Monocyte migration profiles define disease severity in acute COVID-19 and unique features of long COVID.

BACKGROUND: COVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID). METHODS: We assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalisation and up to 9 months of convalescence following COVID-19, respiratory syncytial virus or influenza A. Patients with progressive fibrosing interstitial lung disease were included as a positive control for severe, ongoing lung injury. RESULTS: Monocyte alterations in acute COVID-19 patients included aberrant expression of leukocyte migration molecules, continuing into convalescence (n=142) and corresponding with specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of C-X-C motif chemokine receptor 6 (CXCR6) (p<0.0001) and adhesion molecule P-selectin glycoprotein ligand 1 (p<0.01), alongside preferential migration of monocytes towards the CXCR6 ligand C-X-C motif chemokine ligand 16 (CXCL16) (p<0.05), which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in patients with progressive fibrosing interstitial lung disease (p<0.001), confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited a sustained reduction of the prostaglandin-generating enzyme cyclooxygenase 2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in respiratory syncytial virus or influenza A convalescence. CONCLUSIONS: Our data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.

CD200R1 regulates eosinophilia during pulmonary fungal infection in mice.

CD200 receptor 1(CD200R1) signalling limits myeloid cell responses and reduces autoimmunity, alloimmunity and viral-mediated immunopathology, but has never been examined in the context of eosinophilic inflammation. Susceptibility to lung fungal infection is associated with T-helper 2 (Th2) cytokine dominated responses and strong eosinophilic pathology. Blockade of CD200R1 enhances type I cytokine responses in many infectious and non-infectious settings and so may promote a more protective response to fungal infection. By contrast, we demonstrate that, rather than promoting type I cytokine responses, CD200R1 blockade enhanced eosinophilia in a mouse model of Cryptococcus neoformans infection, whereas CD200R1 agonism reduced lung eosinophilia - with neither strategy completely altering fungal burden. Thus, we reveal a surprising disconnect between pulmonary eosinophilia and cryptococcal burden and dissemination. This research has 2 important implications. Firstly, a lack of CD200R1 signalling enhances immune responses regardless of cytokine polarisation, and secondly reducing eosinophils does not allow protective immunity to develop in susceptible fungal system. Therefore, agonists of CD200R1 may be beneficial for eosinophilic pathologies.

Kidney resident macrophages have distinct subsets and multifunctional roles.

BACKGROUND: The kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. METHODS: Flow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5-/- (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. RESULTS: In wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5-/- (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. CONCLUSIONS: We identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

Neutrophil dysfunction triggers inflammatory bowel disease in G6PC3 deficiency.

The glucose-6-phosphatase catalytic subunit 3 (G6PC3) encodes a ubiquitously expressed enzyme that regulates cytoplasmic glucose availability. Loss-of-function biallelic G6PC3 mutations cause severe congenital neutropenia and a diverse spectrum of extra-hematological manifestations, among which inflammatory bowel disease (IBD) has been anecdotally reported. Neutrophil function and clinical response to granulocyte colony-stimulating factor (G-CSF) and hematopoietic stem cell transplantation (HSCT) were investigated in 4 children with G6PC3 deficiency-associated IBD. G6PC3 deficiency was associated with early-onset IBD refractory to treatment with steroids and infliximab. The symptoms of IBD progressed despite G-CSF treatment. In vitro studies on the patients' blood showed that neutrophils displayed higher levels of activation markers (CD11b, CD66b, and CD14), excessive IL-8 and reactive oxygen species, and increased apoptosis and secondary necrosis. Secondary necrosis was exaggerated after stimulation with Escherichia coli and could be partially rescued with supplemental exogenous glucose. HSCT led to normalization of neutrophil function and remission of gastrointestinal symptoms. We conclude that neutrophils in G6PC3 deficiency release pro-inflammatory mediators when exposed to gut bacteria, associated with intestinal inflammation, despite treatment with G-CSF. HSCT is an effective therapeutic option in patients with G6PC3 deficiency-associated IBD refractory to immune suppressants.

Alterations in T and B cell function persist in convalescent COVID-19 patients.

BACKGROUND: Emerging studies indicate that some coronavirus disease 2019 (COVID-19) patients suffer from persistent symptoms, including breathlessness and chronic fatigue; however, the long-term immune response in these patients presently remains ill-defined. METHODS: Here, we describe the phenotypic and functional characteristics of B and T cells in hospitalized COVID-19 patients during acute disease and at 3-6 months of convalescence. FINDINGS: We report that the alterations in B cell subsets observed in acute COVID-19 patients were largely recovered in convalescent patients. In contrast, T cells from convalescent patients displayed continued alterations with persistence of a cytotoxic program evident in CD8+ T cells as well as elevated production of type 1 cytokines and interleukin-17 (IL-17). Interestingly, B cells from patients with acute COVID-19 displayed an IL-6/IL-10 cytokine imbalance in response to Toll-like receptor activation, skewed toward a pro-inflammatory phenotype. Whereas the frequency of IL-6+ B cells was restored in convalescent patients irrespective of clinical outcome, the recovery of IL-10+ B cells was associated with the resolution of lung pathology. CONCLUSIONS: Our data detail lymphocyte alterations in previously hospitalized COVID-19 patients up to 6 months following hospital discharge and identify 3 subgroups of convalescent patients based on distinct lymphocyte phenotypes, with 1 subgroup associated with poorer clinical outcome. We propose that alterations in B and T cell function following hospitalization with COVID-19 could affect longer-term immunity and contribute to some persistent symptoms observed in convalescent COVID-19 patients. FUNDING: Provided by UKRI, Lister Institute of Preventative Medicine, the Wellcome Trust, The Kennedy Trust for Rheumatology Research, and 3M Global Giving.

A pre-validation transferability study of the GreenScreen HC GADD45a-GFP assay with a metabolic activation system (S9).

A new protocol has recently been developed and validated for the GreenScreen HC GADD45a-GFP genotoxicity reporter assay, enabling the incorporation of an S9 metabolic activation system into the assay. The S9 protocol employs flow-cytometric methodology for the detection of both reporter GFP fluorescence and propidium iodide fluorescence for the estimation of cellular viability. In the spirit of assay validation by bodies such as the European Centre for the Validation of Alternative Methods (ECVAM), the adapted metabolic activation protocol for the GADD45a-GFP assay has been undergoing 'pre-validation'. Results of phases I and II of this pre-validation, namely protocol refinement and protocol transfer, respectively, are presented here. In phase I the protocol was transferred to a second laboratory for initial assessment of method portability and subsequent refinement of the protocol. In phase II, the protocol was then transferred to two further laboratories along with the elaborated standard operating-procedure (SOP) for further assessment of transferability. The three transfer sites then undertook an assessment of the method's reproducibility by testing eight compounds. The outcome of the study was a refined protocol that was found to be highly transferable. It yielded 100% agreement in results between all four laboratories.

Neurturin regulates the lung-resident macrophage inflammatory response to viral infection.

Lung-resident macrophages are crucial to the maintenance of health and in the defence against lower respiratory tract infections. Macrophages adapt to local environmental cues that drive their appropriate function; however, this is often dysregulated in many inflammatory lung pathologies. In mucosal tissues, neuro-immune interactions enable quick and efficient inflammatory responses to pathogenic threats. Although a number of factors that influence the antimicrobial response of lung macrophages are known, the role of neuronal factors is less well understood. Here, we show an intricate circuit involving the neurotrophic factor, neurturin (NRTN) on human lung macrophages that dampens pro-inflammatory cytokine release and modulates the type of matrix metalloproteinases produced in response to viral stimuli. This circuit involves type 1 interferon-induced up-regulation of RET that when combined with the glial cell line-derived neurotrophic factor (GDNF) receptor α2 (GFRα2) allows binding to epithelial-derived NRTN. Our research highlights a non-neuronal immunomodulatory role for NRTN and a novel process leading to a specific antimicrobial immune response by human lung-resident macrophages.

Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19.

COVID-19 pathogenesis is associated with an exaggerated immune response. However, the specific cellular mediators and inflammatory components driving diverse clinical disease outcomes remain poorly understood. We undertook longitudinal immune profiling on both whole blood and peripheral blood mononuclear cells (PBMCs) of hospitalized patients during the peak of the COVID-19 pandemic in the UK. Here, we report key immune signatures present shortly after hospital admission that were associated with the severity of COVID-19. Immune signatures were related to shifts in neutrophil to T cell ratio, elevated serum IL-6, MCP-1 and IP-10, and most strikingly, modulation of CD14+ monocyte phenotype and function. Modified features of CD14+ monocytes included poor induction of the prostaglandin-producing enzyme, COX-2, as well as enhanced expression of the cell cycle marker Ki-67. Longitudinal analysis revealed reversion of some immune features back to the healthy median level in patients with a good eventual outcome. These findings identify previously unappreciated alterations in the innate immune compartment of COVID-19 patients and lend support to the idea that therapeutic strategies targeting release of myeloid cells from bone marrow should be considered in this disease. Moreover, they demonstrate that features of an exaggerated immune response are present early after hospital admission suggesting immune-modulating therapies would be most beneficial at early timepoints.

Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay.

Genotoxicity can be assessed by monitoring expression of a GADD45a-GFP reporter in the human lymphoblastoid cell line TK6. A flow cytometric method has been developed to effectively distinguish GFP fluorescence from coloured and fluorescent test samples as well from the S9 liver extracts used to generate metabolites from pro-genotoxins. The method includes the use of propidium iodide exclusion for the determination of cellular viability. This paper describes the method development, the derivation of decision thresholds for the identification of genotoxins using the method, and presents data from a 56-compound validation study of the method. The results illustrate that the method permitted the detection of the majority of pro-genotoxins tested and, importantly, the high specificity of the GADD45a-GFP assay was maintained.

Sensing of apoptotic cells through Axl causes lung basal cell proliferation in inflammatory diseases.

Epithelial cell proliferation, division, and differentiation are critical for barrier repair following inflammation, but the initial trigger for this process is unknown. Here we define that sensing of apoptotic cells by the TAM receptor tyrosine kinase Axl is a critical indicator for tracheal basal cell expansion, cell cycle reentry, and symmetrical cell division. Furthermore, once the pool of tracheal basal cells has expanded, silencing of Axl is required for their differentiation. Genetic depletion of Axl triggers asymmetrical cell division, leading to epithelial differentiation and ciliated cell regeneration. This discovery has implications for conditions associated with epithelial barrier dysfunction, basal cell hyperplasia, and continued turnover of dying cells in patients with chronic inflammatory pulmonary diseases.

Defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis.

Little is known about the impact of viral infections on lung matrix despite its important contribution to mechanical stability and structural support. The composition of matrix also indirectly controls inflammation by influencing cell adhesion, migration, survival, proliferation and differentiation. Hyaluronan is a significant component of the lung extracellular matrix and production and degradation must be carefully balanced. We have discovered an imbalance in hyaluronan production following resolution of a severe lung influenza virus infection, driven by hyaluronan synthase 2 from epithelial cells, endothelial cells and fibroblasts. Furthermore hyaluronan is complexed with inter-α-inhibitor heavy chains due to elevated TNF-stimulated gene 6 expression and sequesters CD44-expressing macrophages. We show that intranasal administration of exogenous hyaluronidase is sufficient to release inter-α-inhibitor heavy chains, reduce lung hyaluronan content and restore lung function. Hyaluronidase is already used to facilitate dispersion of co-injected materials in the clinic. It is therefore feasible that fibrotic changes following severe lung infection and inflammation could be overcome by targeting abnormal matrix production.

The consequence of matrix dysfunction on lung immunity and the microbiome in COPD.

The pulmonary extracellular matrix (ECM) is a complex network of proteins which primarily defines tissue architecture and regulates various biochemical and biophysical processes. It is a dynamic system comprising two main structures (the interstitial matrix and the basement membrane) which undergo continuous, yet highly regulated, remodelling. This remodelling process is essential for tissue homeostasis and uncontrolled regulation can lead to pathological states including chronic obstructive pulmonary disease (COPD). Altered expression of ECM proteins, as observed in COPD, can contribute to the degradation of alveolar walls and thickening of the small airways which can cause limitations in airflow. Modifications in ECM composition can also impact immune cell migration and retention in the lung with migrating cells becoming entrapped in the diseased airspaces. Furthermore, ECM changes affect the lung microbiome, aggravating and advancing disease progression. A dysbiosis in bacterial diversity can lead to infection, inducing epithelial injury and pro-inflammatory reactions. Here we review the changes noted in the different ECM components in COPD and discuss how an imbalance in microbial commensalism can impact disease development.

Innate Immune Cell Suppression and the Link With Secondary Lung Bacterial Pneumonia.

Secondary infections arise as a consequence of previous or concurrent conditions and occur in the community or in the hospital setting. The events allowing secondary infections to gain a foothold have been studied for many years and include poor nutrition, anxiety, mental health issues, underlying chronic diseases, resolution of acute inflammation, primary immune deficiencies, and immune suppression by infection or medication. Children, the elderly and the ill are particularly susceptible. This review is concerned with secondary bacterial infections of the lung that occur following viral infection. Using influenza virus infection as an example, with comparisons to rhinovirus and respiratory syncytial virus infection, we will update and review defective bacterial innate immunity and also highlight areas for potential new investigation. It is currently estimated that one in 16 National Health Service (NHS) hospital patients develop an infection, the most common being pneumonia, lower respiratory tract infections, urinary tract infections and infection of surgical sites. The continued drive to understand the mechanisms of why secondary infections arise is therefore of key importance.

A crucial role for thiol antioxidants in estrogen-deficiency bone loss.

The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone. Thus, estrogen might prevent bone loss by enhancing oxidant defenses in bone. We found that glutathione and thioredoxin, the major thiol antioxidants, and glutathione and thioredoxin reductases, the enzymes responsible for maintaining them in a reduced state, fell substantially in rodent bone marrow after ovariectomy and were rapidly normalized by exogenous 17-beta estradiol. Moreover, administration of N-acetyl cysteine (NAC) or ascorbate, antioxidants that increase tissue glutathione levels, abolished ovariectomy-induced bone loss, while l-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, caused substantial bone loss. The 17-beta estradiol increased glutathione and glutathione and thioredoxin reductases in osteoclast-like cells in vitro. Furthermore, in vitro NAC prevented osteoclast formation and NF-kappaB activation. BSO and hydrogen peroxide did the opposite. Expression of TNF-alpha, a target for NF-kappaB and a cytokine strongly implicated in estrogen-deficiency bone loss, was suppressed in osteoclasts by 17-beta estradiol and NAC. These observations strongly suggest that estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts. This directly sensitizes osteoclasts to osteoclastogenic signals and entrains ROS-enhanced expression of cytokines that promote osteoclastic bone resorption.

Development of methods to measure humoral immune responses against selected antigens in the common marmoset (Callithrix jacchus) and the effect of pyridostigmine bromide administration.

This methodological study was carried out in preparation for a major long term study, also reported in this volume, which was designed to investigate whether the combination of vaccines and pyridostigmine bromide (PB) could have been responsible for adverse signs and symptoms reported by a number of veterans of the 1990/1991 Gulf conflict. In this context, the marmoset has been used to model aspects of the human immune system. The purposes of this methodological study were to select appropriate immunochemical reagents to measure humoral responses induced in marmosets in response to selected health and hygiene and biological warfare vaccines and to initially assess the effects of PB on the responses recorded. Vaccines were administered at 1/5th of a human dose, and also investigated in combination with the nerve agent pretreatment compound PB. PB dosing was selected to induce an inhibition of erythrocyte acetylcholinesterase by 30%. In order to assess the functionality of the immune system, antibody responses to a neo-antigen (keyhole limpet haemocyanin--KLH), administered some 2 months following the completion of the vaccination schedule, were measured. The present study identified appropriate isotyping reporter reagents which cross-reacted with equivalent marmoset immunoglobulins. Robust antibody responses were identified against anthrax protective antigen (PA), whole cell pertussis vaccine and KLH, while weaker responses were measured against cholera and typhoid vaccines. The killed whole cell plague vaccine induced a response which was at the limit of detection of the assay. Coadministered PB had no discernable effect on immunological responses in this study.