was determined by single-crystal X-ray analysis and the intermolecular interactions were studied by Hirshfeld surface analysis. The interaction of the studied complexes with calf thymus DNA was investigated by UV–Vis absorption titration and further investigation of the possible intercalative mechanism of action was carried out by fluorescence quenching studies with ethidium bromide-DNA adduct monitored via fluorescence emission spectroscopy. Interactions with bovine serum albumin were also evaluated by fluorescence emission spectroscopy. The cytotoxicity and selectivity of 1–3 were studied in vitro on a human breast cancer cell line (MCF-7), a lung cancer cell line (A549), a human glioblastoma cell line (U-118MG) and healthy human fetal lung fibroblast cells (MRC-5). All three complexes showed low to moderate ability to interact with calf thymus DNA, probably through partial intercalation, high and reversible binding to serum albumin and promising cytotoxic effects on the MCF-7 cancer cell line with reasonable selectivity for complexes 1 and 2. Finally, the mitochondrial respiratory capacity of MCF-7 tumour cells was also studied and showed a reduced activity after the addition of the studied complexes.

Alveolar epithelial cells are competent producers of interstitial extracellular matrix with disease relevant plasticity in a human in vitro 3D model

Alveolar epithelial cells (AEC) have been implicated in pathological remodelling. We examined the capacity of AEC to produce extracellular matrix (ECM) and thereby directly contribute towards remodelling in chronic lung diseases. Cryopreserved type 2 AEC (AEC2) from healthy lungs and chronic obstructive pulmonary disease (COPD) afflicted lungs were cultured in decellularized healthy human lung slices for 13 days. Healthy-derived AEC2 were treated with transforming growth factor ß1 (TGF-β1) to evaluate the plasticity of their ECM production. Evaluation of phenotypic markers and expression of matrisome genes and proteins were evaluated by RNA-sequencing, mass spectrometry and immunohistochemistry. The AEC2 displayed an AEC marker profile similar to freshly isolated AEC2 throughout the 13-day culture period. COPD-derived AECs proliferated as healthy AECs with few differences in gene and protein expression while retaining increased expression of disease marker HLA-A. The AEC2 expressed basement membrane components and a complex set of interstitial ECM proteins. TGF-β1 stimuli induced a significant change in interstitial ECM production from AEC2 without loss of specific AEC marker expression. This study reveals a previously unexplored potential of AEC to directly contribute to ECM turnover by producing interstitial ECM proteins, motivating a re-evaluation of the role of AEC2 in pathological lung remodelling.

73 Evaluating the Risks of Prolonged Exposure to Graphene Oxide on Healthy and Streptococcus Pneumoniae Infected 3D Reconstituted Human Lung Cultures

Abstract The growing production of graphene oxide (GO) and GO-based products has led to an increased risk of human exposure and caused scientific and public concern about human health and safety. Even though human exposure to GO can occur via different pathways, the main exposure route for airborn GO is through the lungs, thus placing the respiratory tract at particular risk. Occupational exposure to GO might occur repeatedly over a prolonged period of time but insights on lung toxicity of long-term repeated GO administration is largely lacking. Moreover, there is a risk that GO co-exposure with prevalent lung pathogens (e.g. Streptococcus pneumoniae (SP)) could increase the susceptibility to lung infections. In this study, we aimed to investigate the impact of repeated and prolonged GO exposure in healthy and SP-infected lungs using well-characterized materials with distinct properties and reconstituted primary human bronchial epithelial cultures. Our results revealed that GO had no major toxicity effects on healthy airway epithelium even upon prolonged exposure, but alterations in inflammatory/immune signaling was apparent and showed a correlation with the lateral particle size. GO pre-exposure slightly altered the response of the airway epithelium towards subsequent SP infection as evidenced by a decreased mitochondrial activity, increased bacterial translocation and altered inflammatory/immune responses. Whole genome transcriptomic profiling is ongoing to gain insights in deregulated genes and pathways following chronic GO administration. Our findings on the health risks of GO inhalation in healthy and infected human lung is important to risk assessment and management of this promising material.

132 Colorimetric Assessment of Household Settled Dust Captured on Silicon Nanomembranes

Abstract Humans spend 70-90% of their time indoors, however, there is a significant lack of knowledge regarding human exposure to microplastic particles and fibers (MPs) within the indoor environment. Fibers make up more than 90% of household settled dust worldwide and have been found in indoor air. Studies have identified MPs larger than 50 micrometers in indoor dust, but these are unlikely to be respirable and little information is available regarding smaller airborne particles. It is evident that humans are inhaling these particles as they have been found in excised diseased and healthy human lung tissues. We have developed methods to identify plastic particles in settled household dust larger than one micrometer in diameter by distinguishing plastic from cellulosic, proteinaceous, and inorganic materials using two different stains, Nile Red for plastics, and Trypan Blue for cellulosic materials. Proteinaceous and inorganic materials remain unstained. Household settled dust is collected onto silicon nitride nanomembranes where in situ analysis of particles can be characterized. The innovative use of nanomembranes allow for particle capture and multiple analyses to take place on the same substrate. Particle analysis via colorimetric staining and imaging is followed by polymer identification via Raman spectroscopy and subsequently characterized via scanning electron microscopy for size and surface morphology. Using this innovative approach, microplastic particles larger than one micrometer in diameter have been identified in all settled dust samples.

CD8 +T RM-derived IFNγ is a critical activator of human lung epithelial cells.

Abstract Due to their position in the pulmonary mucosa, CD8 +tissue resident memory T cells (T RM) act as sentinels that rapidly respond to, and mediate protection against, respiratory viruses. In mice, T RMhave been shown to mediate protection at barrier sites by producing cytokines, chemokines, and performing cell lysis. However, in humans, less is known about the effector functions of virus-specific lung CD8 +T RM. Using cells from healthy human lung donors, we first identified and quantified the frequency of antigen-specific cells against common respiratory viruses by performing intracellular cytokine staining. We next investigated the polyfunctionality and residency profiles of responding antigen-specific CD8 +T cells and found that that CD69 +CD103 +T RMcomprise a larger portion of the antigen-specific CD8 +T cells for viruses largely restricted to the respiratory epithelium when compared to cells specific for viruses with a broader tissue tropism. Next, by performing a series of in vitro peptide stimulation and cytokine neutralization experiments, we investigated how antigen-specific CD8 +T cells impact local innate and epithelial cells. When stimulated with their cognate antigen, lung CD8 +T RM-derived IFNγ strongly correlated with epithelial cell activation. Finally, we stimulated lung CD8 +T RMwith cognate antigen +/− anti-IFNγ and performed RNAseq on responding antigen-specific CD8 +T RM(CD25 +CD137 +) and epithelial cells to determine exactly how T RM-derived IFNγ alters the cellular program of these cells. Results of this study suggest that human lung CD8 +T RMact to rapidly reprogram local immune cells, and these data will ultimately help us understand how CD8 +T RMfit into the overall immune response to respiratory viruses. This project is supported by R35 HL150803 and the Emory Center of Excellence for Influenza Research and Response (CEIRR) 75N93019R0028.

Machine Learning-based Prediction of Relative Regional Air Volume Change from Healthy Human Lung CTs

Machine Learning-based Prediction of Relative Regional Air Volume Change from Healthy Human Lung CTs