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1.
J Immunol ; 208(5): 1021-1033, 2022 03 01.
Article in English | MEDLINE | ID: mdl-35173036

ABSTRACT

Lung infections are a perennial leading cause of death worldwide. The lung epithelium comprises three main cell types: alveolar type I (AT1), alveolar type II (AT2), and bronchiolar cells. Constitutively, these three cell types express extremely low amounts of surface MHC class I (MHC I) molecules, that is, <1% of levels found on medullary thymic epithelial cells (ECs). We report that inhalation of the TLR4 ligand LPS upregulates cell surface MHC I by ∼25-fold on the three subtypes of mouse lung ECs. This upregulation is dependent on Nlrc5, Stat1, and Stat2 and caused by a concerted production of the three IFN families. It is nevertheless hampered, particularly in AT1 cells, by the limited expression of genes instrumental in the peptide loading of MHC I molecules. Genes involved in production and response to cytokines and chemokines were selectively induced in AT1 cells. However, discrete gene subsets were selectively downregulated in AT2 or bronchiolar cells following LPS inhalation. Genes downregulated in AT2 cells were linked to cell differentiation and cell proliferation, and those repressed in bronchiolar cells were primarily involved in cilium function. Our study shows a delicate balance between the expression of transcripts maintaining lung epithelium integrity and transcripts involved in Ag presentation in primary lung ECs.


Subject(s)
Alveolar Epithelial Cells/metabolism , Histocompatibility Antigens Class I/metabolism , Interferons/metabolism , Lipopolysaccharides/immunology , Respiratory Mucosa/immunology , Administration, Inhalation , Alveolar Epithelial Cells/immunology , Animals , Antigen Presentation/immunology , Bronchioles/cytology , Bronchioles/metabolism , Cell Differentiation/genetics , Cell Proliferation/genetics , Cilia/physiology , Cytokines/metabolism , Inflammation/pathology , Intracellular Signaling Peptides and Proteins/metabolism , Lung/immunology , Lung/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Up-Regulation
2.
Food Chem Toxicol ; 161: 112852, 2022 Mar.
Article in English | MEDLINE | ID: mdl-35131362

ABSTRACT

While an association between acrylamide (AC) exposure and the risk of developing cancer has been shown in some studies, there are very limited data on the relationship between AC exposure and lung cancer risk. Thus, we investigated the cytotoxic, genotoxic, and carcinogenic effects of AC on human lung bronchial epithelial cell line (BEAS-2B cells). AC (5 and 10 mM) significantly decreased the cell viability for all treatment times. The comet assay results showed that AC (0.5, 1 and 5 mM) increased the DNA tail (%), tail moment and olive tail moment. By using immunofluorescence, we found that AC (0.5, 1 and 5 mM) induced the formation of both phosphorylated form of the histone H2 variant H2AX (gH2AX) and p53-binding protein 1 (53BP1) foci. AC-treated BEAS-2B cells exhibited various morphological and cytoplasmic changes. The transformed cells can induce form foci and significantly increase the number of colonies in soft agar. We showed for the first time that AC could induce DNA strand breaks, cell transformation, and anchorage-independent growth in BEAS-2B cells. Therefore, AC exposure can induce carcinogenesis in lung cells and may be a risk for lung cancer formation. Further studies are necessary to make a possible risk assessment in humans.


Subject(s)
Acrylamide/toxicity , Carcinogenicity Tests , Cell Survival/drug effects , Epithelial Cells/drug effects , Lung/cytology , Mutagenicity Tests , Acrylamide/administration & dosage , Acrylamide/chemistry , Cell Line , Dose-Response Relationship, Drug , Female , Humans , Lung Neoplasms/chemically induced , Male , Molecular Structure , Respiratory Mucosa/cytology
3.
Bioengineered ; 13(2): 3137-3147, 2022 02.
Article in English | MEDLINE | ID: mdl-35037821

ABSTRACT

Asthma is a respiratory disease with complex pathogenesis. Sterol-responsive element-binding proteins 2 (SREBP2) was found to bind to promoter sequences of ABCA1 to suppress ABCA1 promoter activity. This study aimed to explore the expression level of SREBP2 and ATP-binding cassette transporter A1 (ABCA1), and their effects on the development of airway smooth muscle cells (ASMCs) in asthma. ASMCs were treated with different concentrations of TGF-ß1 (0, 0.5, 1, 5 and 10 ng/mL). Short hairpin SREBP2 (shSREBP2), SREBP2, shABCA1 or ABCA1 were transfected into ASMCs. Cell viability, proliferation, apoptosis, migration, and the expression of SREBP2, ABCA1 and related pathway proteins were detected by MTT assay, Brdu staining, flow cytometer, Transwell assay, qRT-PCR, and Western blotting, respectively. The results showed that TGF-ß1 increased the viability, proliferation, migration and inhibited apoptosis in ASMCs. Moreover, TGF-ß1 also decreased the expression of ABCA1, cleaved caspase-3, cleaved PARP, E-cadherin, and increased the expression of vimentin, TLR2, p-p65 and NFATc1. SREBP2 knockdown alleviated these TGF-ß1-induced changes. SREBP2 overexpression inhibited ABCA1 expression and apoptosis, and promoted cell migration and the expression of TLR2, p-p65, NFATc1 in ASMCs. ABCA1 overexpression alleviated these SREBP2-induced promoting and inhibition effects. In conclusion, SREBP2 activates TLR2/NF-κB/NFATc1 regulatory network and promotes TGF-ß1-induced cell movement through inhibiting ABCA1 expression.


Subject(s)
Myocytes, Smooth Muscle , Sterol Regulatory Element Binding Protein 2/physiology , Transforming Growth Factor beta1/pharmacology , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Humans , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/physiology , NF-kappa B/genetics , NF-kappa B/metabolism , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Respiratory System/cytology , Respiratory System/drug effects , Respiratory System/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism
4.
J Fluoresc ; 32(1): 397-404, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34977993

ABSTRACT

Reported here is a new [Cu4I4] cluster-based coordination polymer, namely [Cu4I4(bib)2]n·n(DMF) (1, bib = 1,4-bis(imidazolyl)butane, DMF = N,N'-dimethylformamide), which was synthesized by the self-assemble reaction of CuI, bib and KI under solvothermal conditions. Remarkably, compound 1 shows promising photocatalytic performance toward to the degradation of MB solution under visible light irradiation. For the COPD treatment, the ELISA detection kit was conducted to determine the content of INF-γ released by the respiratory tract mucosal epithelial cells. In addition to this, the activation levels of the NF-κB signaling pathway were still need to be assessed by the real time RT-PCR after the compound treatment.


Subject(s)
Copper/chemistry , Copper/pharmacology , Interferon-gamma/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Animals , Catalysis , Epithelial Cells/metabolism , Humans , Lethal Dose 50 , Mice , NF-kappa B/metabolism , Photochemical Processes , Polymers , Real-Time Polymerase Chain Reaction , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , Signal Transduction , X-Ray Diffraction
5.
Bioengineered ; 12(1): 7694-7703, 2021 12.
Article in English | MEDLINE | ID: mdl-34608825

ABSTRACT

The aim of this study was to explore the effects and action mechanism of Zhike Pingchuan Granule in human bronchial epithelial cells induced by IL-6 or the supernatant of M2. Upon IL-6 stimulation at different doses, Cell Counting Kit-8 (CCK8) assay and flow cytometry were, respectively, utilized to detect the cell viability and apoptosis levels of 16-HBE cells. ELISA and Western blot were, respectively, used to analyze the inflammatory markers and JAK2/STAT3 signals. Immunofluorescence assay was performed to identify M0 and M2 cells. As shown in results, ZKPC perturbed the expression of IL-6 inducible genes important for apoptosis, oxidative and inflammatory response, which was enhanced by JAK2 inhibitor. Besides the inhibitory effects on the phosphorylation levels of JAK2/STAT3, ZKPC markedly increased cell viability and reduced apoptosis in human bronchial epithelial cells (16-HBE) cultured in the supernatant of M2 cells. Collectively, ZKPC could inhibit the IL-6-induced JAK/STAT3 signaling cascade, increase cell viability and decrease apoptosis induced by the supernatant of M2. A more comprehensive understanding of the action mechanism of ZKPC on JAK2/STAT3 signaling pathway in human bronchial epithelial cells induced by IL-6 or M2 supernatant will enable ZKPC development in the control of asthma.


Subject(s)
Apoptosis/drug effects , Drugs, Chinese Herbal/pharmacology , Epithelial Cells/drug effects , Interleukin-6/metabolism , Macrophages/drug effects , Bronchi/cytology , Cell Survival , Cells, Cultured , Humans , Pyrrolidines , Respiratory Mucosa/cytology , Sulfonamides
6.
Kaohsiung J Med Sci ; 38(2): 87-96, 2022 Feb.
Article in English | MEDLINE | ID: mdl-34529353

ABSTRACT

Pyroptosis is a novel proinflammatory programmed cell death process. This study was designed to investigate the functional mechanisms of long noncoding RNA growth arrest-specific transcript 5 (lncRNA GAS5) on lipopolysaccharide (LPS)-induced human bronchial epithelial cell (HBEC) pyroptosis. LPS was used to induce pyroptosis in HBECs, followed by the detection of the expression of GAS5, forkhead box O3 (FOXO3), and nuclear factor E2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway-related factors. Cell viability was evaluated using CCK-8 assay, lactate dehydrogenase (LDH) release was assessed by LDH assay kit and caspase-1 activity by flow cytometry. Furthermore, expression of NOD-like receptor family pyrin domain containing 3 and pyroptosis-related proteins was evaluated using Western blot analysis, while enzyme-linked immunosorbent assay was used to determine the levels of inflammatory factors. The interaction between GAS5 and FOXO3 was confirmed using bioinformatic prediction, RNA immunoprecipitation assay, RNA pull-down, and dual-luciferase reporter gene assay. Treatment of HBECs with LPS upregulated the expression of GAS5 and FOXO3, resulting in the inactivation of the Nrf2/HO-1 signaling pathway. On the other hand, inhibition of both GAS5 and FOXO3 promoted cell viability, reduced LDH release, pyroptosis, and inflammatory response in LPS-induced HBECs. Furthermore, FOXO3 could interact with GAS5, while FOXO3 overexpression reversed the inhibitory effect of GAS5 knockdown on cell pyroptosis. Thus, mechanistically, inhibition of FOXO3 activates the Nrf2/HO-1 pathway to suppress LPS-induced pyroptosis in HBECs. This study revealed that GAS5 knockdown attenuates FOXO3 expression thereby activating the Nrf2/HO-1 pathway to inhibit LPS-induced pyroptosis in HBECs. These findings may contribute to identifying novel targets that inhibit pyroptosis in HBECs.


Subject(s)
Bronchi/cytology , Epithelial Cells , Forkhead Box Protein O3/physiology , Pyroptosis , RNA, Long Noncoding/physiology , RNA, Small Nucleolar/genetics , Respiratory Mucosa/cytology , Cells, Cultured , Epithelial Cells/drug effects , Humans , Lipopolysaccharides/pharmacology , Pyroptosis/drug effects
7.
J Ethnopharmacol ; 283: 114694, 2022 Jan 30.
Article in English | MEDLINE | ID: mdl-34601084

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: The flower buds of Tussilago farfara L. (Abbreviated as FTF) were widely used in traditional Chinese medicine (TCM) to treat respiratory diseases, including asthma, dry throat, great thirst, turbid saliva, stinky pus, and coughs caused by various causes. AIM OF STUDY: The aim of study is to explore the efficiency of FTF in vitro and in vivo for the treatment of lung inflammation, and to illustrate the possible mechanisms of FTF in treating inflammation-related respiratory diseases targeting NOD-like receptor 3 (NLRP3) inflammasome, nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear transcription factor-κB (NF-κB). METHODS: Lung inflammation model in vivo was induced by exposure of mice to cigarette smoke (CS) for two weeks. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), inflammatory factors, and histology in lung tissues were investigated in presence or absence of ethanol extract of the flower buds of T. farfara L. (FTF-EtOH). In the cell-based models, nitric oxide (NO) assay, flow cytometry assay, enzyme-linked immunosorbent assay (Elisa), and glutathione (GSH) assay were used to explore the anti-inflammatory and anti-oxidant effects of FTF-EtOH. Possible anti-inflammatory mechanisms of FTF targeting NLRP3 inflammasome, Nrf2, and NF-κB have been determined using western blot, quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), immunofluorescence assay, nuclear and cytoplasmic extraction, and ubiqutination assay. RESULTS: FTF-EtOH suppressed CS-induced overproduction of inflammatory factors [e.g., tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)], and upregulation of the content of intracellular MDA in the lung homogenate of mice. In cell-based models, FTF-EtOH reduced the lipopolysaccharide (LPS)-induced overproduction of inflammatory factors, and attenuated the CS extract-induced overgeneration of reactive oxygen species (ROS). Furthermore, FTF-EtOH up-regulated Nrf2 and its downstream genes through enhancing the stability of Nrf2 protein, and inhibited the activation of NF-κB and NLRP3 inflammasome, which have been confirmed by detecting the protein levels in the mouse model. CONCLUSIONS: FTF-EtOH effectively attenuated lung inflammation in vitro and in vivo. The protection of FTF-EtOH against inflammation was produced by activation of Nrf2 and inhibitions of NF-κB and NLRP3 inflammasome. These datas definitely support the ethnopharmacological use of FTF as an anti-inflammatory drug for treating respiratory diseases in TCM.


Subject(s)
Inflammation/drug therapy , Lung Diseases/drug therapy , Phytotherapy , Plant Extracts/therapeutic use , Smoke/adverse effects , Tussilago/chemistry , Animals , Cell Line , Cell Survival/drug effects , Epithelial Cells/drug effects , Flowers/chemistry , Humans , Inflammation/chemically induced , Lung Diseases/chemically induced , Male , Mice , Mice, Inbred C57BL , Plant Extracts/chemistry , Respiratory Mucosa/cytology , Tobacco
8.
Elife ; 102021 08 13.
Article in English | MEDLINE | ID: mdl-34463615

ABSTRACT

Background: SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. Methods: We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Results: Infected ALO monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2→AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Conclusions: Findings validate a human lung model of COVID-19, which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines. Funding: This work was supported by the National Institutes for Health (NIH) grants 1R01DK107585-01A1, 3R01DK107585-05S1 (to SD); R01-AI141630, CA100768 and CA160911 (to PG) and R01-AI 155696 (to PG, DS and SD); R00-CA151673 and R01-GM138385 (to DS), R01- HL32225 (to PT), UCOP-R00RG2642 (to SD and PG), UCOP-R01RG3780 (to P.G. and D.S) and a pilot award from the Sanford Stem Cell Clinical Center at UC San Diego Health (P.G, S.D, D.S). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. L.C.A's salary was supported in part by the VA San Diego Healthcare System. This manuscript includes data generated at the UC San Diego Institute of Genomic Medicine (IGC) using an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929).


Subject(s)
Adult Stem Cells , COVID-19 , Lung/pathology , Models, Biological , Organoids , Adult Stem Cells/virology , COVID-19/pathology , COVID-19/virology , Female , Humans , Lung/cytology , Lung/virology , Male , Middle Aged , Organoids/virology , Pulmonary Alveoli/cytology , Pulmonary Alveoli/virology , Respiratory Mucosa/cytology , Respiratory Mucosa/virology
9.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Article in English | MEDLINE | ID: mdl-34548396

ABSTRACT

Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, "tethered" mucus layer in chronic muco-obstructive lung diseases.


Subject(s)
Epithelial Cells/metabolism , Mucin 5AC/chemistry , Mucin 5AC/metabolism , Mucin-5B/chemistry , Mucin-5B/metabolism , Respiratory Mucosa/metabolism , Cells, Cultured , Epithelial Cells/cytology , Humans , Respiratory Mucosa/cytology
10.
Am J Physiol Lung Cell Mol Physiol ; 321(6): L1072-L1088, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34612064

ABSTRACT

Human organ-on-a-chip models are powerful tools for preclinical research that can be used to study the mechanisms of disease and evaluate new targets for therapeutic intervention. Lung-on-a-chip models have been one of the most well-characterized designs in this field and can be altered to evaluate various types of respiratory disease and to assess treatment candidates prior to clinical testing. These systems are capable of overcoming the flaws of conventional two-dimensional (2-D) cell culture and in vivo animal testing due to their ability to accurately recapitulate the in vivo microenvironment of human tissue with tunable material properties, microfluidic integration, delivery of precise mechanical and biochemical cues, and designs with organ-specific architecture. In this review, we first describe an overview of currently available lung-on-a-chip designs. We then present how recent innovations in human stem cell biology, tissue engineering, and microfabrication can be used to create more predictive human lung-on-a-chip models for studying respiratory disease. Finally, we discuss the current challenges and future directions of lung-on-a-chip designs for in vitro disease modeling with a particular focus on immune and multiorgan interactions.


Subject(s)
Alveolar Epithelial Cells/physiology , Models, Biological , Respiratory Mucosa/physiology , Respiratory Tract Diseases/physiopathology , Alveolar Epithelial Cells/cytology , Animals , Drug Evaluation, Preclinical , Humans , Lab-On-A-Chip Devices , Respiratory Mucosa/cytology , Tissue Engineering
11.
Microbiol Spectr ; 9(2): e0126021, 2021 10 31.
Article in English | MEDLINE | ID: mdl-34612698

ABSTRACT

Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.


Subject(s)
Bronchioles/virology , Bronchoalveolar Lavage Fluid/virology , COVID-19/pathology , Endogenous Retroviruses/growth & development , Respiratory Mucosa/virology , Bronchioles/cytology , Endogenous Retroviruses/isolation & purification , Epithelial Cells/virology , Humans , Inflammation/virology , Leukocytes, Mononuclear/virology , Respiratory Mucosa/cytology , SARS-CoV-2 , Transcriptome/genetics , Up-Regulation
12.
Nat Cell Biol ; 24(1): 10-23, 2022 01.
Article in English | MEDLINE | ID: mdl-34969962

ABSTRACT

Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-ß1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1hi/TGFB1hi fibroblasts. Our study indicates that hAEC2 loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through an hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.


Subject(s)
Cell Transdifferentiation/physiology , Epithelial Cells/cytology , Idiopathic Pulmonary Fibrosis/pathology , Keratin-5/metabolism , Pulmonary Alveoli/cytology , Respiratory Mucosa/cytology , Alveolar Epithelial Cells/metabolism , Animals , Bone Morphogenetic Proteins/metabolism , Cell Differentiation , Cells, Cultured , Epidermal Cells/cytology , Fibroblasts/cytology , Humans , Mesoderm/cytology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Signal Transduction/physiology , Single-Cell Analysis , Transforming Growth Factor beta1/metabolism
13.
J Immunol ; 208(2): 407-419, 2022 01 15.
Article in English | MEDLINE | ID: mdl-34965963

ABSTRACT

Tuberculosis consistently causes more deaths worldwide annually than any other single pathogen, making new effective vaccines an urgent priority for global public health. Among potential adjuvants, STING-activating cyclic dinucleotides (CDNs) uniquely stimulate a cytosolic sensing pathway activated only by pathogens. Recently, we demonstrated that a CDN-adjuvanted protein subunit vaccine robustly protects against tuberculosis infection in mice. In this study, we delineate the mechanistic basis underlying the efficacy of CDN vaccines for tuberculosis. CDN vaccines elicit CD4 T cells that home to lung parenchyma and penetrate into macrophage lesions in the lung. Although CDNs, like other mucosal vaccines, generate B cell-containing lymphoid structures in the lungs, protection is independent of B cells. Mucosal vaccination with a CDN vaccine induces Th1, Th17, and Th1-Th17 cells, and protection is dependent upon both IL-17 and IFN-γ. Single-cell RNA sequencing experiments reveal that vaccination enhances a metabolic state in Th17 cells reflective of activated effector function and implicate expression of Tnfsf8 (CD153) in vaccine-induced protection. Finally, we demonstrate that simply eliciting Th17 cells via mucosal vaccination with any adjuvant is not sufficient for protection. A vaccine adjuvanted with deacylated monophosphoryl lipid A (MPLA) failed to protect against tuberculosis infection when delivered mucosally, despite eliciting Th17 cells, highlighting the unique promise of CDNs as adjuvants for tuberculosis vaccines.


Subject(s)
Adjuvants, Immunologic/pharmacology , Interleukin-17/immunology , Mycobacterium tuberculosis/immunology , Th17 Cells/immunology , Tuberculosis Vaccines/immunology , Tuberculosis, Pulmonary/prevention & control , Animals , CD30 Ligand/metabolism , Interferon-gamma/immunology , Lung/cytology , Lung/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Tuberculosis, Pulmonary/immunology , Vaccination
14.
Nat Immunol ; 23(1): 23-32, 2022 01.
Article in English | MEDLINE | ID: mdl-34937933

ABSTRACT

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Nasopharynx/immunology , Nose/cytology , Respiratory Mucosa/immunology , SARS-CoV-2/immunology , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/pathology , Granulocytes/immunology , HLA-DR Antigens/metabolism , Humans , Killer Cells, Natural/immunology , Memory T Cells/immunology , Monocytes/immunology , Nasopharynx/cytology , Nasopharynx/virology , Neutrophils/immunology , Nose/immunology , Nose/virology , Prospective Studies , Respiratory Mucosa/cytology , Respiratory Mucosa/virology
15.
STAR Protoc ; 2(4): 100892, 2021 12 17.
Article in English | MEDLINE | ID: mdl-34746861

ABSTRACT

Neutrophils are difficult to study, particularly in tissues, due to their short half-life and propensity for activation. We describe an organotypic airway model that uses patient airway fluid to enable the transmigration of blood neutrophils to acquire an airway-like phenotype in order to better understand their contribution to airway diseases. In particular, we showcase how conditioned neutrophils modulate their bacteria-killing abilities. For complete details on the use and execution of this protocol, please refer to Margaroli et al. (2021).


Subject(s)
Cell Culture Techniques/methods , Neutrophils , Respiratory Mucosa , Bacteria/immunology , Cell Movement/physiology , Cell Transdifferentiation , Cells, Cultured , Humans , Microbial Viability/immunology , Models, Biological , Neutrophils/cytology , Neutrophils/immunology , Neutrophils/physiology , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Respiratory Mucosa/physiology
16.
Microbiol Spectr ; 9(2): e0019221, 2021 10 31.
Article in English | MEDLINE | ID: mdl-34704784

ABSTRACT

Pseudomonas aeruginosa is a ubiquitous human pathogen that causes severe infections. Although antibiotics, such as tobramycin, are currently used for infection therapy, their antibacterial activity has resulted in the emergence of multiple antibiotic-resistant bacteria. The 6-gingerol analog, a structural derivative of the main component of ginger, is a quorum sensing (QS) inhibitor. However, it has a lower biofilm inhibitory activity than antibiotics and the possibility to cause toxicity in humans. Therefore, novel and more effective approaches for decreasing dosing concentration and increasing biofilm inhibitory activity are required to alleviate P. aeruginosa infections. In this study, a 6-gingerol analog was combined with tobramycin to treat P. aeruginosa infections. The combined treatment of 6-gingerol analog and tobramycin showed strong inhibitory activities on biofilm formation and the production of QS-related virulence factors of P. aeruginosa compared to single treatments. Furthermore, the combined treatment alleviated the infectivity of P. aeruginosa in an insect model using Tenebrio molitor larvae without inducing any cytotoxic effects in human lung epithelial cells. The 6-gingerol analog showed these inhibitory activities at much lower concentrations when used in combination with tobramycin. Adjuvant effects were observed through increased QS-disrupting processes rather than through antibacterial action. In particular, improved RhlR inactivation by this combination is a possible target for therapeutic development in LasR-independent chronic infections. Therefore, the combined treatment of 6-gingerol analog and tobramycin may be considered an effective method for treating P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is a pathogen that causes various infectious diseases through quorum-sensing regulation. Although antibiotics are mainly used to treat P. aeruginosa infections, they cause the emergence of resistant bacteria in humans. To compensate for the disadvantages of antibiotics and increase their effectiveness, natural products were used in combination with antibiotics in this study. We discovered that combined treatment with 6-gingerol analog from naturally-derived ginger substances and tobramycin resulted in more effective reductions of biofilm formation and virulence factor production in P. aeruginosa than single treatments. Our findings support the notion that when 6-gingerol analog is combined with tobramycin, the effects of the analog can be exerted at much lower concentrations. Furthermore, its improved LasR-independent RhlR inactivation may serve as a key target for therapeutic development in chronic infections. Therefore, the combined treatment of 6-gingerol analog and tobramycin is suggested as a novel alternative for treating P. aeruginosa infections.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Catechols/therapeutic use , Fatty Alcohols/therapeutic use , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Tobramycin/therapeutic use , Anti-Bacterial Agents/adverse effects , Biofilms/drug effects , Biofilms/growth & development , Catechols/adverse effects , Cell Line , Cell Proliferation/drug effects , Drug Resistance, Bacterial/genetics , Drug Resistance, Multiple, Bacterial/genetics , Epithelial Cells/drug effects , Fatty Alcohols/adverse effects , Humans , Pseudomonas aeruginosa/genetics , Quorum Sensing/drug effects , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects , Tobramycin/adverse effects
17.
J Cyst Fibros ; 20(6): e129-e139, 2021 11.
Article in English | MEDLINE | ID: mdl-34657818

ABSTRACT

BACKGROUND: In cystic fibrosis, the respiratory epithelium is the target tissue of both the genetic abnormality of the disease and of external aggressions, notably by pathogens (Pseudomonas aeruginosa). A detailed characterisation of the cystic fibrosis bronchial epithelium is however lacking, as most previous studies focused on the nasal epithelium or on cell lines. This study aimed to characterise the abnormal phenotype and epithelial-to-mesenchymal transition in cystic fibrosis bronchial epithelium and to evaluate in cell cultures whether abnormalities persist ex vivo. METHODS: Explant lung tissues (n = 44) were assessed for bronchial epithelial cell phenotyping by immunostaining. Human bronchial epithelial cells were derived from basal cells isolated from cystic fibrosis patients or control donors and cultured in air-liquid interface for 2, 4 or 6 weeks. RESULTS: Enhanced mucin 5AC and decreased ß-tubulin expression were observed in cystic fibrosis airways reflecting a decreased ciliated/goblet cell ratio, associated with increased number of vimentin-positive cells, indicating epithelial-to-mesenchymal transition process. These features were recapitulated in vitro, in cystic fibrosis-derived reconstituted epithelium. However, they were not induced by CFTR inhibition or Pseudomonas infection, and most abnormalities tended to disappear in long-term culture (6 weeks) except for increased fibronectin release, an epithelial-to-mesenchymal transition marker. CONCLUSIONS: This study provides new insights into airway epithelial changes in cystic fibrosis, which are imprinted through an acquired mechanism that we could not relate to CFTR function.


Subject(s)
Bronchi/cytology , Cystic Fibrosis/metabolism , Respiratory Mucosa/cytology , Adult , Biomarkers/metabolism , Cell Differentiation , Female , Humans , Male , Middle Aged , Mucin 5AC/metabolism , Tubulin/metabolism
18.
Toxicol Lett ; 353: 100-106, 2021 Dec 15.
Article in English | MEDLINE | ID: mdl-34653535

ABSTRACT

A major challenge in nanoparticle (NP) research is to elucidate how NPs activate initial targets in cells, leading to cytotoxicity and inflammation. We have previously shown that silica (Si)NPs induce pro-inflammatory responses in bronchial epithelial cells (BEAS-2B) via mechanisms involving transforming growth factor (TGF)-α release, and activation of MAP-kinase p38 and JNK besides NF-κB (p65). In the present study, the roles of scavenger receptors (SRs) in SiNP-induced cytokine responses in BEAS-2B cells were examined by siRNA silencing. Cells exposed to Si10 and Si50 (nominal sizes 10 and 50 nm) showed marked interleukin (IL)-6, CXCL8, IL-1α, IL-1ß responses. Transient knockdown of SR-B1, LOX-1 and CXCL16 reduced the Si10- and Si50-induced cytokine responses, to a different magnitude dependent on the particle size, SR and cytokine. Si10-induced TGF-α responses were also markedly reduced by knockdown of SR-B1 and CXCL16. Furthermore, the role of SR-B1 in Si10-induced phosphorylations of p65 and MAP-kinases p38 and JNK were examined, and no significant reductions were observed upon knockdown of SR-B1. In conclusion, LOX-1 and CXCL16 and especially SR-B1 seem to have important roles in mediating cytokine responses and TGF-α release due to SiNP exposure in BEAS-2B cells, without a down-stream role of MAP-kinase and NF-κB.


Subject(s)
Bronchi/cytology , Cytokines/metabolism , Epithelial Cells/drug effects , Nanoparticles/toxicity , Silicon Dioxide/metabolism , Bronchi/drug effects , Cell Line , Cell Survival/drug effects , Cytokines/genetics , Gene Expression Regulation/drug effects , Humans , Respiratory Mucosa/cytology
19.
Viruses ; 13(8)2021 08 12.
Article in English | MEDLINE | ID: mdl-34452468

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air-liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.


Subject(s)
Bronchi/immunology , Bronchi/virology , Immunity, Innate , Respiratory Mucosa/immunology , Respiratory Mucosa/virology , SARS-CoV-2/immunology , Aged , Aging/immunology , Bronchi/cytology , Bronchi/metabolism , COVID-19/immunology , Cell Death/genetics , Cells, Cultured , Cellular Senescence/genetics , Cytokines/biosynthesis , Cytokines/genetics , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/virology , Female , Humans , Inflammation , Interferons/biosynthesis , Interferons/genetics , Male , RNA-Seq , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , SARS-CoV-2/physiology , Signal Transduction/genetics
20.
Toxicol Lett ; 351: 53-64, 2021 Oct 15.
Article in English | MEDLINE | ID: mdl-34454013

ABSTRACT

Benzo[a]pyrene(B[a]P) is a known human carcinogen. The ability of B[a]P to form stable DNA adducts has been repeatedly demonstrated. However, the relationship between DNA adduct formation and cell damage and its underlying molecular mechanisms are less well understood. In this study, we determined the cytotoxicity of benzo[a]pyrenediolepoxide, a metabolite of B[a]P, in human bronchial epithelial cells (BEAS-2B). The formation of BPDE-DNA adducts was quantified using a dot blot. DNA damage resulting from the formation of BPDE-DNA adducts was detected by chromatin immuneprecipitation sequencing (ChIP-Seq), with minor modifications, using specific antibodies against BPDE. In total, 1846 differentially expressed gene loci were detected between the treatment and control groups. The distribution of the BPDE-bound regions indicated that BPDE could covalently bind with both coding and non-coding regions to cause DNA damage. However, the majority of binding occurred at protein-coding genes. Furthermore, among the BPDE-bound genes, we found 16 protein-coding genes related to DNA damage repair. We explored the response to BPDE exposure at the transcriptional level using qRT-PCR and observed a strong inhibition of EIF4A3. We then established an EIF4A3 overexpression cell model and performed comet assays, which revealed that the levels of DNA damage in EIF4A3-overexpressing cells were lower than those in normal cells following BPDE exposure. This suggests that the BPDE-DNA adduct-induced reduction in EIF4A3 expression contributed to the DNA damage induced by BPDE exposure in BEAS-2B cells. These novel findings indicate that ChIP-Seq combined with BPDE specific antibody may be used for exploring the underlying mechanism of DNA adduct-induced genomic damage.


Subject(s)
Benzo(a)pyrene/toxicity , DEAD-box RNA Helicases/metabolism , DNA Adducts , DNA Damage/drug effects , Epithelial Cells/drug effects , Eukaryotic Initiation Factor-4A/metabolism , Cell Line , Cloning, Molecular , DEAD-box RNA Helicases/genetics , Eukaryotic Initiation Factor-4A/genetics , Gene Expression Regulation/drug effects , Humans , Respiratory Mucosa/cytology
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