Cannabis smoke suppresses antiviral immune responses to influenza A in mice.

Rationale: Despite its increasingly widespread use, little is known about the impact of cannabis smoking on the response to viral infections like influenza A virus (IAV). Many assume that cannabis smoking will disrupt antiviral responses in a manner similar to cigarette smoking; however, since cannabinoids exhibit anti-inflammatory effects, cannabis smoke exposure may impact viral infection in distinct ways. Methods: Male and female BALB/c mice were exposed daily to cannabis smoke and concurrently intranasally instilled with IAV. Viral burden, inflammatory mediator levels (multiplex ELISA), lung immune cells populations (flow cytometry) and gene expression patterns (RNA sequencing) were assessed in the lungs. Plasma IAV-specific antibodies were measured via ELISA. Results: We found that cannabis smoke exposure increased pulmonary viral burden while decreasing total leukocytes, including macrophages, monocytes and dendritic cell populations in the lungs. Furthermore, infection-induced upregulation of certain inflammatory mediators (interferon-γ and C-C motif chemokine ligand 5) was blunted by cannabis smoke exposure, which in females was linked to the transcriptional downregulation of pathways involved in innate and adaptive immune responses. Finally, plasma levels of IAV-specific IgM and IgG1 were significantly decreased in cannabis smoke-exposed, infected mice compared to infected controls, only in female mice. Conclusions: Overall, cannabis smoke exposure disrupted host-defence processes, leading to increased viral burden and dampened inflammatory signalling. These results suggest that cannabis smoking is detrimental to the maintenance of pulmonary homeostasis during viral infection and highlight the need for data regarding the impact on immune competency in humans.

Potentiation of long-acting ß2-agonist and glucocorticoid responses in human airway epithelial cells by modulation of intracellular cAMP.

INTRODUCTION: Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP). HYPOTHESIS: Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy. METHODS: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA. RESULTS: Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES. CONCLUSION: Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses.

Type I interferons (IFNs) are our first line of defense against virus infection. Recent studies have suggested the ability of SARS-CoV-2 proteins to inhibit IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wild-type SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are scarce. Here we demonstrate that SARS-CoV-2 infection induces a type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Furthermore, we show that physiological levels of IFNα detected in patients with moderate COVID-19 is sufficient to suppress SARS-CoV-2 replication in human airway cells.

Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment.

NK cells are central to anti-tumor immunity and recently showed efficacy for treating hematologic malignancies. However, their dysfunction in the hostile tumor microenvironment remains a pivotal barrier for cancer immunotherapies against solid tumors. Using cancer patient samples and proteomics, we found that human NK cell dysfunction in the tumor microenvironment is due to suppression of glucose metabolism via lipid peroxidation-associated oxidative stress. Activation of the Nrf2 antioxidant pathway restored NK cell metabolism and function and resulted in greater anti-tumor activity in vivo. Strikingly, expanded NK cells reprogrammed with complete metabolic substrate flexibility not only sustained metabolic fitness but paradoxically augmented their tumor killing in the tumor microenvironment and in response to nutrient deprivation. Our results uncover that metabolic flexibility enables a cytotoxic immune cell to exploit the metabolic hostility of tumors for their advantage, addressing a critical hurdle for cancer immunotherapy.

Development and validation of an open-source, disposable, 3D-printed in vitro environmental exposure system for Transwell culture inserts.

Accessible in vitro models recapitulating the human airway that are amenable to study whole cannabis smoke exposure are needed for immunological and toxicological studies that inform public health policy and recreational cannabis use. In the present study, we developed and validated a novel three-dimensional (3D)-printed in vitro exposure system (IVES) that can be directly applied to study the effect of cannabis smoke exposure on primary human bronchial epithelial cells. Using commercially available design software and a 3D printer, we designed a four-chamber Transwell insert holder for exposures to whole smoke. COMSOL Multiphysics software was used to model gas distribution, concentration gradients, velocity profile and shear stress within IVES. Following simulations, primary human bronchial epithelial cells cultured at the air-liquid interface on Transwell inserts were exposed to whole cannabis smoke using a modified version of the Foltin puff procedure. Following 24 h, outcome measurements included cell morphology, epithelial barrier function, lactate dehydrogenase (LDH) levels, cytokine expression and gene expression. Whole smoke delivered through IVES possesses velocity profiles consistent with uniform gas distribution across the four chambers and complete mixing. Airflow velocity ranged between 1.0 and 1.5 µm·s-1 and generated low shear stresses (<<1 Pa). Human airway epithelial cells exposed to cannabis smoke using IVES showed changes in cell morphology and disruption of barrier function without significant cytotoxicity. Cannabis smoke elevated interleukin-1 family cytokines and elevated CYP1A1 and CYP1B1 expression relative to control, validating IVES smoke exposure impacts in human airway epithelial cells at a molecular level. The growing legalisation of cannabis on a global scale must be paired with research related to potential health impacts of lung exposures. IVES represents an accessible, open-source, exposure system that can be used to model varying types of cannabis smoke exposures with human airway epithelial cells grown under air-liquid interface culture conditions.

Expression of endocannabinoid system components in human airway epithelial cells: impact of sex and chronic respiratory disease status.

Cannabis smoking is the dominant route of delivery, with the airway epithelium functioning as the site of first contact. The endocannabinoid system is responsible for mediating the physiological effects of inhaled phytocannabinoids. The expression of the endocannabinoid system in the airway epithelium and contribution to normal physiological responses remains to be defined. To begin to address this knowledge gap, a curated dataset of 1090 unique human bronchial brushing gene expression profiles was created. The dataset included 616 healthy subjects, 136 subjects with asthma, and 338 subjects with COPD. A 32-gene endocannabinoid signature was analysed across all samples with sex and disease-specific analyses performed. Immunohistochemistry and immunoblots were performed to probe in situ and in vitro protein expression. CB1, CB2, and TRPV1 protein signal is detectable in human airway epithelial cells in situ and in vitro, justifying examining the downstream endocannabinoid pathway. Sex status was associated with differential expression of 7 of 32 genes. In contrast, disease status was associated with differential expression of 21 of 32 genes in people with asthma and 26 of 32 genes in people with COPD. We confirm at the protein level that TRPV1, the most differentially expressed candidate in our analyses, was upregulated in airway epithelial cells from people with asthma relative to healthy subjects. Our data demonstrate that the endocannabinoid system is expressed in human airway epithelial cells with expression impacted by disease status and minimally by sex. The data suggest that cannabis consumers may have differential physiological responses in the respiratory mucosa.

ABCF1 Regulates dsDNA-induced Immune Responses in Human Airway Epithelial Cells.

Background: The airway epithelium represents a critical component of the human lung that helps orchestrate defenses against respiratory tract viral infections, which are responsible for more than 2.5 million deaths/year globally. Innate immune activities of the airway epithelium rely on Toll-like receptors (TLRs), nucleotide binding and leucine-rich-repeat pyrin domain containing (NLRP) receptors, and cytosolic nucleic acid sensors. ATP Binding Cassette (ABC) transporters are ubiquitous across all three domains of life-Archaea, Bacteria, and Eukarya-and expressed in the human airway epithelium. ABCF1, a unique ABC family member that lacks a transmembrane domain, has been defined as a cytosolic nucleic acid sensor that regulates CXCL10, interferon-ß expression, and downstream type I interferon responses. We tested the hypothesis that ABCF1 functions as a dsDNA nucleic acid sensor in human airway epithelial cells important in regulating antiviral responses. Methods: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue, while confirmatory transcript and protein expression was performed in human airway epithelial cells. Functional experiments were performed with siRNA methods in a human airway epithelial cell line. Complementary transcriptomic analyses were performed to explore the contributions of ABCF1 to gene expression patterns. Results: Using archived human lung and human airway epithelial cells, we confirm expression of ABCF1 gene and protein expression in these tissue samples, with a role for mediating CXCL10 production in response to dsDNA viral mimic challenge. Although, ABCF1 knockdown was associated with an attenuation of select genes involved in the antiviral responses, Gene Ontology analyses revealed a greater interaction of ABCF1 with TLR signaling suggesting a multifactorial role for ABCF1 in innate immunity in human airway epithelial cells. Conclusion: ABCF1 is a candidate cytosolic nucleic acid sensor and modulator of TLR signaling that is expressed at gene and protein levels in human airway epithelial cells. The precise level where ABCF1 protein functions to modulate immune responses to pathogens remains to be determined but is anticipated to involve IRF-3 and CXCL10 production.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

Effect of long-acting ß-agonists/glucocorticoids on human airway epithelial cell cytokine, transcriptomic and oxidative stress responses to cannabis smoke.

LABA/GC intervention in airway epithelial cells exposed to cannabis smoke reduces levels of pro-inflammatory (CXCL8) and antiviral (CXCL10) mediators, while transcriptomic signatures of neutrophil-mediated immunity and oxidative stress remain elevated http://bit.ly/2qiSQhH.

Transcriptomic and barrier responses of human airway epithelial cells exposed to cannabis smoke.

Globally, many jurisdictions are legalizing or decriminalizing cannabis, creating a potential public health issue that would benefit from experimental evidence to inform policy, government regulations, and user practices. Tobacco smoke exposure science has created a body of knowledge that demonstrates the conclusive negative impacts on respiratory health; similar knowledge remains to be established for cannabis. To address this unmet need, we performed in vitro functional and transcriptomic experiments with a human airway epithelial cell line (Calu-3) exposed to cannabis smoke, with tobacco smoke as a positive control. Demonstrating the validity of our in vitro model, tobacco smoke induced gene expression profiles that were significantly correlated with gene expression profiles from published tobacco exposure datasets from bronchial brushings and primary human airway epithelial cell cultures. Applying our model to cannabis smoke, we demonstrate that cannabis smoke induced functional and transcriptional responses that overlapped with tobacco smoke. Ontology and pathway analysis revealed that cannabis smoke induced DNA replication and oxidative stress responses. Functionally, cannabis smoke impaired epithelial cell barrier function, antiviral responses, and increased inflammatory mediator production. Our study reveals striking similarities between cannabis and tobacco smoke exposure on impairing barrier function, suppressing antiviral pathways, potentiating of pro-inflammatory mediators, and inducing oncogenic and oxidative stress gene expression signatures. Collectively our data suggest that cannabis smoke exposure is not innocuous and may possess many of the deleterious properties of tobacco smoke, warranting additional studies to support public policy, government regulations, and user practices.

The impact of cigarette smoke exposure, COPD, or asthma status on ABC transporter gene expression in human airway epithelial cells.

ABC transporters are conserved in prokaryotes and eukaryotes, with humans expressing 48 transporters divided into 7 classes (ABCA, ABCB, ABCC, ABCD, ABDE, ABCF, and ABCG). Throughout the human body, ABC transporters regulate cAMP levels, chloride secretion, lipid transport, and anti-oxidant responses. We used a bioinformatic approach complemented with in vitro experimental methods for validation of the 48 known human ABC transporters in airway epithelial cells using bronchial epithelial cell gene expression datasets available in NCBI GEO from well-characterized patient populations of healthy subjects and individuals that smoke cigarettes, or have been diagnosed with COPD or asthma, with validation performed in Calu-3 airway epithelial cells. Gene expression data demonstrate that ABC transporters are variably expressed in epithelial cells from different airway generations, regulated by cigarette smoke exposure (ABCA13, ABCB6, ABCC1, and ABCC3), and differentially expressed in individuals with COPD and asthma (ABCA13, ABCC1, ABCC2, ABCC9). An in vitro cell culture model of cigarette smoke exposure was able to recapitulate select observed in situ changes. Our work highlights select ABC transporter candidates of interest and a relevant in vitro model that will enable a deeper understanding of the contribution of ABC transporters in the respiratory mucosa in lung health and disease.

Mutations in Mitochondrial DNA From Pancreatic Ductal Adenocarcinomas Associate With Survival Times of Patients and Accumulate as Tumors Progress.

Somatic mutations have been found in the mitochondria in different types of cancer cells, but it is not clear whether these affect tumorigenesis or tumor progression. We analyzed mitochondrial genomes of 268 early-stage, resected pancreatic ductal adenocarcinoma tissues and paired non-tumor tissues. We defined a mitochondrial somatic mutation (mtSNV) as a position where the difference in heteroplasmy fraction between tumor and normal sample was ≥0.2. Our analysis identified 304 mtSNVs, with at least 1 mtSNV in 61% (164 of 268) of tumor samples. The noncoding control region had the greatest proportion of mtSNVs (60 of 304 mutations); this region contains sites that regulate mitochondrial DNA transcription and replication. Frequently mutated genes included ND5, RNR2, and CO1, plus 29 mutations in transfer RNA genes. mtSNVs in 2 separate mitochondrial genes (ND4 and ND6) were associated with shorter overall survival time. This association appeared to depend on the level of mtSNV heteroplasmy. Non-random co-occurrence between mtSNVs and mutations in nuclear genes indicates interactions between nuclear and mitochondrial DNA. In an analysis of primary tumors and metastases from 6 patients, we found tumors to accumulate mitochondrial mutational mutations as they progress.

Mitochondrial mutations drive prostate cancer aggression.

Nuclear mutations are well known to drive tumor incidence, aggression and response to therapy. By contrast, the frequency and roles of mutations in the maternally inherited mitochondrial genome are poorly understood. Here we sequence the mitochondrial genomes of 384 localized prostate cancer patients, and identify a median of one mitochondrial single-nucleotide variant (mtSNV) per patient. Some of these mtSNVs occur in recurrent mutational hotspots and associate with aggressive disease. Younger patients have fewer mtSNVs than those who diagnosed at an older age. We demonstrate strong links between mitochondrial and nuclear mutational profiles, with co-occurrence between specific mutations. For example, certain control region mtSNVs co-occur with gain of the MYC oncogene, and these mutations are jointly associated with patient survival. These data demonstrate frequent mitochondrial mutation in prostate cancer, and suggest interplay between nuclear and mitochondrial mutational profiles in prostate cancer.In prostate cancer, the role of mutations in the maternally-inherited mitochondrial genome are not well known. Here, the authors demonstrate frequent, age-dependent mitochondrial mutation in prostate cancer. Strong links between mitochondrial and nuclear mutational profiles are associated with clinical aggressivity.