A bronchial gene signature specific for severe COPD that is retained in the nose.

Introduction: A subset of COPD patients develops advanced disease with severe airflow obstruction, hyperinflation and extensive emphysema. We propose that the pathogenesis in these patients differs from mild-moderate COPD and is reflected by bronchial gene expression. The aim of the present study was to identify a unique bronchial epithelial gene signature for severe COPD patients. Methods: We obtained RNA sequencing data from bronchial brushes from 123 ex-smokers with severe COPD, 23 with mild-moderate COPD and 23 non-COPD controls. We identified genes specific to severe COPD by comparing severe COPD to non-COPD controls, followed by removing genes that were also differentially expressed between mild-moderate COPD and non-COPD controls. Next, we performed a pathway analysis on these genes and evaluated whether this signature is retained in matched nasal brushings. Results: We identified 219 genes uniquely differentially expressed in severe COPD. Interaction network analysis identified VEGFA and FN1 as the key genes with the most interactions. Genes were involved in extracellular matrix regulation, collagen binding and the immune response. Of interest were 10 genes (VEGFA, DCN, SPARC, COL6A2, MGP, CYR61, ANXA6, LGALS1, C1QA and C1QB) directly connected to fibronectin 1 (FN1). Most of these genes were lower expressed in severe COPD and showed the same effect in nasal brushings. Conclusions: We found a unique severe COPD bronchial gene signature with key roles for VEGFA and FN1, which was retained in the upper airways. This supports the hypothesis that severe COPD, at least partly, comprises a different pathology and supports the potential for biomarker development based on nasal brushes in COPD.

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

The protein kinase R modifies gut physiology to limit colitis.

Here we investigate the function of the innate immune molecule protein kinase R (PKR) in intestinal inflammation. To model a colitogenic role of PKR, we determine the physiological response to dextran sulfate sodium (DSS) of wild-type and two transgenic mice strains mutated to express either a kinase-dead PKR or to ablate expression of the kinase. These experiments recognize kinase-dependent and -independent protection from DSS-induced weight loss and inflammation, against a kinase-dependent increase in the susceptibility to DSS-induced injury. We propose these effects arise through PKR-dependent alteration of gut physiology, evidenced as altered goblet cell function and changes to the gut microbiota at homeostasis that suppresses inflammasome activity by controlling autophagy. These findings establish that PKR functions as both a protein kinase and a signaling molecule in instituting immune homeostasis in the gut.

Surfactant protein D is a biomarker of influenza-related pediatric lung injury.

BACKGROUND: Biomarkers that can risk-stratify children with influenza virus lower respiratory infection may identify patients for targeted intervention. Early elevation of alveolar-related proteins in the bloodstream in these patients could indicate more severe lung damage portending worse outcomes. METHODS: We used a mouse model of human influenza infection and evaluated relationships between lung pathophysiology and surfactant protein D (SP-D), SP-A, and Club cell protein 16 (CC16). We then measured SP-A, SP-D, and CC16 levels in plasma samples from 94 children with influenza-associated acute respiratory failure (PICFLU cohort), excluding children with underlying conditions explaining disease severity. We tested for associations between levels of circulating proteins and disease severity including the diagnosis of acute respiratory distress syndrome (ARDS), mechanical ventilator, intensive care unit and hospital days, and hospital mortality. RESULTS: Circulating SP-D showed a greater increase than SP-A and CC16 in mice with increased alveolar-vascular permeability following influenza infection. In the PICFLU cohort, SP-D was associated with moderate-severe ARDS diagnosis (p = 0.01) and with mechanical ventilator (r = 0.45, p = 0.002), ICU (r = 0.44, p = 0.002), and hospital days (r = 0.37, p = 0.001) in influenza-infected children without bacterial coinfection. Levels of SP-D were lower in children with secondary bacterial pneumonia (p = 0.01) and not associated with outcomes. CC16 and SP-A levels did not differ with bacterial coinfection and were not consistently associated with severe outcomes. CONCLUSIONS: SP-D has potential as an early circulating biomarker reflecting a degree of lung damage caused directly by influenza virus infection in children. Secondary bacterial pneumonia alters SP-D biomarker performance.

Lysophosphatidic acid species are associated with exacerbation in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) exacerbations are heterogenous and profoundly impact the disease trajectory. Bioactive lipid lysophosphatidic acid (LPA) has been implicated in airway inflammation but the significance of LPA in COPD exacerbation is not known. The aim of the study was to investigate the utility of serum LPA species (LPA16:0, 18:0, 18:1, 18:2, 20:4) as biomarkers of COPD exacerbation. PATIENTS AND METHODS: LPA species were measured in the baseline placebo sera of a COPD randomized controlled trial. Tertile levels of each LPA were used to assign patients into biomarker high, medium, and low subgroups. Exacerbation rate and risk were compared among the LPA subgroups. RESULTS: The levels of LPA species were intercorrelated (rho 0.29-0.91). Patients with low and medium levels of LPA (LPA16:0, 20:4) had significantly higher exacerbation rate compared to the respective LPA-high patients [estimated rate per patient per year (95% CI)]: LPA16:0-low = 1.2 (0.8-1.9) (p = 0.019), LPA16:0-medium = 1.3 (0.8-2.0) (p = 0.013), LPA16:0-high = 0.5 (0.2-0.9); LPA20:4-low = 1.4 (0.9-2.1) (p = 0.0033), LPA20:4-medium = 1.2 (0.8-1.8) (p = 0.0089), LPA20:4-high = 0.4 (0.2-0.8). These patients also had earlier time to first exacerbation (hazard ratio (95% CI): LPA16:0-low = 2.6 (1.1-6.0) (p = 0.028), LPA16:0-medium = 2.7 (1.2-6.3) (p = 0.020); LPA20.4-low = 2.8 (1.2-6.6) (p = 0.017), LPA20:4-medium = 2.7 (1.2-6.4) (p = 0.021). Accordingly, these patients had a significant increased exacerbation risk compared to the respective LPA-high subgroups [odd ratio (95% CI)]: LPA16:0-low = 3.1 (1.1-8.8) (p = 0.030), LPA16:0-medium = 3.0 (1.1-8.3) (p = 0.031); LPA20:4-low = 3.8 (1.3-10.9) (p = 0.012), LPA20:4-medium = 3.3 (1.2-9.5) (p = 0.025). For the other LPA species (LPA18:0, 18:1, 18:2), the results were mixed; patients with low and medium levels of LPA18:0 and 18:2 had increased exacerbation rate, but only LPA18:0-low patients had significant increase in exacerbation risk and earlier time to first exacerbation compared to the LPA18:0-high subgroup. CONCLUSIONS: The study provided evidence of association between systemic LPA levels and exacerbation in COPD. Patients with low and medium levels of specific LPA species (LPA16:0, 20:4) had increased exacerbation rate, risk, and earlier time to first exacerbation. These non-invasive biomarkers may aid in identifying high risk patients with dysregulated LPA pathway to inform risk management and drug development.

High serum granulocyte-colony stimulating factor characterises neutrophilic COPD exacerbations associated with dysbiosis.

INTRODUCTION: COPD exacerbations are heterogeneous and can be triggered by bacterial, viral, or noninfectious insults. Exacerbations are also heterogeneous in neutrophilic or eosinophilic inflammatory responses. A noninvasive peripheral biomarker of COPD exacerbations characterised by bacterial/neutrophilic inflammation is lacking. Granulocyte-colony stimulating factor (G-CSF) is a key cytokine elevated during bacterial infection and mediates survival, proliferation, differentiation and function of neutrophils. OBJECTIVE: We hypothesised that high peripheral G-CSF would be indicative of COPD exacerbations with a neutrophilic and bacterial phenotype associated with microbial dysbiosis. METHODS: Serum G-CSF was measured during hospitalised exacerbation (day 0 or D0) and after 30 days of recovery (Day30 or D30) in 37 subjects. In a second cohort, serum and sputum cytokines were measured in 59 COPD patients during stable disease, at exacerbation, and at 2-weeks and 6-weeks following exacerbation. RESULTS: Serum G-CSF was increased during exacerbation in a subset of patients. These exacerbations were enriched for bacterial but not viral or type-2 biologies. The median serum G-CSF level was 1.6-fold higher in bacterial exacerbation compared to nonbacterial exacerbation (22 pg·mL-1 versus 13 pg·mL-1, p=0.0007). Serum G-CSF classified bacterial exacerbations with an area under the curve (AUC) for the receiver operating characteristic (ROC) curve equal to 0.76. Exacerbations with a two-fold or greater increase in serum G-CSF were characterised by neutrophilic inflammation, with increased sputum and blood neutrophils, and high sputum interleukin (IL)-1ß, IL-6 and serum amyloid A1 (SAA1) levels. These exacerbations were preceded by dysbiosis, with decreased microbiome diversity and enrichment of respiratory pathogens such as Haemophilus and Moraxella. Furthermore, serum G-CSF at exacerbation classified neutrophilic-dysbiotic exacerbations (AUC for the ROC curve equal to 0.75). CONCLUSIONS: High serum G-CSF enriches for COPD exacerbations characterised by neutrophilic inflammation with underlying bacterial dysbiosis.

Serum Lysophosphatidic Acid Measurement by Liquid Chromatography-Mass Spectrometry in COPD Patients.

Lysophospholipids are bioactive signaling molecules derived from cell membrane glycerophospholipids or sphingolipids and are highly regulated under normal physiological conditions. Lysophosphatidic acids (LPAs) are a class of lysophospholipids that act on G-protein-coupled receptors to exert a variety of cellular functions. Dysregulation of phospholipase activity and consequently LPA synthesis in serum have been linked to inflammation, such as seen in chronic obstructive pulmonary disease (COPD). The accurate measurement of phospholipids is critical for evaluating their dysregulation in disease. In this study, we optimized experimental parameters for the sensitive measurement of LPAs. We validated the method based on matrix, linearity, accuracy, precision, and stability. An investigation into sample extraction processes emphasized that the common practice of including low concentration of hydrochloric acid in the extraction buffer causes an overestimation of lipid recovery. The liquid chromatography gradient was optimized to separate various lysophospholipid classes. After optimization, detection limits of LPA were sufficiently sensitive for subsequent analysis, ranging from 2 to 8 nM. The validated workflow was applied to a cohort of healthy donor and COPD patient sera. Eight LPA species were identified, and five unique species of LPA were quantified. Most LPA species increased significantly in COPD patients compared to healthy donors. The correlation between LPAs and other demographic parameters was further investigated in a sample set of over 200 baseline patient sera from a COPD clinical trial. For the first time, LPAs other than the two most abundant and readily detectable moieties are quantified in COPD patients using validated methods, opening the door to downstream biomarker evaluation in respiratory disease.

Exercise-induced alterations in phospholipid hydrolysis, airway surfactant, and eicosanoids and their role in airway hyperresponsiveness in asthma.

The mechanisms responsible for driving endogenous airway hyperresponsiveness (AHR) in the form of exercise-induced bronchoconstriction (EIB) are not fully understood. We examined alterations in airway phospholipid hydrolysis, surfactant degradation, and lipid mediator release in relation to AHR severity and changes induced by exercise challenge. Paired induced sputum (n = 18) and bronchoalveolar lavage (BAL) fluid (n = 11) were obtained before and after exercise challenge in asthmatic subjects. Samples were analyzed for phospholipid structure, surfactant function, and levels of eicosanoids and secreted phospholipase A2 group 10 (sPLA2-X). A primary epithelial cell culture model was used to model effects of osmotic stress on sPLA2-X. Exercise challenge resulted in increased surfactant degradation, phospholipase activity, and eicosanoid production in sputum samples of all patients. Subjects with EIB had higher levels of surfactant degradation and phospholipase activity in BAL fluid. Higher basal sputum levels of cysteinyl leukotrienes (CysLTs) and prostaglandin D2 (PGD2) were associated with direct AHR, and both the postexercise and absolute change in CysLTs and PGD2 levels were associated with EIB severity. Surfactant function either was abnormal at baseline or became abnormal after exercise challenge. Baseline levels of sPLA2-X in sputum and the absolute change in amount of sPLA2-X with exercise were positively correlated with EIB severity. Osmotic stress ex vivo resulted in movement of water and release of sPLA2-X to the apical surface. In summary, exercise challenge promotes changes in phospholipid structure and eicosanoid release in asthma, providing two mechanisms that promote bronchoconstriction, particularly in individuals with EIB who have higher basal levels of phospholipid turnover.

RNase L activates the NLRP3 inflammasome during viral infections.

The NLRP3 inflammasome assembles in response to danger signals, triggering self-cleavage of procaspase-1 and production of the proinflammatory cytokine IL-1ß. Although virus infection activates the NLRP3 inflammasome, the underlying events remain incompletely understood. We report that virus activation of the NLRP3 inflammasome involves the 2',5'-oligoadenylate (2-5A) synthetase(OAS)/RNase L system, a component of the interferon-induced antiviral response that senses double-stranded RNA and activates endoribonuclease RNase L to cleave viral and cellular RNAs. The absence of RNase L reduces IL-1ß production in influenza A virus-infected mice. RNA cleavage products generated by RNase L enhance IL-1ß production but require the presence of 2',3'-cyclic phosphorylated termini characteristic of RNase L activity. Additionally, these cleavage products stimulate NLRP3 complex formation with the DExD/H-box helicase, DHX33, and mitochondrial adaptor protein, MAVS, which are each required for effective NLRP3 inflammasome activation. Thus, RNA cleavage events catalyzed by RNase L are required for optimal inflammasome activation during viral infections.

RNase L targets distinct sites in influenza A virus RNAs.

UNLABELLED: Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. IMPORTANCE: RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease.

Cell-type-specific effects of RNase L on viral induction of beta interferon.

The interferon (IFN)-inducible antiviral state is mediated in part by the 2',5'-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-ß by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-ß by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-ß production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-ß in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-ß induction that could affect tissue protection and survival during viral infections. IMPORTANCE Type I interferons (IFNs) such as IFN-ß are essential antiviral cytokines that are often required for animal survival following infections by highly pathogenic viruses. Therefore, host factors that regulate type I IFN production are critically important for animal and human health. Previously we reported that the OAS/RNase L pathway amplifies antiviral innate immunity by enhancing IFN-ß production in mouse embryonic fibroblasts and in virus-infected mice. Here we report that high basal levels of OAS/RNase L in macrophages reduce, rather than increase, virus induction of IFN-ß. RNA damage and apoptosis caused by RNase L were the likely reasons for the decreased IFN-ß production in virus-infected macrophages. Our studies suggest that during viral infections, the OAS/RNase L pathway can either enhance or suppress IFN production, depending on the cell type. IFN regulation by RNase L is suggested to contribute to tissue protection and survival during viral infections.

RNase-L deficiency exacerbates experimental colitis and colitis-associated cancer.

BACKGROUND: The endoribonuclease RNase-L is a type-I interferon (IFN)-regulated component of the innate immune response that functions in antiviral, antibacterial, and antiproliferative activities. RNase-L produces RNA agonists of RIG-I-like receptors, sensors of cytosolic pathogen-associated RNAs that induce cytokines including IFN-ß. IFN-ß and RIG-I-like receptors signaling mediate protective responses against experimental colitis and colitis-associated cancer and contribute to gastrointestinal homeostasis. Therefore, we investigated a role for RNase-L in murine colitis and colitis-associated cancer and its association with RIG-I-like receptors signaling in response to bacterial RNA. METHODS: Colitis was induced in wild type-deficient and RNase-L-deficient mice (RNase-L⁻/⁻) by administration of dextran sulfate sodium (DSS). Colitis-associated cancer was induced by DSS and azoxymethane (AOM). Histological analysis and immunohistochemistry were performed on colon tissue to analyze immune cell infiltration and tissue damage after induction of colitis. Expression of cytokines was measured by quantitative real-time-PCR and ELISA. RESULTS: DSS-treated RNase-L⁻/⁻ mice exhibited a significantly higher clinical score, delayed leukocyte infiltration, reduced expression of IFN-ß, tumor necrosis factor α, interleukin-1ß, and interleukin-18 at early times post-DSS exposure, and increased mortality as compared with wild-type mice. DSS/AOM-treated RNase-L⁻/⁻ mice displayed an increased tumor burden. Bacterial RNA triggered IFN-ß production in an RNase-L-dependent manner and provided a potential mechanism by which RNase-L contributes to the gastrointestinal immune response to microbiota and protects against experimental colitis and colitis-associated cancer. CONCLUSIONS: RNase-L promotes the innate immune response to intestinal damage and ameliorates murine colitis and colitis-associated cancer. The RNase-L-dependent production of IFN-ß stimulated by bacterial RNA may be a mechanism to protect against gastrointestinal inflammatory disease.

RNase L triggers autophagy in response to viral infections.

Autophagy is a programmed homeostatic response to diverse types of cellular stress that disposes of long-lived proteins, organelles, and invading microbes within double-membraned structures called autophagosomes. The 2',5'-oligoadenylate/RNase L system is a virus-activated host RNase pathway that disposes of or processes viral and cellular single-stranded RNAs. Here we report that activation of RNase L during viral infections induces autophagy. Accordingly, infections with encephalomyocarditis virus or vesicular stomatitis virus led to higher levels of autophagy in wild-type mouse embryonic fibroblasts (MEF) than in RNase L-null MEF. Similarly, direct activation of RNase L with a 2',5'-oligoadenylate resulted in p62(SQSTM1) degradation, LC3BI/LC3BII conversion, and appearance of autophagosomes. To determine the effect of RNase L-mediated autophagy on viral replication, we compared viral yields in wild-type and RNase L-null MEF in the absence or presence of either chemical inhibitors of autophagy (bafilomycin A1 or 3-methyladenine) or small interfering RNA (siRNA) against ATG5 or beclin-1. At a low multiplicity of infection, induction of autophagy by RNase L during the initial cycle of virus growth contributed to the suppression of virus replication. However, in subsequent rounds of infection, autophagy promoted viral replication, reducing the antiviral effect of RNase L. Our results indicate a novel function of RNase L as an inducer of autophagy that affects viral yields.

New insights into the role of RNase L in innate immunity.

The interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway blocks infections by some types of viruses through cleavage of viral and cellular single-stranded RNA. Viruses induce type I IFNs that initiate signaling to the OAS genes. OAS proteins are pathogen recognition receptors for the viral pathogen-associated molecular pattern, double-stranded RNA. Double-stranded RNA activates OAS to produce p(x)5'A(2'p5'A)(n); x = 1-3; n > 2 (2-5A) from ATP. Upon binding 2-5A, RNase L is converted from an inactive monomer to a potently active dimeric endoribonuclease for single-stranded RNA. RNase L contains, from N- to C-terminus, a series of 9 ankyrin repeats, a linker, several protein kinase-like motifs, and a ribonuclease domain homologous to Ire1 (involved in the unfolded protein response). In the past few years, it has become increasingly apparent that RNase L and OAS contribute to innate immunity in many ways. For example, small RNA cleavage products produced by RNase L during viral infections can signal to the retinoic acid-inducible-I like receptors to amplify and perpetuate signaling to the IFN-ß gene. In addition, RNase L is now implicated in protecting the central nervous system against viral-induced demyelination. A role in tumor suppression was inferred by mapping of the RNase L gene to the hereditary prostate cancer 1 (HPC1) gene, which in turn led to discovery of the xenotropic murine leukemia-related virus. A broader role in innate immunity is suggested by involvement of RNase L in cytokine induction and endosomal pathways that suppress bacterial infections. These newly described findings about RNase L could eventually provide the basis for developing broad-spectrum antimicrobial drugs.

Protein kinase R-dependent regulation of interleukin-10 in response to double-stranded RNA.

The double-stranded RNA-activated protein kinase R (PKR) is an important component of antiviral defense. PKR participates in different signaling pathways in response to various stimuli to regulate translation via phosphorylation of the eukaryotic initiation factor 2alpha, and transcription via activating NF-kappaB and IRF-1, to induce pro-inflammatory cytokines. Here we show PKR regulates interleukin-10 induction in response to double-stranded RNA, bacterial lipopolysaccaride, and Sendai virus infection. Using chemical inhibitors, dominant negative constructs, and genetic knockouts, we demonstrate that the PKR-mediated interleukin-10 induction engages JNK and NF-kappaB. Together, our data demonstrate the role of PKR in regulating an anti-inflammatory cytokine. The findings have significance in antiviral as well as broader innate immune responses.