The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.

COVID-19 therapeutics: Challenges and directions for the future.

The emergence of SARS-CoV-2 triggering the COVID-19 pandemic ranks as arguably the greatest medical emergency of the last century. COVID-19 has highlighted health disparities both within and between countries and will leave a lasting impact on global society. Nonetheless, substantial investment in life sciences over recent decades has facilitated a rapid scientific response with innovations in viral characterization, testing, and sequencing. Perhaps most remarkably, this permitted the development of highly effective vaccines, which are being distributed globally at unprecedented speed. In contrast, drug treatments for the established disease have delivered limited benefits so far. Innovative and rapid approaches in the design and execution of large-scale clinical trials and repurposing of existing drugs have saved many lives; however, many more remain at risk. In this review we describe challenges and unmet needs, discuss existing therapeutics, and address future opportunities. Consideration is given to factors that have hindered drug development in order to support planning for the next pandemic challenge and to allow rapid and cost-effective development of new therapeutics with equitable delivery.

Regulatory B cells in respiratory health and diseases.

B cells are critical mediators of humoral immune responses in the airways through antibody production, antigen presentation, and cytokine secretion. In addition, a subset of B cells, known as regulatory B cells (Bregs), exhibit immunosuppressive functions via diverse regulatory mechanisms. Bregs modulate immune responses via the secretion of IL-10, IL-35, and tumor growth factor-ß (TGF-ß), and by direct cell contact. The balance between effector and regulatory B cell functions is critical in the maintenance of immune homeostasis. The importance of Bregs in airway immune responses is emphasized by the different respiratory disorders associated with abnormalities in Breg numbers and function. In this review, we summarize the role of immunosuppressive Bregs in airway inflammatory diseases and highlight the importance of this subset in the maintenance of respiratory health. We propose that improved understanding of signals in the lung microenvironment that drive Breg differentiation can provide novel therapeutic avenues for improved management of respiratory diseases.

Identification of Siglec-1-negative alveolar macrophages with proinflammatory phenotypes in chronic obstructive pulmonary disease.

Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. However, subpopulations of AMs participating in chronic inflammation have been poorly characterized. We previously reported that Siglec-1 expression on AMs, which is important for bacteria engulfment, was decreased in COPD. Here, we show that Siglec-1-negative AMs isolated from COPD lung tissues exhibit a proinflammatory phenotype and are associated with poor clinical outcomes in patients with COPD. Using flow cytometry, we segregated three subsets of AMs based on the expression of Siglec-1 and their side scattergram (SSC) and forward scattergram (FSC) properties: Siglec-1+SSChiFSChi, Siglec-1-SSChiFSChi, and Siglec-1-SSCloFSClo subsets. The Siglec-1-SSCloFSClo subset number was increased in COPD. RNA sequencing revealed upregulation of multiple proinflammatory signaling pathways and emphysema-associated matrix metalloproteases in the Siglec-1-SSCloFSClo subset. Gene set enrichment analysis indicated that the Siglec-1-SSCloFSClo subset adopted intermediate phenotypes between monocytes and mature alveolar macrophages. Functionally, these cells produced TNF-α, IL-6, and IL-8 at baseline, and these cytokines were significantly increased in response to viral RNA. The increase in Siglec-1-negative AMs in induced sputum is associated with future exacerbation risk and lung function decline in patients with COPD. Collectively, the novel Siglec-1-SSCloFSClo subset of AMs displays proinflammatory properties, and their emergence in COPD airways may be associated with poor clinical outcomes.NEW & NOTEWORTHY Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. We find that Siglec-1-negative alveolar macrophages have a wide range of proinflammatory landscapes and a protease-expressing phenotype. Moreover, this subset is associated with the pathogenesis of COPD and responds to viral stimuli.

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.

IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.

Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

The clock gene Bmal1 inhibits macrophage motility, phagocytosis, and impairs defense against pneumonia.

The circadian clock regulates many aspects of immunity. Bacterial infections are affected by time of day, but the mechanisms involved remain undefined. Here we show that loss of the core clock protein BMAL1 in macrophages confers protection against pneumococcal pneumonia. Infected mice show both reduced weight loss and lower bacterial burden in circulating blood. In vivo studies of macrophage phagocytosis reveal increased bacterial ingestion following Bmal1 deletion, which was also seen in vitro. BMAL1-/- macrophages exhibited marked differences in actin cytoskeletal organization, a phosphoproteome enriched for cytoskeletal changes, with reduced phosphocofilin and increased active RhoA. Further analysis of the BMAL1-/- macrophages identified altered cell morphology and increased motility. Mechanistically, BMAL1 regulated a network of cell movement genes, 148 of which were within 100 kb of high-confidence BMAL1 binding sites. Links to RhoA function were identified, with 29 genes impacting RhoA expression or activation. RhoA inhibition restored the phagocytic phenotype to that seen in control macrophages. In summary, we identify a surprising gain of antibacterial function due to loss of BMAL1 in macrophages, associated with a RhoA-dependent cytoskeletal change, an increase in cell motility, and gain of phagocytic function.

Glucocorticoids rapidly inhibit cell migration through a novel, non-transcriptional HDAC6 pathway.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.This article has an associated First Person interview with the first author of the paper.

The histone methyltransferase Ezh2 restrains macrophage inflammatory responses.

Robust inflammatory responses are critical to survival following respiratory infection, with current attention focused on the clinical consequences of the Coronavirus pandemic. Epigenetic factors are increasingly recognized as important determinants of immune responses, and EZH2 is a prominent target due to the availability of highly specific and efficacious antagonists. However, very little is known about the role of EZH2 in the myeloid lineage. Here, we show EZH2 acts in macrophages to limit inflammatory responses to activation, and in neutrophils for chemotaxis. Selective genetic deletion in macrophages results in a remarkable gain in protection from infection with the prevalent lung pathogen, pneumococcus. In contrast, neutrophils lacking EZH2 showed impaired mobility in response to chemotactic signals, and resulted in increased susceptibility to pneumococcus. In summary, EZH2 shows complex, and divergent roles in different myeloid lineages, likely contributing to the earlier conflicting reports. Compounds targeting EZH2 are likely to impair mucosal immunity; however, they may prove useful for conditions driven by pulmonary neutrophil influx, such as adult respiratory distress syndrome.

Decreased expression of airway epithelial Axl is associated with eosinophilic inflammation in severe asthma.

BACKGROUND: Airway epithelium-derived cytokines are critical to provoke and perpetuate type 2 inflammation in asthma. Yet it is poorly understood how this epithelial cell-driven inflammatory response is negatively regulated. We previously reported that Axl receptor tyrosine kinase was expressed by basal cells in the airway epithelium and had a role in defining their stem cell identity. However, whether and how Axl regulates airway type 2 inflammation remains unknown. METHODS: We performed immunofluorescence staining to compare Axl expression in airway epithelium between non-asthmatic subjects, mild-moderate asthma and severe asthma. We confirmed this result by interrogating public databases of global gene expression in endobronchial biopsies. We then quantified eosinophil numbers infiltrating into the trachea of wild-type or Axl-knockout mice that were intranasally treated with house dust mite extracts (HDM). Cell-based assays using siRNA targeting Axl were further performed to identify molecules involved in Axl-mediated regulation of inflammation. RESULTS: Histological assessments and transcriptome analyses revealed decreases in protein and mRNA of Axl in airway basal cells of severe asthmatics. This reduction of Axl expression was correlated with infiltration of eosinophils and mast cells in severe asthmatics. Eosinophil infiltration was more evident in the trachea of Axl-knockout mice in response to repetitive HDM administration. siRNA-mediated knockdown of Axl increased mRNA and protein expression of granulocyte macrophage-colony stimulating factor (GM-CSF) in human bronchial epithelial cells. CONCLUSIONS: Axl kinase expressed by basal cells may suppress excessive eosinophilic inflammation via inhibition of GM-CSF in the airway. Axl reduction has clinical implications for the pathogenesis of severe asthma.

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.

A critical function for CD200 in lung immune homeostasis and the severity of influenza infection.

The lung must maintain a high threshold of immune 'ignorance' to innocuous antigens to avoid inflammatory disease that depends on the balance of positive inflammatory signals and repressor pathways. We demonstrate here that airway macrophages had higher expression of the negative regulator CD200 receptor (CD200R) than did their systemic counterparts. Lung macrophages were restrained by CD200 expressed on airway epithelium. Mice lacking CD200 had more macrophage activity and enhanced sensitivity to influenza infection, which led to delayed resolution of inflammation and, ultimately, death. The administration of agonists that bind CD200R, however, prevented inflammatory lung disease. Thus, CD200R is critical for lung macrophage immune homeostasis in the resting state and limits inflammatory amplitude and duration during pulmonary influenza infection.

Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Pulmonary airway epithelial cells (AECs) form a critical interface between host and environment. We investigated the role of the circadian clock using mice bearing targeted deletion of the circadian gene brain and muscle ARNT-like 1 (Bmal1) in AECs. Pulmonary neutrophil infiltration, biomechanical function, and responses to influenza infection were all disrupted. A circadian time-series RNA sequencing study of laser-captured AECs revealed widespread disruption in genes of the core circadian clock and output pathways regulating cell metabolism (lipids and xenobiotics), extracellular matrix, and chemokine signaling, but strikingly also the gain of a novel rhythmic transcriptome in Bmal1-targeted cells. Many of the rhythmic components were replicated in primary AECs cultured in air-liquid interface, indicating significant cell autonomy for control of pulmonary circadian physiology. Finally, we found that metabolic cues dictate phasing of the pulmonary clock and circadian responses to immunologic challenges. Thus, the local circadian clock in AECs is vital in lung health by coordinating major cell processes such as metabolism and immunity.-Zhang, Z. Hunter, L., Wu, G., Maidstone, R., Mizoro, Y., Vonslow, R., Fife, M., Hopwood, T., Begley, N., Saer, B., Wang, P., Cunningham, P., Baxter, M., Durrington, H., Blaikley, J. F., Hussell, T., Rattray, M., Hogenesch, J. B., Gibbs, J., Ray, D. W., Loudon, A. S. I. Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Axl and MerTK receptor tyrosine kinases maintain human macrophage efferocytic capacity in the presence of viral triggers.

The requirement to remove apoptotic cells is equally important in homeostasis and inflammatory disease. In particular, during viral infections large quantities of infected cells undergo apoptosis and need to be efficiently cleared by phagocytes to prevent secondary necrosis. Although specific roles of several apoptotic cell sensors, such as the TAM (Tyro3, Axl, MerTK) receptor family, have been characterized in mouse models, little is known about their regulation and involvement in apoptotic cell uptake (efferocytosis) by human macrophages under inflammatory conditions. We show that whereas pro-inflammatory stimuli consistently downregulated MerTK expression in human monocyte-derived macrophages (MDMs), stimuli indicative of a viral infection, interferon-α (IFN-α) and the TLR3 ligand poly(I:C), specifically induced Axl expression and promoted binding of the bridging molecule Gas6. Axl induction by IFN-α and poly(I:C) was associated with higher MDM efferocytic capacity compared to cells treated with other pro-inflammatory stimuli, such as LPS and IFN-γ. While MerTK blocking antibody uniformly suppressed apoptotic cell uptake by MDMs, Axl blocking antibody significantly reduced efferocytosis by poly(I:C)-stimulated MDMs, but not by resting MDMs. Our observations demonstrate that Axl induction during viral infections contributes to maintaining macrophage capacity to engulf apoptotic cells, which may have important consequences for resolution of anti-viral immune responses.

Endothelin-1 mediates Aspergillus fumigatus-induced airway inflammation and remodelling.

BACKGROUND: Asthma is a chronic inflammatory condition of the airways and patients sensitized to airborne fungi such as Aspergillus fumigatus have more severe asthma. Thickening of the bronchial subepithelial layer is a contributing factor to asthma severity for which no current treatment exists. Airway epithelium acts as an initial defence barrier to inhaled spores, orchestrating an inflammatory response and contributing to subepithelial fibrosis. OBJECTIVE: We aimed to analyse the production of pro-fibrogenic factors by airway epithelium in response to A fumigatus, in order to propose novel anti-fibrotic strategies for fungal-induced asthma. METHODS: We assessed the induction of key pro-fibrogenic factors, TGF-ß1, TGF-ß2, periostin and endothelin-1, by human airway epithelial cells and in mice exposed to A fumigatus spores or secreted fungal factors. RESULTS: Aspergillus fumigatus specifically caused production of endothelin-1 by epithelial cells in vitro but not any of the other pro-fibrogenic factors assessed. A fumigatus also induced endothelin-1 in murine lungs, associated with extensive inflammation and airway remodelling. Using a selective endothelin-1 receptor antagonist, we demonstrated for the first time that endothelin-1 drives many features of airway remodelling and inflammation elicited by A fumigatus. CONCLUSION: Our findings are consistent with the hypothesis that elevated endothelin-1 levels contribute to subepithelial thickening and highlight this factor as a possible therapeutic target for difficult-to-treat fungal-induced asthma.

CD200 receptor restriction of myeloid cell responses antagonizes antiviral immunity and facilitates cytomegalovirus persistence within mucosal tissue.

CD200 receptor (CD200R) negatively regulates peripheral and mucosal innate immune responses. Viruses, including herpesviruses, have acquired functional CD200 orthologs, implying that viral exploitation of this pathway is evolutionary advantageous. However, the role that CD200R signaling plays during herpesvirus infection in vivo requires clarification. Utilizing the murine cytomegalovirus (MCMV) model, we demonstrate that CD200R facilitates virus persistence within mucosal tissue. Specifically, MCMV infection of CD200R-deficient mice (CD200R(-/-)) elicited heightened mucosal virus-specific CD4 T cell responses that restricted virus persistence in the salivary glands. CD200R did not directly inhibit lymphocyte effector function. Instead, CD200R(-/-) mice exhibited enhanced APC accumulation that in the mucosa was a consequence of elevated cellular proliferation. Although MCMV does not encode an obvious CD200 homolog, productive replication in macrophages induced expression of cellular CD200. CD200 from hematopoietic and non-hematopoietic cells contributed independently to suppression of antiviral control in vivo. These results highlight the CD200-CD200R pathway as an important regulator of antiviral immunity during cytomegalovirus infection that is exploited by MCMV to establish chronicity within mucosal tissue.

OX40L blockade is therapeutic in arthritis, despite promoting osteoclastogenesis.

An immune response is essential for protection against infection, but, in many individuals, aberrant responses against self tissues cause autoimmune diseases such as rheumatoid arthritis (RA). How to diminish the autoimmune response while not augmenting infectious risk is a challenge. Modern targeted therapies such as anti-TNF or anti-CD20 antibodies ameliorate disease, but at the cost of some increase in infectious risk. Approaches that might specifically reduce autoimmunity and tissue damage without infectious risk would be important. Here we describe that TNF superfamily member OX40 ligand (OX40L; CD252), which is expressed predominantly on antigen-presenting cells, and its receptor OX40 (on activated T cells), are restricted to the inflamed joint in arthritis in mice with collagen-induced arthritis and humans with RA. Blockade of this pathway in arthritic mice reduced inflammation and restored tissue integrity predominantly by inhibiting inflammatory cytokine production by OX40L-expressing macrophages. Furthermore, we identify a previously unknown role for OX40L in steady-state bone homeostasis. This work shows that more targeted approaches may augment the "therapeutic window" and increase the benefit/risk in RA, and possibly other autoimmune diseases, and are thus worth testing in humans.