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Aside from centrally induced trained immunity in the bone marrow (BM) and peripheral blood by parenteral vaccination or infection, evidence indicates that mucosal-resident innate immune memory can develop via a local inflammatory pathway following mucosal exposure. However, whether mucosal-resident innate memory results from integrating distally generated immunological signals following parenteral vaccination/infection is unclear. Here we show that subcutaneous Bacillus Calmette–Guérin (BCG) vaccination can induce memory alveolar macrophages (AMs) and trained immunity in the lung. Although parenteral BCG vaccination trains BM progenitors and circulating monocytes, induction of memory AMs is independent of circulating monocytes. Rather, parenteral BCG vaccination, via mycobacterial dissemination, causes a time-dependent alteration in the intestinal microbiome, barrier function and microbial metabolites, and subsequent changes in circulating and lung metabolites, leading to the induction of memory macrophages and trained immunity in the lung. These data identify an intestinal microbiota-mediated pathway for innate immune memory development at distal mucosal tissues and have implications for the development of next-generation vaccine strategies against respiratory pathogens.

S1: RT-PCR confirms expression of E6 and E7 in TC1 S2: CD4+ T cell responses after E6E7 vaccination S3: E6E7 vaccination induces T-cell degranulation Tumor free S4: Tumor volume curves in E6E7 vaccinated mice S5: TCM frequency in mice after TC1 tumor clearance S6: PCA analysis of TC1 tumors S7: Images of MG1-GFP infected HPV+ primary tumor

Localised autoimmune responses have been reported in patients with severe eosinophilic asthma, characterised by eosinophil degranulation and airway infections.To determine the presence of autoantibodies against macrophage scavenger receptors within the airways and their effects on macrophage function and susceptibility to infection.Anti-eosinophil peroxidase (EPX), anti-macrophage receptor with collagenous structure (MARCO) immunoglobulin G (IgG) titers, and type 1/2 (T1/T2) cytokines were measured in 221 sputa from 143 well-characterized patients with severe asthma. Peripheral monocytes and monocyte-derived macrophages (MDMs) isolated from healthy controls were treated with immunoprecipitated immunoglobulins (IP-Igs) from sputa with high anti-MARCO titers or non-specific IgG to assess uptake of Streptococcus pneumoniae or response to the bacterial product lipopolysaccharide (LPS).Anti-MARCO IgG was detected in 36% of patients, with significantly higher titers (up to 1:16) in patients with mixed granulocytic sputa, indicative of airway infections. Multivariate regression analysis confirmed increased frequency of degranulation (free eosinophil granules), increased blood eosinophils (indicative of high T2 burden), increased sputum total cell count, peripheral blood leukocytes (indicative of infection) and lymphopenia were associated with increased anti-MARCO IgG titers; IL-15 (OR:1.79; CI-1.19-2.70), IL-13 (OR:1.06; CI-1.02-1.12) and IL-12p70 (OR:3.34; CI 1.32-8.40) were the associated cytokines. Patients with anti-MARCO antibodies had higher chances of subsequent infective vs. eosinophilic exacerbations (P=0.01). MDMs treated with IP-Igs (anti-MARCO+ sputa) had reduced bacterial uptake by 39±15% and significantly reduced release of IL-10 and GM-CSF (P0.05) in response to a lipopolysaccharide stimulus.Autoantibodies against macrophage scavenger receptors in eosinophilic asthma airways may impede effective host defenses and lead to recurrent infective bronchitis.

Chronic obstructive pulmonary disease (COPD) is a complex and progressive respiratory disease. Autoimmune processes have been hypothesized to contribute to disease progression; however, the presence of autoantibodies in the serum has been variable. Given that COPD is a lung disease, we sought to investigate whether autoantibodies in sputum supernatant would better define pulmonary autoimmune processes. Matched sputum and serum samples were obtained from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study and at the Guangzhou Institute of Respiratory Health (GIRH). Samples were collected from patients with varying severity of COPD, asymptomatic smokers, and healthy control subjects. IgG and IgM autoantibodies were detected in sputum and serum of all subjects in both cohorts using a broad-spectrum autoantigen array. No differences were observed in sputum autoantibodies between COPD and asymptomatic smokers in either cohort. In contrast, 16% of detectable sputum IgG autoantibodies were decreased in subjects with COPD compared to healthy controls in the ADEPT cohort. Compared to asymptomatic smokers, approximately 13% of detectable serum IgG and 40% of detectable serum IgM autoantibodies were differentially expressed in GIRH COPD subjects. Of the differentially expressed specificities, anti-nuclear autoantibodies were predominately decreased. A weak correlation between increased serum IgM anti-tissue autoantibodies and a measure of airspace enlargement was observed. The differential expression of specificities varied between the cohorts. In closing, using a comprehensive autoantibody array, we demonstrate that autoantibodies are present in subjects with COPD, asymptomatic smokers, and healthy controls. Cohorts displayed high levels of heterogeneity, precluding the utilization of autoantibodies for diagnostic purposes.

Discovery of the microbiota-gut–brain axis has led to proposed microbe-based therapeutic strategies in mental health, including the use of mood-altering bacterial species, termed psychobiotics. However, we still have limited understanding of the key signaling pathways engaged by specific organisms in modulating brain function, and evidence suggests that bacteria with broadly similar neuroactive and immunomodulatory actions can drive different behavioral outcomes. We sought to identify pathways distinguishing two psychoactive bacterial strains that seemingly engage similar gut–brain signaling pathways but have distinct effects on behaviour. We used RNAseq to identify mRNAs differentially expressed in the blood and hippocampus of mice following Lacticaseibacillus rhamnosus JB-1, and Limosilactobacillus reuteri 6475 treatment and performed Gene Set Enrichment Analysis (GSEA) to identify enrichment in pathway activity. L. rhamnosus, but not L. reuteri treatment altered several pathways in the blood and hippocampus, and the rhamnosus could be clearly distinguished based on mRNA profile. In particular, L. rhamnosus treatment modulated the activity of interferon signaling, JAK/STAT, and TNF-alpha via NF-KB pathways. Our results highlight that psychobiotics can induce complex changes in host gene expression, andin understanding these changes, we may help fine-tune selection of psychobiotics for treating mood disorders.

Type 1 alveolar epithelial cells (AT1s) and type 2 alveolar epithelial cells (AT2s) regulate the structural integrity and function of alveoli. AT1s mediate gas exchange, whereas AT2s serve multiple functions, including surfactant secretion and alveolar repair through proliferation and differentiation into AT1s as progenitors. However, mechanisms regulating AT2 proliferation and differentiation remain unclear. Here we demonstrate that Gremlin, an intrinsic inhibitor of bone morphogenetic protein (BMP), induces AT2 proliferation and differentiation. Transient overexpression of Gremlin in rat lungs by adenovirus vector delivery suppressed BMP signaling, induced proliferation of AT2s and the production of Bmp2, which in turn led to the recovery of BMP signaling and induced AT2 differentiation into AT1s. Gremlin was upregulated in a bleomycin-induced lung injury model. TGF-β and IL-1β induced Gremlin expression in fibroblasts. Taken together, our findings implicate that Gremlin expression during lung injury leads to precisely timed inhibition of BMP signaling and activates AT2s, leading to alveolar repair.

2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a direct role in both metabolism and protein stability. Oxidation of the metabolite cysteamine produces hypotaurine, while oxidation of N-terminal cysteines targets protein substrates for N-degron pathway-mediated degradation. Despite these known functions, the (patho)physiological roles of ADO remain obscure. By analyzing TCGA datasets we discovered that high ADO expression is associated with poor outcome for patients with hepatocellular carcinoma (HCC) (HR 1.2, p Citation Format: Sandy C-E Lee, Andrea H. Pyo, Helia Mohammadi, Ji Zhang, Anna Dvorkin-Gheva, Lucie Malbeteau, Stephen Chung, Shahbaz Khan, Thomas Kislinger, Julie A. Reisz, Courtney Jones, Marianne Koritzinsky. ADO is essential for redox homeostasis in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4783.

Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and excessive extracellular matrix deposition. Endoplasmic reticulum (ER) stress initiates the unfolded protein response (UPR), a cellular stress response pathway that has been implicated in both inflammatory and fibrotic processes. Here, we sought to investigate the role of the 13 kDa FK506-binding protein (FKBP13), an ER stress-inducible molecular chaperone, in various forms of pulmonary fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy samples from 24 patients with idiopathic pulmonary fibrosis (IPF) and 17 control subjects. FKBP13 expression was found to be elevated in the fibrotic regions of IPF lung tissues, and within this cohort, was correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also increased in lung biopsies of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using FKBP13-/- mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 21) and resolution (Day 50) phase. FKBP13-/- mice showed increased infiltration of inflammatory cells and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of fibrosis at Day 50. These changes were associated with an increased number of cells that stained positive for TUNEL and cleaved caspase 3 in the FKBP13-/- lungs, indicating a heightened cellular sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity or progression of interstitial lung diseases, and that it has a biologically relevant role in protecting mice against bleomycin-induced injury, inflammation and fibrosis.

Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

[Background] Beyond their specific experiment, video records of behavior have future value—for example, as inputs for new experiments or for yet unknown types of analysis of behavior—similar to tissue or blood sample banks in life sciences where clinically derived or otherwise well-described experimental samples are stored to be available for some unknown potential future purpose., [Findings] Research using an animal model of obsessive-compulsive disorder employed a standardized paradigm where the behavior of rats in a large open field was video recorded for 55 minutes on each test. From 43 experiments, there are 19,976 such trials that amount to over 2 years of continuous recording. In addition to videos, there are 2 video-derived raw data objects: XY locomotion coordinates and plots of animal trajectory. To motivate future use, the 3 raw data objects are annotated with a general schema—one that abstracts the data records from their particular experiment while providing, at the same time, a detailed list of independent variables bearing on behavioral performance. The raw data objects are deposited as 43 datasets but constitute, functionally, a library containing 1 large dataset., [Conclusions] Size and annotation schema give the library high reuse potential: in applications using machine learning techniques, statistical evaluation of subtle factors, simulation of new experiments, or as educational resource. Ultimately, the library can serve both as the seed and as the test bed to create a machine-searchable virtual library of linked open datasets for behavioral performance in defined conditions., Research using the animal model of OCD was supported by grants to Henry Szechtman from the Canadian Institutes of Health Research (CIHR) operating grants MOP-64424 and MT-12852, Ontario Mental Health Foundation (OMHF) Type A grant “Psychobiology of Security Motivation in an Animal Model of OCD,” and Natural Sciences and Engineering Research Council of Canada (NSERC) operating grant RGPIN A0544.

BackgroundAutoimmunity has been reported in patients with severe COVID-19. We investigated whether antinuclear/extractable-nuclear antibodies (ANAs) were present up to a year after infection, and if they were associated with the development of clinically relevant Post-Acute Sequalae of COVID-19 (PASC) symptoms.MethodsA rapid assessment line immunoassay was used to measure circulating levels of ANA/ENAs in 106 convalescent COVID-19 patients with varying acute phase severities at 3, 6, and 12 months post-recovery. Patient-reported fatigue, cough, and dyspnea were recorded at each timepoint. Multivariable logistic regression model and receiver-operating curves (ROC) were used to test the association of autoantibodies with patient-reported outcomes and pro-inflammatory cytokines.ResultsCompared to age- and sex-matched healthy controls (n=22) and those who had other respiratory infections (n=34), patients with COVID-19 had higher detectable ANAs at 3 months post-recovery (pInterpretationPersistently positive ANAs at 12 months post-COVID are associated with persisting symptoms and inflammation (TNFα) in a subset of COVID-19 survivors. This finding indicates the need for further investigation into the role of autoimmunity in PASC.

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/β-catenin signaling., The Wnt molecular subgroup of medulloblastoma is associated with better prognosis than the other molecular subgroups. Here, the authors show that activating Wnt signaling impairs tumor development and improves survival in Group 3 and Group 4 medulloblastoma preclinical models.

Tumor-targeted chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR-T cells) have demonstrated striking clinical success, but their use has been associated with a constellation of toxicities. A better understanding of the pathogenesis of these toxicities is required to improve the safety profile of CAR-T cells. Herein, we describe a xenograft model of off-tumor CAR-T cell-associated toxicity. Human CAR-T cells targeted against HER2 using a small-protein binding domain induced acute, dose-dependent toxicities in mice. The inclusion of a CD28 or 4-1BB co-stimulatory domain in the CAR was required to produce toxicity; however, co-stimulation through CD28 was most toxic on a per-cell basis. CAR-T cell activation in the lungs and heart was associated with a systemic cytokine storm. The severity of observed toxicities was dependent upon the peripheral blood mononuclear cell (PBMC) donor used as a T cell source and paralleled the CD4+-to-CD8+ T cell ratio in the adoptive transfer product. CD4+ CAR-T cells were determined to be the primary contributors to CAR-T cell-associated toxicity. However, donor-specific differences persisted after infusion of a purified CD4+ CAR-T cell product, indicating a role for additional variables. This work highlights the contributions of CAR-T cell-intrinsic variables to the pathogenesis of off-tumor toxicity., Graphical Abstract, Chimeric antigen receptor-engineered T cells (CAR-T cells) are capable of striking anti-tumor efficacy. However, their use has been associated with a constellation of toxicities. Herein, Bramson and colleagues describe a xenograft model of off-tumor CAR-T cell toxicity in a murine host. This model is used to investigate how variables intrinsic to a human CAR-T cell product, including PBMC donor, contribute to severity of toxicity.

Chronic lung disease accounts for a significant global burden with respect to death, disability, and health-care costs. Due to the heterogeneous nature and limited treatment options for these diseases, it is imperative that the cellular and molecular mechanisms underlying the disease pathophysiology are further understood. The lung is a complex organ with a diverse cell population, and each cell type will likely have different roles in disease initiation, progression, and resolution. The effectiveness of a given therapeutic agent may depend on the net effect on each of these cell types. Over the past decade, it has been established that endoplasmic reticulum stress and the unfolded protein response are involved in the development of several chronic lung diseases. These conserved cellular pathways are important for maintaining cellular proteostasis, but their aberrant activation can result in pathology. This review discusses the current understanding of endoplasmic reticulum stress and the unfolded protein response at the cellular level in the development and progression of various chronic lung diseases. We highlight the need for increased understanding of the specific cellular contributions of unfolded protein response activation to these pathologies and suggest that the development of cell-specific targeted therapies is likely required to further decrease disease progression and to promote resolution of chronic lung disease.

Asthma, an inflammatory disorder of the airways, is one of the most common chronic illnesses worldwide and is associated with significant morbidity. There is growing recognition of an association between asthma and mood disorders including post-traumatic stress disorder (PTSD) and major depressive disorder (MDD). Although there are several hypotheses regarding the relationship between asthma and mental health, there is little understanding of underlying mechanisms and causality. In the current study we utilized publicly available datasets of human blood mRNA collected from patients with severe and moderate asthma, MDD, and PTSD. We performed differential expression (DE) analysis and Gene Set Enrichment Analysis (GSEA) on diseased subjects against the healthy subjects from their respective datasets, compared the results between diseases, and validated DE genes and gene sets with 4 more independent datasets. Our analysis revealed that commonalities in blood transcriptomic changes were only found between the severe form of asthma and mood disorders. Gene expression commonly regulated in PTSD and severe asthma, included ORMDL3 a gene known to be associated with asthma risk and STX8, which is involved in TrkA signalling. We also identified several pathways commonly regulated to both MDD and severe asthma. This study reveals gene and pathway regulation that potentially drives the comorbidity between severe asthma, PTSD, and MDD and may serve as foci for future research aimed at gaining a better understanding of both the relationship between asthma and PTSD, and the pathophysiology of the individual disorders.

SummaryBackgroundAdenoviral (Ad)-vectored vaccines are typically administered via intramuscular injection to humans, but this route of delivery is unable to induce respiratory mucosal immunity which requires respiratory mucosal route of vaccination. However, inhaled aerosol delivery of Ad-vectored vaccines has remained poorly characterized and its ability to induce respiratory mucosal immunity in humans is still unknown. The goal of our study was to evaluate and compare the safety and immunogenicity of a human serotype 5 Ad-based tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to healthy humans via inhaled aerosol or intramuscular injection.MethodsIn this open-labeled phase 1b trial, 31 healthy adults between 18 and 55 years of age with a history of BCG vaccination were enrolled at McMaster University Medical Centre, Hamilton, Ontario, Canada. AdHu5Ag85A was administered by a single-dose aerosol using the Aeroneb® Solo Vibrating Mesh Nebulizer or by intramuscular (IM) injection; 11 in the low dose (LD, 1×106 PFU) aerosol group, 11 in the high dose (HD, 2×106 PFU) aerosol group and 9 in the IM (1×108 PFU) group. The primary outcome was safety of a single administration of vaccine delivered to the respiratory tract by aerosol or by IM injection. The vaccine-related local and systemic adverse events were collected from participants from a self-completed diary for 14 days after vaccination and at scheduled follow-up visits. Routine laboratory biochemical and haematological tests were measured at 2, 4 and 12 weeks after vaccination and lung function was measured at 2, 4, 8 and 12 weeks after vaccination. The secondary outcome was comparison of immunogenicity among the different routes and aerosol dose groups. Immunogenicity to aerosol or IM vaccination was measured both in the peripheral blood and bronchoalveolar lavage samples by Luminex, and cell surface and intracellular cytokine immunostaining. Anti-AdHu5 antibodies and neutralization titers were determined before and after vaccination using ELISA and bioassay, respectively. This trial is registered with ClinicalTrial.gov, NCT02337270.ResultsThe aerosol droplets generated by Aeroneb® Solo Nebulizer were mostly InterpretationInhaled aerosol delivery of Ad-vectored vaccine is a safe, economical and superior way to elicit respiratory mucosal immunity. The results of this study encourage further development of aerosol vaccine strategies against not only TB but also other respiratory pathogens including COVID-19.FundingThe Canadian Institutes for Health Research and the Natural Sciences and Engineering Research Council of Canada.Research in contextEvidence before this studyWe searched PubMed for published research articles without language or date restrictions by using search terms “adenovirus”, “aerosol” and “clinical trial” or “virus”, “vaccine”, “aerosol”, and “clinical trial”. Our search results indicate that adenoviral-vectored vaccine has rarely been delivered via inhaled aerosol into the respiratory tract of healthy humans. Adenoviral-vectored TB or HIV vaccine was delivered via inhaled aerosol to non-human primates, providing support for its further development for human application. An aerosolized adenoviral vector expressing CFTR was tested as a gene replacement therapeutic in cystic fibrosis patients. Recently, although an adenoviral-vectored COVID-19 vaccine was delivered via aerosol to humans, there were no evidence presented that it induced local mucosal immunity. Given the increased recognition of its value, inhaled aerosol has been explored in humans with non-adenoviral, viral vaccines against respiratory infections of global importance including measles and TB. However, since different aerosol technologies were used in these studies and aerosol characteristics including delivery efficiency vary according to the viral platform, aerosol delivery technology and its efficiency in inducing respiratory mucosal immunity remain to be established for administering adenoviral-vectored vaccine to healthy humans.Added value of this studyThis represents the first study to demonstrate the characteristics of a single-dose aerosolized human serotype 5 adenoviral-vectored vaccine, and its safety and immunogenicity in BCG-vaccinated healthy humans. It is also the first to show the superiority of inhaled aerosol immunization with this type of vaccine platform, over intramuscular (IM) route of immunization, in inducing respiratory mucosal immunity. Robust adaptive immune memory responses were induced in the respiratory tract with an aerosol vaccine dose up to 100 times smaller than the dose for IM immunization. Besides local mucosal immunity induced by aerosol immunization, it also induces levels of systemic immunity comparable to those by IM immunization. Also for the first time, we show that contrast to IM immunization, aerosol immunization does not increase either local or systemic anti-adenoviral neutralizing antibodies.Implications of all the available evidenceCollectively our findings show the technical feasibility, safety and potency of needle/pain-free delivery of a recombinant adenoviral-vectored vaccine to the respiratory tract of healthy adults. This vaccine strategy differs from parenteral route of immunization in its potency to elicit much desired respiratory mucosal immunity consisting of trained macrophages and tissue-resident memory T cells. The study provides the proof of concept to endorse inhaled aerosol vaccine strategies against pulmonary TB. Of further importance, as a number of currently authorized COVID-19 vaccines are also adenoviral-vectored, our study offers important technological details and scientific rationale for the development of inhalable next-generation COVID-19 vaccines aiming to induce all-around protective respiratory mucosal immunity in humans.

BackgroundCigarette smokers are at increased risk of acquiring influenza, developing severe disease and requiring hospitalisation/intensive care unit admission following infection. However, immune mechanisms underlying this predisposition are incompletely understood, and therapeutic strategies for influenza are limited.MethodsWe used a mouse model of concurrent cigarette smoke exposure and H1N1 influenza infection, colony-stimulating factor (CSF)3 supplementation/receptor (CSF3R) blockade and single-cell RNA sequencing (scRNAseq) to investigate this relationship.ResultsCigarette smoke exposure exacerbated features of viral pneumonia such as oedema, hypoxaemia and pulmonary neutrophilia. Smoke-exposed infected mice demonstrated an increase in viral (v)RNA, but not replication-competent viral particles, relative to infection-only controls. Interstitial rather than airspace neutrophilia positively predicted morbidity in smoke-exposed infected mice. Screening of pulmonary cytokines using a novel dysregulation score identified an exacerbated expression of CSF3 and interleukin-6 in the context of smoke exposure and influenza. Recombinant (r)CSF3 supplementation during influenza aggravated morbidity, hypothermia and oedema, while anti-CSF3R treatment of smoke-exposed infected mice improved alveolar–capillary barrier function. scRNAseq delineated a shift in the distribution of Csf3+ cells towards neutrophils in the context of cigarette smoke and influenza. However, although smoke-exposed lungs were enriched for infected, highly activated neutrophils, gene signatures of these cells largely reflected an exacerbated form of typical influenza with select unique regulatory features.ConclusionThis work provides novel insight into the mechanisms by which cigarette smoke exacerbates influenza infection, unveiling potential therapeutic targets (e.g. excess vRNA accumulation, oedematous CSF3R signalling) for use in this context, and potential limitations for clinical rCSF3 therapy during viral infectious disease.

Placental extravillous trophoblast (EVT) invasion is essential in establishing proper blood supply to the fetus during pregnancy. However, traditional 2D in vitro systems do not model the in vivo invasion process in an anatomically-relevant manner. Our objectives were to develop a 3D spheroid model that would allow better emulation of placental invasion in vitro and to characterize the transcriptomic and functional outcomes. HTR8/SVneo EVT cells were self-assembled into 3D spheroids using ultra-low attachment plates. Transcriptomic profiling followed by gene set enrichment and gene ontology analyses revealed major global transcriptomic differences, with significant up-regulations in EVTs cultured as 3D spheroids in canonical pathways and biological processes such as immune response, angiogenesis, response to stimulus, wound healing, and others. These findings were further validated by RT-qPCR, showing significant up-regulations in genes and/or proteins related to epithelial-mesenchymal transition, cell-cell contact, angiogenesis, and invasion/migration. A high-throughput, spheroid invasion assay was applied to reveal the dynamic invasion of EVTs away from the spheroid core into extracellular matrix. Lastly, lipopolysaccharide, dexamethasone, or Δ9-tetrahydrocannabinol exposure was found to impact the invasion of EVT spheroids. Altogether, we present a well-characterized, 3D spheroid model of EVT invasion and demonstrate its potential use in drug and toxin screening during pregnancy.

BackgroundLocal airway autoimmune responses may contribute to steroid dependence and persistent eosinophilia in severe asthma. Auto-IgG antibodies directed against granule proteins such as eosinophil peroxidase (EPX), macrophage scavenger receptor with collagenous structure (MARCO) and nuclear/extranuclear antigens (antinuclear antibodies (ANAs)) have been reported. Our objective was to describe the prevalence and clinical characteristics of asthmatic patients with airway autoreactivity, and to assess if this could be predicted from clinical history of autoreactivity.MethodsWe analysed anti-EPX, anti-MARCO and ANAs in 218 sputum samples collected prospectively from 148 asthmatic patients, and evaluated their association with lung function parameters, blood/airway inflammation, severity indices and exacerbations. Additionally, 107 of these patients consented to fill out an autoimmune checklist to determine personal/family history of systemic autoimmune disease and symptoms.ResultsOut of the 148 patients, 59 (40%) were anti-EPX IgG+, 53 (36%) were anti-MARCO IgG+and 64 out of 129 (50%) had ≥2 nuclear/extranuclear autoreactivities. A composite airway autoreactivity score (CAAS) demonstrated that 82 patients (55%) had ≥2 airway autoreactivities (considered as CAAS+). Increased airway eosinophil degranulation (OR 15.1, 95% CI 1.1–199.4), increased blood leukocytes (OR 3.5, 95% CI 1.3–10.1) and reduced blood lymphocytes (OR 0.19, 95% CI 0.04–0.84) predicted CAAS+. A third of CAAS+patients reported an exacerbation, associated with increased anti-EPX and/or anti-MARCO IgG (p+asthmatic patients reported sicca symptoms (p=0.02). Current anti-inflammatory (inhaled/oral corticosteroids and/or adjunct anti-interleukin-5 biologics) treatment does not attenuate airway autoantibodies, irrespective of eosinophil suppression.ConclusionWe report 55% of moderate–severe asthmatic patients to have airway autoreactivity that persists despite anti-inflammatory treatment and is associated with exacerbations.

Reductive power in the cytosol of pancreatic cancer cells drives a dependency on the endoplasmic reticulum protein PRDX4., There is an urgent need to identify vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here, we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces H2O2 metabolized by peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs–governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. Hence, this study has identified NOX4 as a protein that paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.

Background The T cell Antigen Couper (TAC) is a chimeric receptor that redirects the endogenous T cell receptor (TCR) against a tumor target via an extracellular antigen-binding domain to induce activation and oncolysis. TAC-engineered T cells (TAC-T cells) showed a similar capacity to activate T cells against the tumor-associated antigen HER2 as their classical chimeric antigen receptor (CAR)-engineered counterparts in vitro. However, in a xenograft model, anti-HER2 CAR-T cells gave rise to lethal off-target toxicity while TAC-T cells were efficacious and well-tolerated, despite utilizing the same antigen-binding domain.1 2 Here, we describe differences in T cell activation by TAC (canonical via endogenous TCR) and CAR (non-canonical independent of TCR) that make CAR-T cells less discriminate towards an off-target stimulus than TAC-T cells. Methods Paired sets of TAC- and CAR-engineered human T cells, utilizing a variety of antigen-binding domains, were compared in vitro to determine their propensity for tonic signaling and requirements for triggering T cell activation. Results Transcriptional profiling of CAR- and TAC- T cells in the absence of antigenic stimulus revealed an elevated basal activation status in CAR-T cells. Unstimulated CAR-T cells displayed elevated expression levels of activation and exhaustion markers, as well as basal cytokine production, versus their TAC-T cell counterparts. The degree of basal activation varied with the binding domain incorporated into the CAR, where some binding domains triggered functional exhaustion. Regardless of the binding domain, unstimulated TAC-T cells were indistinguishable from control T cells that expressed no synthetic receptor. Further, TAC-T cells displayed no evidence of functional exhaustion. TCR knock-out studies confirmed that TAC receptors signal via the endogenous TCR, whereas CAR signaling is TCR-independent. Consistent with TCR-dependent signaling, ligation of TAC receptors resulted in the formation of conventional immunological synapses, whereas ligation of CARs produced unconventional synapses. Despite these functional differences, CARs and TAC receptors demonstrated a similar capacity to activate T cells against antigen-positive tumor cell targets. However, CAR-T cells displayed reactivity to antigen-negative cells, due to interaction with a cross-reactive antigen; TAC-T cells displayed no reactivity to antigen-negative cells. Conclusions Tonic signaling in CAR-T cells reduces their activation threshold and increases their propensity to be activated by cross-reactive antigen. In contrast, TAC receptors do not deliver tonic signals, which increases the stringency of activation and reduces the likelihood of off-target responses. This feature of the TAC platform is advantageous to safeguard against the unexpected cross-reactivity that may occur when a new antigen-binding domain is deployed in vivo. Ethics Approval Use of human materials was approved by the Hamilton Integrated Research Ethics Board (HiREB). References Helsen C, Hammill JA, Lau VWC, Mwawasi KA, Afsahi A, Bezverbnaya K, Newhook L, Hayes DL, Aarts C, Bojovic B, Denisova GF, Kwiecien JM, Brain I, Derocher H, Milne K, Nelson BH, Bramson JL. The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity. Nat Commun 2018;9:3049. Hammill JA, Kwiecien JM, Dvorkin-Gheva A, Lau VWC, Baker C, Wu Y, Bezverbnaya K, Aarts C, Helsen CW, Denisova GF, Derocher H, Milne K, Nelson BH, Bramson JL. A cross-reactive small protein binding domain provides a model to study off-tumor CAR-T cell toxicity. Mol Ther Oncolytics 2020;17:278–292.

In December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises ACE2 and 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 alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection., ACE2 gene and protein expression is low to absent in airway and alveolar epithelial cells in human lungs. Our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung or other receptors for SARS-CoV-2.

In December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilizes ACE2 and 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 localization of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analyzed 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, 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. 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 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 alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

Background Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR). Methods We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. Results We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. Conclusions Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.