The role of innate lymphoid cells in mucosal inflammation in paediatric viral bronchiolitis

Background Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and acute lower respiratory tract infections (LRTI) among children worldwide. RSV is the leading cause of childhood hospitalisation and a financial burden on national health systems. No vaccine that protects against RSV infection is available. Children with bronchiolitis are at high risk of developing wheeze and asthma later in childhood. Recent data suggest that innate immunity plays a vital role in childhood, especially when maternal antibodies' levels are low and adaptive immune responses are immature. Innate lymphoid cells (ILCs) are among the most recently discovered innate immune cell types that could play a pivotal role in innate responses to RSV bronchiolitis. ILCs have been classified into three groups: ILC1, ILC2 and ILC3 depending on their function, transcription factor expression and phenotype. Although ILC2 numbers have been found to be elevated in bronchiolitis, the phenotypic and functional differences between ILCs from healthy infants and those with bronchiolitis have not been explored. Hypothesis I propose that ILC frequencies and function are associated with disease severity and aetiology in children with acute lower respiratory tract infection. Sub-hypothesis 1 ILCs from neonates are functionally different from healthy adult subjects. Sub-hypothesis 2 Alterations in ILC frequencies and numbers in blood and airway samples are associated with disease severity, pathogen type and load. Sub-hypothesis 3 Alterations in ILC function are associated with disease severity, pathogen type and load. Methods To determine differences in ILCs in early life, lymphocyte populations, including ILC subset frequencies and numbers in blood samples collected from adults and neonates were investigated by flow cytometry. To study ILCs in bronchiolitis, blood and upper and lower airway samples were collected from infants with and without a respiratory viral infection. An optimised multicolour immunophenotyping panel (OMIP), intracellular staining panels (ICS) and other systems of analytical approach such as UMAP (Uniform Manifold Approximation and Projection), heatmaps, correlograms and principal component analysis (PCA) were used. ILCs were defined using the following gating strategy: live, single, CD45+, lineage negative (CD3, CD14, CD16, CD56, CD1a, CD123 FcεR1α)- and CD127+ cells. ILC1 cells were defined as CD117−CRTH2−, ILC2 cells were defined as CRTH2+ CD117int and ILC3s were defined as CD117+CRTH2−. At the same time, other lymphocyte and granulocyte populations were enumerated. Alongside cellular work, using the Meso Scale Discovery (MSD) platform, 29 immune mediators and cytokines were measured in airway samples. Results I successfully optimised flow cytometry panels to determine frequencies and function of ILCs in small volumes of difficult to obtain clinical samples. Results suggest that ILC numbers were higher in whole blood obtained from neonates compared to healthy adults (P=0.0032), however, they produced significantly less IFN-γ (P=0.0015). Furthermore, there were significantly lower numbers of NKT (P=0.0001) and MAIT cells (P=0.0031) in neonates compared to healthy adults. In bronchiolitis, ILC numbers were significantly decreased in peripheral blood (P=0.005) and airways samples (P=0.025) obtained from bronchiolitic compared to control subjects; however, their capacity for IFN-γ production was similar between those groups. Interestingly, there was a significantly decreased production of IFN-γ in CD4+ (P=0.042) and CD8+ T cells (P=0.006) from bronchiolitic compared to control patients. Finally, levels of almost all mediators were higher in bronchiolitic babies than controls, including both type-1, type-2 and type-17 associated cytokines. Only IL-4 production in lower and upper airways was increased in rhinovirus- (RV) but not RSV-associated bronchiolitis (P=0.006). Conclusions Small volumes of whole blood and paediatric respiratory samples can be used for enumerating ILCs (NK cells, ILC1, ILC2, ILC3), granulocytes (eosinophils and neutrophils), T – cells (CD4+ and CD8+), MAIT cells and NKT-like cells. The comparison of neonate and adult functional responses suggests that innate lymphoid cells are mature in early life and might play a pivotal role in the neonatal type-2 bias via the production of IL-13. Bronchiolitis was characterised not only by CD3+ T cell but also by CD3- lymphopenia. ILCs numbers were much lower in infants with bronchiolitis compared to healthy controls; however, ILC had the ability to produce IFN-γ. My data suggest that RSV and RV can lead to different phenotypes of bronchiolitis. Finally, I demonstrated the importance of studying the lower and upper airways simultaneously, discovering that inflammatory signatures were different between these two compartments. For the first time all members of the ILC family were studied in viral bronchiolitis. Determining why some infants develop bronchiolitis and the pathogenesis of lower airway disease in infants could lead to a better understanding of early life immunity and the development of new biomarkers and clinical prophylactics.Open Acces

Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery. IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials

Glucocorticoids impair type I IFN signalling and enhance rhinovirus replication

Inhaled corticosteroids (ICS) are recommended treatments for all degrees of asthma severity and in combination with bronchodilators are indicated for COPD patients with a history of frequent exacerbations. However, the long-term side effects of glucocorticoids (GCs) may include increased risk of respiratory infections, including viral triggered exacerbations. Rhinovirus (RV) infection is the main trigger of asthma and COPD exacerbations. Thus, we sought to explore the influence of GCs on viral replication. We demonstrate the ICS fluticasone propionate (FP) and two selective non-steroidal (GRT7) and steroidal (GRT10) glucocorticoid receptor (GR) agonists significantly suppress pro-inflammatory (IL-6 and IL-8) and antiviral (IFN-λ1) cytokine production and the expression of the interferon-stimulated genes (ISGs) OAS and viperin in RV-infected bronchial epithelial cells, with a consequent increase of viral replication. We also show that FP, GRT7 and GRT10 inhibit STAT1 Y701 and/or STAT2 Y690 phosphorylation and ISG mRNA induction following cell stimulation with recombinant IFN-β. In addition, we investigated the effects of the ICS budesonide (BD) and the long-acting β2 agonist (LABA) formoterol, alone or as an ICS/LABA combination, on RV-induced ISG expression and viral replication. Combination of BD/formoterol increases the suppression of OAS and viperin mRNA observed with both BD and formoterol alone, but an increase in viral RNA was only observed with BD treatment and not with formoterol. Overall, we provide evidence of an impairment of the innate antiviral immune response by GC therapy and the potential for GCs to enhance viral replication. These findings could have important clinical implications

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination.

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript