Primary human bronchial epithelial cells during tuberculosis infection

Infection with Mycobacterium tuberculosis (Mtb) is acquired through the lungs after inhalation of aerosolised bacteria. Epidemiological studies have shown that infection can only be detected in 30-50% of individuals after exposure, suggesting that the immune system can mediate clearance of or resistance to infection. Early responses to infection in an Mtb-naïve host are thought to be championed by the innate immune cells in the lungs, such as alveolar macrophages. However, the majority of cells exposed to inhaled air are epithelial, which line the entirety of the respiratory tract. Their function is not well understood in tuberculosis and almost no studies of primary airway epithelial cells have been undertaken. To identify their contribution to the lung immune responses to Mtb infection, human primary bronchial epithelial cells were interrogated in vitro and ex vivo. Primary bronchial epithelial cells (PBEC) were poor responders to low doses of Mtb in vitro and were markedly less susceptible to infection than macrophages. Large bacterial burden triggered cell death and IL8 release in an NADPH-oxidase dependent manner. Additionally, PBECs were potent responders to myeloid-mediated Mtb-driven inflammation. Myeloid-epithelial cross-talk was, in part, dependent on interleukin 1β and type I interferon-signalling and enhanced the antimicrobial host immune response and neutrophil influx in vitro. Through the assessment of the whole transcriptome and mediator profile of the human airway epithelial lining ex vivo, the in vitro inflammatory epithelial signature could be partially confirmed in patients with suspected pulmonary tuberculosis and Mtb-exposed individuals. Additionally, healthy recently infected Mtb-exposed individuals, showed signs of ongoing low-level immune activation in their nasal and bronchial epithelial mucosa. Taken together, this is the first comprehensive analysis of the human primary airway epithelial response to Mtb-infection and offers new insights into their emerging role in human tuberculosis.Open Acces

Executable network of SARS-CoV-2-host interaction predicts drug combination treatments

The COVID-19 pandemic has pushed healthcare systems globally to a breaking point. The urgent need for effective and affordable COVID-19 treatments calls for repurposing combinations of approved drugs. The challenge is to identify which combinations are likely to be most effective and at what stages of the disease. Here, we present the first disease-stage executable signalling network model of SARS-CoV-2-host interactions used to predict effective repurposed drug combinations for treating early- and late stage severe disease. Using our executable model, we performed in silico screening of 9870 pairs of 140 potential targets and have identified nine new drug combinations. Camostat and Apilimod were predicted to be the most promising combination in effectively supressing viral replication in the early stages of severe disease and were validated experimentally in human Caco-2 cells. Our study further demonstrates the power of executable mechanistic modelling to enable rapid pre-clinical evaluation of combination therapies tailored to disease progression. It also presents a novel resource and expandable model system that can respond to further needs in the pandemic

HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory T cells

HIV-1 replicates in CD4+ T cells, leading to AIDS. Determining how HIV-1 shapes its niche to create a permissive environment is central to informing efforts to limit pathogenesis, disturb reservoirs, and achieve a cure. A key roadblock in understanding HIV-T cell interactions is the requirement to activate T cells in vitro to make them permissive to infection. This dramatically alters T cell biology and virus-host interactions. Here we show that HIV-1 cell-to-cell spread permits efficient, productive infection of resting memory T cells without prior activation. Strikingly, we find that HIV-1 infection primes resting T cells to gain characteristics of tissue-resident memory T cells (TRM), including upregulating key surface markers and the transcription factor Blimp-1 and inducing a transcriptional program overlapping the core TRM transcriptional signature. This reprogramming is driven by Vpr and requires Vpr packaging into virions and manipulation of STAT5. Thus, HIV-1 reprograms resting T cells, with implications for viral replication and persistence

Preclinical and randomized phase I studies of plitidepsin in adults hospitalized with COVID-19

Plitidepsin, a marine-derived cyclic-peptide, inhibits SARS-CoV-2 replication at nanomolar concentrations by targeting the host protein eukaryotic translation elongation factor 1A. Here, we show that plitidepsin distributes preferentially to lung over plasma, with similar potency against across several SARS-CoV-2 variants in preclinical studies. Simultaneously, in this randomized, parallel, open-label, proof-of-concept study (NCT04382066) conducted in 10 Spanish hospitals between May and November 2020, 46 adult hospitalized patients with confirmed SARS-CoV-2 infection received either 1.5 mg (n = 15), 2.0 mg (n = 16), or 2.5 mg (n = 15) plitidepsin once daily for 3 d. The primary objective was safety; viral load kinetics, mortality, need for increased respiratory support, and dose selection were secondary end points. One patient withdrew consent before starting procedures; 45 initiated treatment; one withdrew because of hypersensitivity. Two Grade 3 treatment-related adverse events were observed (hypersensitivity and diarrhea). Treatment-related adverse events affecting more than 5% of patients were nausea (42.2%), vomiting (15.6%), and diarrhea (6.7%). Mean viral load reductions from baseline were 1.35, 2.35, 3.25, and 3.85 log10 at days 4, 7, 15, and 31. Nonmechanical invasive ventilation was required in 8 of 44 evaluable patients (16.0%); six patients required intensive care support (13.6%), and three patients (6.7%) died (COVID-19-related). Plitidepsin has a favorable safety profile in patients with COVID-19.This work was supported by grants from the Government of Spain (PIE_INTRAMURAL_ LINEA 1 - 202020E079; PIE_INTRAMURAL_CSIC-202020E043). The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols pharmaceutical. We also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). N Izquierdo-Useros has nonrestrictive funding from PharmaMar to study the antiviral effect of Plitidepsin. NJ Krogan was funded by grants from the National Institutes of Health (P50AI150476, U19AI135990, U19AI135972, R01AI143292, R01AI120694, and P01AI063302); by the Excellence in Research Award (ERA) from the Laboratory for Genomics Research (LGR), a collaboration between the University of California, San Francisco (UCSF), University of California, Berkley (UCB), and GlaxoSmithKline (GSK) (#133122P); by the Roddenberry Foundation, and gifts from QCRG philanthropic donors. This work was supported by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement #HR0011-19-2-0020. The views, opinions, and/or findings contained in this material are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. This research was partly funded by Center for Research for Influenza Pathogenesis and Transmission (CRIPT), a National Institute of Allergy and Infectious Diseases (NIAID) supported Center of Excellence for Influenza Research and Response (CEIRS, contract # 75N93021C00014), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grants U19AI142733, U19AI135972, and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to A García-Sastre. S Yildiz received funding from a Swiss National Foundation Early Postdoc Mobility fellowship (P2GEP3_184202).Peer reviewe

Plitidepsin has a positive therapeutic index in adult patients with COVID-19 requiring hospitalization

Plitidepsin is a marine-derived cyclic-peptide that inhibits SARS-CoV-2 replication at low nanomolar concentrations by the targeting of host protein eEF1A (eukaryotic translation-elongation-factor-1A). We evaluated a model of intervention with plitidepsin in hospitalized COVID-19 adult patients where three doses were assessed (1.5, 2 and 2.5 mg/day for 3 days, as a 90-minute intravenous infusion) in 45 patients (15 per dose-cohort). Treatment was well tolerated, with only two Grade 3 treatment-related adverse events observed (hypersensitivity and diarrhea). The discharge rates by Days 8 and 15 were 56.8% and 81.8%, respectively, with data sustaining dose-effect. A mean 4.2 log10 viral load reduction was attained by Day 15. Improvement in inflammation markers was also noted in a seemingly dose-dependent manner. These results suggest that plitidepsin impacts the outcome of patients with COVID-19.This study has been funded by Pharmamar, S.A. (Madrid, Spain). This work was supported by grants from the Government of Spain (PIE_INTRAMURAL_ LINEA 1 - 202020E079; PIE_INTRAMURAL_CSIC-202020E043). The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols pharmaceutical. We also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). N.I.U. has non-restrictive funding from PharmaMar to study the antiviral effect of Plitidepsin. N.J.K. was funded by grants from the National Institutes of Health (P50AI150476, U19AI135990, U19AI135972, R01AI143292, R01AI120694, and P01AI063302); by the Excellence in Research Award (ERA) from the Laboratory for Genomics Research (LGR), a collaboration between UCSF, UCB, and GSK (#133122P); by the Roddenberry Foundation, and gifts from QCRG philanthropic donors. This work was supported by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement #HR0011-19-2-0020. The views, opinions, and/or findings contained in this material are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. This research was partly funded by CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grants U19AI142733, U19AI135972 and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to AG-S. S.Y. received funding from a Swiss National Foundation (SNF) Early Postdoc Mobility fellowship (P2GEP3_184202).N

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children

Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1β and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myeloid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb

Reuschl et al. Evolution of enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants.

SARS-CoV-2 human adaptation resulted in distinct lineages with enhanced transmissibility called variants of concern (VOCs). Omicron is the first VOC to evolve distinct globally dominant subvariants. Here we compared their replication in human cell lines and primary airway cultures and measured host responses to infection. We discovered subvariants BA.4 and BA.5 have improved their suppression of innate immunity when compared with earlier subvariants BA.1 and BA.2. This correlated with increased expression of viral innate antagonists Orf6 and nucleocapsid, reminiscent of VOCs Alpha to Delta. Increased Orf6 levels suppressed host innate responses to infection by decreasing IRF3 and STAT1 signaling measured by transcription factor phosphorylation and nuclear translocation. Our data suggest convergent evolution of enhanced innate immune antagonist expression is a common pathway of human adaptation and link Omicron subvariant dominance to improved innate immune evasion.</p

<i>Socs3fl/fl LysM cre</i> mice show higher susceptibility to infection with <i>M. tuberculosis</i>.

<p><i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> littermate controls were sacrificed at indicated time points after aerosol infection with M. tuberculosis and colony forming units (CFU) per lung (A) and spleen (B) were assessed. The CFU per lung of individual mice and the median per group (n≥4) at the indicated time points after infection are depicted. Differences in CFU are significant (*p&lt;0.05 and **p&lt;0.01 Mann Whitney U test).Gross-pathology photograph of the lungs from Socs3^fl/fl and Socs3^fl/fl LysM cre mice 8 weeks after infection with M. tuberculosis (C). Histopathological scoring of hematoxylin-eosin stained paraffin lung sections from <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> mice measured 4 weeks after infection with <i>M. tuberculosis</i> (D). The mean % lung area with granulomas or free of lesions ± SEM is displayed. Differences with controls are significant (n = 8 per group, *p&lt;0.05 Student t test). The cumulative mortality of <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> mice (n = 10) after aerosol infection with <i>M. tuberculosis</i> is depicted (E). Survival curves are different (Log-rank test p&lt;0.005). CFU per lung, spleen and liver in <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> mice (n≥5 per group) were assessed 6 weeks after infection with 10⁶ BCG i.v. (F). The median CFU and interquartile range per group are depicted. Differences in CFU are significant (**p&lt;0.01 Mann Whitney U test). The mean percentage of Gr1+F4/80- neutrophils in the lung of <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> mice (n = 5 per group) 3 weeks after infection with <i>M. tuberculosis</i> ± SEM was determined by FACS analysis (G). The accumulation of myeloid peroxidase (Mpo) transcripts in lungs from mice at 0, 3 or 8 weeks after <i>M. tuberculosis</i> infection (n≥5 per group) was determined by real time PCR. The mean fold <i>Mpo</i> mRNA increase ± SEM in is depicted (H).</p