SERS Microsensors for the Study of pH Regulation in Cystic Fibrosis Patient-Derived Airway Cultures.

Acidification of the airway surface liquid in the respiratory system could play a role in the pathology of Cystic Fibrosis, but its low volume and proximity to the airway epithelium make it a challenging biological environment in which to noninvasively collect pH measurements. To address this challenge, we explored surface enhanced Raman scattering microsensors (SERS-MS), with a 4-mercaptobenzoic acid (MBA) pH reporter molecule, as pH sensors for the airway surface liquid of patient-derived in vitro models of the human airway. Using air-liquid interface (ALI) cultures to model the respiratory epithelium, we show that SERS-MS facilitates the optical measurement of trans-epithelial pH gradients between the airway surface liquid and the basolateral culture medium. SERS-MS also enabled the successful quantification of pH changes in the airway surface liquid following stimulation of the Cystic Fibrosis transmembrane conductance regulator (CFTR, the apical ion channel that is dysfunctional in Cystic Fibrosis airways). Finally, the influence of CFTR mutations on baseline airway surface liquid pH was explored by using SERS-MS to measure the pH in ALIs grown from Cystic Fibrosis and non-Cystic Fibrosis donors.

Latent gammaherpesvirus infection enhances type I IFN response and reduces virus spread in an influenza A virus co-infection model.

Infections with persistent or latent viruses alter host immune homeostasis and have potential to affect the outcome of concomitant acute viral infections such as influenza A virus (IAV). Gammaherpesviruses establish life-long infections and require an on-going immune response to control reactivation. We have used a murine model of co-infection to investigate the response to IAV infection in mice latently infected with the gammaherpesvirus MHV-68. Over the course of infection, latently infected BALB/c mice showed less weight loss, clinical signs, pulmonary cellular infiltration and expression of inflammatory mediators than naïve mice infected with IAV and had significantly more activated CD8+ T cells in the lungs. Four days after IAV infection, virus spread in the lungs of latently infected animals was significantly lower than in naïve animals. By 7 days after IAV infection latently infected lungs express elevated levels of cytokines and chemokines indicating they are primed to respond to the secondary infection. Investigation at an early time point showed that 24 h after IAV infection co-infected animals had higher expression of IFNß and Ddx58 (RIG-I) and a range of ISGs than mice infected with IAV alone suggesting that the type I IFN response plays a role in the protective effect. This effect was mouse strain dependent and did not occur in 129/Sv/Ev mice. These results offer insight into innate immune mechanisms that could be utilized to protect against IAV infection and highlight on-going and persistent viral infections as a significant factor impacting the severity of acute respiratory infections.

The senescent secretome drives PLVAP expression in cultured human hepatic endothelial cells to promote monocyte transmigration.

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease.

CAGE sequencing reveals CFTR-dependent dysregulation of type I IFN signaling in activated cystic fibrosis macrophages.

An intense, nonresolving airway inflammatory response leads to destructive lung disease in cystic fibrosis (CF). Dysregulation of macrophage immune function may be a key facet governing the progression of CF lung disease, but the underlying mechanisms are not fully understood. We used 5' end centered transcriptome sequencing to profile P. aeruginosa LPS-activated human CF macrophages, showing that CF and non-CF macrophages deploy substantially distinct transcriptional programs at baseline and following activation. This includes a significantly blunted type I IFN signaling response in activated patient cells relative to healthy controls that was reversible upon in vitro treatment with CFTR modulators in patient cells and by CRISPR-Cas9 gene editing to correct the F508del mutation in patient-derived iPSC macrophages. These findings illustrate a previously unidentified immune defect in human CF macrophages that is CFTR dependent and reversible with CFTR modulators, thus providing new avenues in the search for effective anti-inflammatory interventions in CF.

SERS microsensors for pH measurements in the lumen and ECM of stem cell derived human airway organoids.

Patient derived organoids have the potential to improve the physiological relevance of in vitro disease models. However, the 3D architecture of these self-assembled cellular structures makes probing their biochemistry more complex than in traditional 2D culture. We explore the application of surface enhanced Raman scattering microsensors (SERS-MS) to probe local pH gradients within patient derived airway organoid cultures. SERS-MS consist of solid polymer cores decorated with surface immobilised gold nanoparticles which are functionalised with pH sensitive reporter molecule 4-mercaptobenzoic acid (MBA). We demonstrate that by mixing SERS-MS into the extracellular matrix (ECM) of airway organoid cultures the probes can be engulfed by expanding organoids and report on local pH in the organoid lumen and ECM.

An Inhaled Galectin-3 Inhibitor in COVID-19 Pneumonitis: A Phase Ib/IIa Randomized Controlled Clinical Trial (DEFINE).

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).

Randomised controlled trial of intravenous nafamostat mesylate in COVID pneumonitis: Phase 1b/2a experimental study to investigate safety, Pharmacokinetics and Pharmacodynamics.

BACKGROUND: Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate. METHODS: We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis. FINDINGS: Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 - 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43-18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55-95% CI 0.31-0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry. INTERPRETATION: In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events. FUNDING: DEFINE was funded by LifeArc (an independent medical research charity) under the STOPCOVID award to the University of Edinburgh. We also thank the Oxford University COVID-19 Research Response Fund (BRD00230).

Macrophages from gut-corrected CF mice express human CFTR and lack a pro-inflammatory phenotype.

Macrophages represent prominent immune orchestrators of cystic fibrosis (CF) inflammation and, as such, are an ever-increasing focus of CF research with several reports of intrinsic immune dysfunction related to loss of CFTR activity in macrophages themselves. Animal models of CF have contributed, in no small part, to a deepening of our understanding of the pathophysiology of the disease and towards therapeutic development. A commonly-used animal model in CF research is the Cftrtm1Unc Tg(FABP-hCFTR) mouse, which displays gut-specific expression of a human CFTR transgene in order to rescue the high rate of early mortality in Cftr-null mice associated with severe intestinal obstruction. We find significant variation in the response to inflammatory challenge of patient macrophages and cells derived from the Cftrtm1Unc Tg(FABP-hCFTR) mouse and show that macrophages derived from this mouse exhibit aberrant expression of human CFTR. This may contribute to the absence of inflammatory changes in this model.

High Purity Isolation of Low Density Neutrophils Casts Doubt on Their Exceptionality in Health and Disease.

Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and NDNs and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4+ or CD8+ T cell proliferation or IFNγ production compared with NDNs. LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.

The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types.

Neutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues during health and disease. They are able to form stable interactions, with profound effects on the phenotype and function of the T cells. However, the outcome of these effects are frequently contradictory; in some systems neutrophils suppress T cell proliferation, in others they are activatory or present antigen directly. Published protocols modelling these interactions in vitro do not reflect the full range of interactions found in vivo; they do not examine how activated and naïve T cells differentially respond to neutrophils, or whether de-granulating or resting neutrophils induce different outcomes. Here, we established a culture protocol to ask these questions with human T cells and autologous neutrophils. We find that resting neutrophils suppress T cell proliferation, activation and cytokine production but that de-granulating neutrophils do not, and neutrophil-released intracellular contents enhance proliferation. Strikingly, we also demonstrate that T cells early in the activation process are susceptible to suppression by neutrophils, while later-stage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allows nuanced analysis of the outcome of interaction of these cells and may explain the contradictory results observed previously.

Neutrophil Extracellular Traps in Inflammatory Bowel Disease: Pathogenic Mechanisms and Clinical Translation.

The Inflammatory Bowel Diseases (IBD), Ulcerative Colitis (UC) and Crohn's Disease (CD) are characterised by chronic non-resolving gut mucosal inflammation involving innate and adaptive immune responses. Neutrophils, usually regarded as first responders in inflammation, are a key presence in the gut mucosal inflammatory milieu in IBD. Here, we review the role of neutrophil extracellular trap (NET) formation as a potential effector disease mechanism. NETs are extracellular webs of chromatin, microbicidal proteins and oxidative enzymes that are released by neutrophils to contain pathogens. NETs contribute to the pathogenesis of several immune-mediated diseases such as systemic lupus erythematosus and rheumatoid arthritis; and recently, as a major tissue damaging process involved in the host response to severe acute respiratory syndrome coronavirus 2 infection. NETs are pertinent as a defence mechanism at the gut mucosal interphase exposed to high levels of bacteria, viruses and fungi. On the other hand, NETs can also potentiate and perpetuate gut inflammation. In this review, we discuss the broad protective vs. pathogenic roles of NETs, explanatory factors that could lead to an increase in NET formation in IBD and how NETs may contribute to gut inflammation and IBD-related complications. Finally, we summarise therapeutic opportunities to target NETs in IBD.

The neutrophil antimicrobial peptide cathelicidin promotes Th17 differentiation.

The host defence peptide cathelicidin (LL-37 in humans, mCRAMP in mice) is released from neutrophils by de-granulation, NETosis and necrotic death; it has potent anti-pathogen activity as well as being a broad immunomodulator. Here we report that cathelicidin is a powerful Th17 potentiator which enhances aryl hydrocarbon receptor (AHR) and RORγt expression, in a TGF-ß1-dependent manner. In the presence of TGF-ß1, cathelicidin enhanced SMAD2/3 and STAT3 phosphorylation, and profoundly suppressed IL-2 and T-bet, directing T cells away from Th1 and into a Th17 phenotype. Strikingly, Th17, but not Th1, cells were protected from apoptosis by cathelicidin. We show that cathelicidin is released by neutrophils in mouse lymph nodes and that cathelicidin-deficient mice display suppressed Th17 responses during inflammation, but not at steady state. We propose that the neutrophil cathelicidin is required for maximal Th17 differentiation, and that this is one method by which early neutrophilia directs subsequent adaptive immune responses.

Concurrent Infection With the Filarial Helminth Litomosoides sigmodontis Attenuates or Worsens Influenza A Virus Pathogenesis in a Stage-Dependent Manner.

Filarial helminths infect approximately 120 million people worldwide initiating a type 2 immune response in the host. Influenza A viruses stimulate a virulent type 1 pro-inflammatory immune response that in some individuals can cause uncontrolled immunopathology and fatality. Although coinfection with filariasis and influenza is a common occurrence, the impact of filarial infection on respiratory viral infection is unknown. The aim of this study was to determine the impact of pre-existing filarial infection on concurrent infection with influenza A virus. A murine model of co-infection was established using the filarial helminth Litomosoides sigmodontis and the H1N1 (A/WSN/33) influenza A virus (IAV). Co-infection was performed at 3 different stages of L. sigmodontis infection (larval, juvenile adult, and patency), and the impact of co-infection was determined by IAV induced weight loss and clinical signs, quantification of viral titres, and helminth counts. Significant alterations of IAV pathogenesis, dependent upon stage of infection, was observed on co-infection with L. sigmodontis. Larval stage L. sigmodontis infection alleviated clinical signs of IAV co-infection, whilst more established juvenile adult infection also significantly delayed weight loss. Viral titres remained unaltered at either infection stage. In contrast, patent L. sigmdodontis infection led to a reversal of age-related resistance to IAV infection, significantly increasing weight loss and clinical signs of infection as well as increasing IAV titre. These data demonstrate that the progression of influenza infection can be ameliorated or worsened by pre-existing filarial infection, with the outcome dependent upon the stage of filarial infection.

Human cystic fibrosis monocyte derived macrophages display no defect in acidification of phagolysosomes when measured by optical nanosensors.

BACKGROUND: Defective macrophage phagolysosomal acidification is implicated in numerous lung diseases including Cystic Fibrosis (CF) and may contribute to defective pathogen killing. Conflicting reports relating to phagolysosomal pH in CF macrophages have been published, in part related to the use of pH-sensitive fluorescent probes where potential inadequacies in experimental design can be a contributing factor (e.g. employing probes with incorrect pKa for the cellular compartment of interest). We developed a reliable method to quantify macrophage phagolysosomal pH using surface-enhanced Raman spectroscopy-based nanosensors. METHODS: Monocyte-derived macrophages from CF and healthy control participants were incubated with nanosensors. Live cell imaging identified phagocytosed nanosensors, and surface-enhanced Raman spectroscopy was performed using para-mercaptobenzoic acid functionalised gold nanoparticles which produce Raman spectra that change predictably with their environmental pH. Conventional fluorescence spectroscopy was carried out in comparison. Nanosensor localisation to phagolysosomes was confirmed by transmission electron microscopy. RESULTS: Nanosensors were actively phagocytosed by macrophages into phagolysosomes and acidification occurred rapidly and remained stable for at least 60 min. There was no difference in phagolysosomal pH between healthy control and CF macrophages (5.41 ±â€¯0.11 vs. 5.41 ±â€¯0.20, p > .9999), further confirmed by inhibiting Cystic Fibrosis Transmembrane Conductance Regulator in healthy control monocyte-derived macrophages. CONCLUSIONS: Optical nanosensors accurately measure macrophage phagolysosomal pH and demonstrate no phagolysosomal acidification defect in human CF monocyte-derived macrophages. Further studies using alveolar macrophages could extend the impact of our findings. Nanosensors represent a novel and precise means to measure organelle functions with widespread potential for the study and monitoring of several lung diseases.

Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation. METHODS: Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR. RESULTS: CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF. CONCLUSIONS: CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity.

UNLABELLED: Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE: Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.