Recruited atypical Ly6G+ macrophages license alveolar regeneration after lung injury.

The lung is constantly exposed to airborne pathogens and particles that can cause alveolar damage. Hence, appropriate repair responses are essential for gas exchange and life. Here, we deciphered the spatiotemporal trajectory and function of an atypical population of macrophages after lung injury. Post-influenza A virus (IAV) infection, short-lived monocyte-derived Ly6G-expressing macrophages (Ly6G+ Macs) were recruited to the alveoli of lung perilesional areas. Ly6G+ Macs engulfed immune cells, exhibited a high metabolic potential, and clustered with alveolar type 2 epithelial cells (AT2s) in zones of active epithelial regeneration. Ly6G+ Macs were partially dependent on granulocyte-macrophage colony-stimulating factor and interleukin-4 receptor signaling and were essential for AT2-dependent alveolar regeneration. Similar macrophages were recruited in other models of injury and in the airspaces of lungs from patients with suspected pneumonia. This study identifies perilesional alveolar Ly6G+ Macs as a spatially restricted, short-lived macrophage subset promoting epithelial regeneration postinjury, thus representing an attractive therapeutic target for treating lung damage.

[Lung marginated neutrophils : more than immune defenders, gardians of lung endothelial homeostasis ?] / Neutrophiles marginés pulmonaires :au-delà du système de défense, des gardiens de l'homéostasie endothéliale pulmonaire ?

The fields of neutrophil and endothelial cell biology are being deeply revised. While lung marginated neutrophils have been identified decades ago, their roles in the healthy adult lung are still contentious. Furthermore, while it is now clear that the lung constitutes an important immunological niche, the role of lung endothelial cells has been neglected so far. A better understanding of the role of short-lived neutrophils in contributing to lung endothelial cell physiology will improve our understanding of lung endothelial cell fate and heterogeneity under homeostasis and inflammation. Furthermore, it will provide new mechanistic insights on lung marginated neutrophil function and crosstalk with endothelial cells and provide robust foundations for devising therapeutic approaches in which endothelial cell (dys)functions are involved.

Le domaine de la biologie des neutrophiles et des cellules endothéliales est en pleine révision. Si les neutrophiles marginés pulmonaires ont été identifiés il y a plusieurs décennies, leur rôle au niveau du poumon adulte sain reste controversé. De plus, alors qu'il est maintenant reconnu que le poumon constitue une niche immunologique importante, le rôle des cellules endothéliales au niveau de ces niches a, jusqu'à présent, été négligé. Une meilleure compréhension du rôle des neutrophiles marginés dans un poumon sain ainsi que de leur contribution à la physiologie des cellules endothéliales permettrait d'améliorer nos connaissances concernant la biologie et l'hétérogénéité des cellules endothéliales en conditions d'homéostasie et inflammatoires. Enfin, un aperçu mécanistique des relations entre les neutrophiles marginés pulmonaires et les cellules endothéliales constituerait une base solide à l'élaboration de nouvelles stratégies thérapeutiques lors de dysfonctionnements de l'endothélium.

Journey of monocytes and macrophages upon influenza A virus infection.

Influenza A virus (IAV) infections pose a global health challenge that necessitates a comprehensive understanding of the host immune response to devise effective therapeutic interventions. As monocytes and macrophages play crucial roles in host defence, inflammation, and repair, this review explores the intricate journey of these cells during and after IAV infection. First, we highlight the dynamics and functions of lung-resident macrophage populations post-IAV. Second, we review the current knowledge of recruited monocytes and monocyte-derived cells, emphasising their roles in viral clearance, inflammation, immunomodulation, and tissue repair. Third, we shed light on the consequences of IAV-induced macrophage alterations on long-term lung immunity. We conclude by underscoring current knowledge gaps and exciting prospects for future research in unravelling the complexities of macrophage responses to respiratory viral infections.

Lung Interstitial Macrophages Can Present Soluble Antigens and Induce Foxp3+ Regulatory T Cells.

Lung macrophages constitute a sophisticated surveillance and defense system that contributes to tissue homeostasis and host defense and allows the host to cope with the myriad of insults and antigens to which the lung mucosa is exposed. As opposed to alveolar macrophages, lung interstitial macrophages (IMs) express high levels of Type 2 major histocompatibility complex (MHC-II), a hallmark of antigen-presenting cells. Here, we showed that lung IMs, like dendritic cells, possess the machinery to present soluble antigens in an MHC-II-restricted way. Using ex vivo ovalbumin (OVA)-specific T cell proliferation assays, we found that OVA-pulsed IMs could trigger OVA-specific CD4+ T cell proliferation and Foxp3 expression through MHC-II-, IL-10-, and transforming growth factor ß-dependent mechanisms. Moreover, we showed that IMs efficiently captured locally instilled antigens in vivo, did not migrate to the draining lymph nodes, and enhanced local interactions with CD4+ T cells in a model of OVA-induced allergic asthma. These results support that IMs can present antigens to CD4+ T cells and trigger regulatory T cells, which might attenuate lung immune responses and have functional consequences for lung immunity and T cell-mediated disorders.

MafB-restricted local monocyte proliferation precedes lung interstitial macrophage differentiation.

Resident tissue macrophages (RTMs) are differentiated immune cells that populate distinct niches and exert important tissue-supportive functions. RTM maintenance is thought to rely either on differentiation from monocytes or on RTM self-renewal. Here, we used a mouse model of inducible lung interstitial macrophage (IM) niche depletion and refilling to investigate the development of IMs in vivo. Using time-course single-cell RNA-sequencing analyses, bone marrow chimeras and gene targeting, we found that engrafted Ly6C+ classical monocytes proliferated locally in a Csf1 receptor-dependent manner before differentiating into IMs. The transition from monocyte proliferation toward IM subset specification was controlled by the transcription factor MafB, while c-Maf specifically regulated the identity of the CD206+ IM subset. Our data provide evidence that, in the mononuclear phagocyte system, the ability to proliferate is not merely restricted to myeloid progenitor cells and mature RTMs but is also a tightly regulated capability of monocytes developing into RTMs in vivo.

INPP5K controls the dynamic structure and signaling of wild-type and mutated, leukemia-associated IL-7 receptors.

The downstream signaling of the interleukin-7 (IL-7) receptor (IL-7R) plays important physiological and pathological roles, including the differentiation of lymphoid cells and proliferation of acute lymphoblastic leukemia cells. Gain-of-function mutations in the IL-7Rα chain, the specific component of the receptor for IL-7, result in constitutive, IL-7-independent signaling and trigger acute lymphoblastic leukemia. Here, we show that the loss of the phosphoinositide 5-phosphatase INPP5K is associated with increased levels of the INPP5K substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and causes an altered dynamic structure of the IL-7 receptor. We discovered that the IL-7Rα chain contains a very conserved positively charged polybasic amino acid sequence in its cytoplasmic juxtamembrane region; this region establish stronger ionic interactions with negatively charged PtdIns(4,5)P2 in the absence of INPP5K, freezing the IL-7Rα chain structure. This dynamic structural alteration causes defects in IL-7R signaling, culminating in decreased expressions of EBF1 and PAX5 transcription factors, in microdomain formation, cytoskeletal reorganization, and bone marrow B-cell differentiation. Similar alterations after the reduced INPP5K expression also affected mutated, constitutively activated IL-7Rα chains that trigger leukemia development, leading to reduced cell proliferation. Altogether, our results indicate that the lipid 5-phosphatase INPP5K hydrolyzes PtdIns(4,5)P2, allowing the requisite conformational changes of the IL-7Rα chain for optimal signaling.

Neutrophil Extracellular Traps Are Found in Bronchoalveolar Lavage Fluids of Horses With Severe Asthma and Correlate With Asthma Severity.

Asthma encompasses a spectrum of heterogenous immune-mediated respiratory disorders sharing a similar clinical pattern characterized by cough, wheeze and exercise intolerance. In horses, equine asthma can be subdivided into severe or moderate asthma according to clinical symptoms and the extent of airway neutrophilic inflammation. While severe asthmatic horses are characterized by an elevated neutrophilic inflammation of the lower airways, cough, dyspnea at rest and high mucus secretion, horses with moderate asthma show a milder neutrophilic inflammation, exhibit intolerance to exercise but no labored breathing at rest. Yet, the physiopathology of different phenotypes of equine asthma remains poorly understood and there is a need to elucidate the underlying mechanisms tailoring those phenotypes in order to improve clinical management and elaborate novel therapeutic strategies. In this study, we sought to quantify the presence of neutrophil extracellular traps (NETs) in bronchoalveolar lavage fluids (BALF) of moderate or severe asthmatic horses and healthy controls, and assessed whether NETs correlated with disease severity. To this end, we evaluated the amounts of NETs by measuring cell-free DNA and MPO-DNA complexes in BALF supernatants or by quantifying NETs release by BALF cells by confocal microscopy. We were able to unequivocally identify elevated NETs levels in BALF of severe asthmatic horses as compared to healthy controls or moderate asthmatic horses. Moreover, we provided evidence that BALF NETs release was a specific feature seen in severe equine asthma, as opposed to moderate asthma, and correlated with disease severity. Finally, we showed that NETs could act as a predictive factor for severe equine asthma. Our study thus uniquely identifies NETs in BALF of severe asthmatic horses using three distinct methods and supports the idea that moderate and severe equine asthma do not rely on strictly similar pathophysiological mechanisms. Our data also suggest that NETs represent a relevant biomarker, a putative driver and a potential therapeutic target in severe asthma disease.

Methods to Detect Neutrophil Extracellular Traps in Asthma.

Neutrophil extracellular traps (NETs) have the ability to regulate many aspects of asthma pathology. NETs can be detected either in bronchoalveolar lavage fluids (BALF) or in lung biopsies. Here, we describe methods to quantify NETs in BALF, namely the quantification of cell-free DNA, or of myeloperoxidase (MPO) or neutrophil elastase (NE) complexed with cell-free DNA. We also explain how to detect NETs in lung biopsies by two distinct techniques. The first technique is based on quantification of the citrullinated form of histone 3 (Cit-H3 , a specific component of NET) by western blot on tissue protein extracts. The second technique is based on the visualization of extracellular structures composed of MPO co-localizing with Cit-H3 in tissue sections by confocal microscopy. Finally, we describe a method allowing for quantification of NET volume in lung sections.

Identification and Quantitation of Neutrophil Extracellular Traps in Human Tissue Sections.

Neutrophils are one of the first innate immune cells recruited to tissues during inflammation. An important function of neutrophils relies on their ability to release extracellular structures, known as Neutrophil Extracellular Traps or NETs, into their environment. Detecting such NETs in humans has often proven challenging for both biological fluids and tissues; however, this can be achieved by quantitating NET components (e.g., DNA or granule/histone proteins) or by directly visualizing them by microscopy, respectively. Direct visualization by confocal microscopy is preferably performed on formalin-fixed paraffin-embedded (FFPE) tissue sections stained with a fluorescent DNA dye and antibodies directed against myeloperoxidase (MPO) and citrullinated histone 3 (Cit-H3), two components of NETs, following paraffin removal, antigen retrieval, and permeabilization. NETs are defined as extracellular structures that stain double-positive for MPO and Cit-H3. Here, we propose a novel software-based objective method for NET volume quantitation in tissue sections based on the measurement of the volume of structures exhibiting co-localization of Cit-H3 and MPO outside the cell. Such a technique not only allows the unambiguous identification of NETs in tissue sections but also their quantitation and relationship with surrounding tissues. Graphic abstract: Graphical representation of the methodology used to stain and quantitate NETs in human lung tissue.

Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy.

BACKGROUND: High-mobility group box 1 (HMGB1) is a multifunctional redox-sensitive protein involved in various intracellular (eg, chromatin remodeling, transcription, autophagy) and extracellular (inflammation, autoimmunity) processes. Regarding its role in cancer development/progression, paradoxical results exist in the literature and it is still unclear whether HMGB1 mainly acts as an oncogene or a tumor suppressor. METHODS: HMGB1 expression was first assessed in tissue specimens (n=359) of invasive breast, lung and cervical cancer and the two distinct staining patterns detected (nuclear vs cytoplasmic) were correlated to the secretion profile of malignant cells, patient outcomes and the presence of infiltrating immune cells within tumor microenvironment. Using several orthotopic, syngeneic mouse models of basal-like breast (4T1, 67NR and EpRas) or non-small cell lung (TC-1) cancer, the efficacy of several HMGB1 inhibitors alone and in combination with immune checkpoint blockade antibodies (anti-PD-1/PD-L1) was then investigated. Isolated from retrieved tumors, 14 immune cell (sub)populations as well as the activation status of antigen-presenting cells were extensively analyzed in each condition. Finally, the redox state of HMGB1 in tumor-extruded fluids and the influence of different forms (oxidized, reduced or disulfide) on both dendritic cell (DC) and plasmacytoid DC (pDC) activation were determined. RESULTS: Associated with an unfavorable prognosis in human patients, we clearly demonstrated that targeting extracellular HMGB1 elicits a profound remodeling of tumor immune microenvironment for efficient cancer therapy. Indeed, without affecting the global number of (CD45+) immune cells, drastic reductions of monocytic/granulocytic myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes, a higher M1/M2 ratio of macrophages as well as an increased activation of both DC and pDC were continually observed following HMGB1 inhibition. Moreover, blocking HMGB1 improved the efficacy of anti-PD-1 cancer monoimmunotherapy. We also reported that a significant fraction of HMGB1 encountered within cancer microenvironment (interstitial fluids) is oxidized and, in opposite to its reduced isoform, oxidized HMGB1 acts as a tolerogenic signal in a receptor for advanced glycation endproducts-dependent manner. CONCLUSION: Collectively, we present evidence that extracellular HMGB1 blockade may complement first-generation cancer immunotherapies by remobilizing antitumor immune response.

Neutrophil extracellular traps infiltrate the lung airway, interstitial, and vascular compartments in severe COVID-19.

Infection with SARS-CoV-2 is causing a deadly and pandemic disease called coronavirus disease-19 (COVID-19). While SARS-CoV-2-triggered hyperinflammatory tissue-damaging and immunothrombotic responses are thought to be major causes of respiratory failure and death, how they relate to lung immunopathological changes remains unclear. Neutrophil extracellular traps (NETs) can contribute to inflammation-associated lung damage, thrombosis, and fibrosis. However, whether NETs infiltrate particular compartments in severe COVID-19 lungs remains to be clarified. Here we analyzed postmortem lung specimens from four patients who succumbed to COVID-19 and four patients who died from a COVID-19-unrelated cause. We report the presence of NETs in the lungs of each COVID-19 patient. NETs were found in the airway compartment and neutrophil-rich inflammatory areas of the interstitium, while NET-prone primed neutrophils were present in arteriolar microthrombi. Our results support the hypothesis that NETs may represent drivers of severe pulmonary complications of COVID-19 and suggest that NET-targeting approaches could be considered for the treatment of uncontrolled tissue-damaging and thrombotic responses in COVID-19.

Epithelial RABGEF1 deficiency promotes intestinal inflammation by dysregulating intrinsic MYD88-dependent innate signaling.

Intestinal epithelial cells (IECs) contribute to the regulation of intestinal homeostasis and inflammation through their interactions with the environment and host immune responses. Yet our understanding of IEC-intrinsic regulatory pathways remains incomplete. Here, we identify the guanine nucleotide exchange factor RABGEF1 as a regulator of intestinal homeostasis and innate pathways dependent on IECs. Mice with IEC-specific Rabgef1 deletion (called Rabgef1IEC-KO mice) developed a delayed spontaneous colitis associated with the local upregulation of IEC chemokine expression. In mouse models of colitis based on Interleukin-10 deficiency or dextran sodium sulfate (DSS) exposure, we found that IEC-intrinsic RABGEF1 deficiency exacerbated development of intestinal pathology and dysregulated IEC innate pathways and chemokine expression. Mechanistically, we showed that RABGEF1 deficiency in mouse IECs in vitro was associated with an impairment of early endocytic events, an increased activation of the p38 mitogen-activated protein kinase (MAPK)-dependent pathway, and increased chemokine secretion. Moreover, we provided evidence that the development of spontaneous colitis was dependent on microbiota-derived signals and intrinsic MYD88-dependent pathways in vivo. Our study identifies mouse RABGEF1 as an important regulator of intestinal inflammation, MYD88-dependent IEC-intrinsic signaling, and chemokine production. This suggests that RABGEF1-dependent pathways represent interesting therapeutic targets for inflammatory conditions in the gut.

Locally instructed CXCR4hi neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps.

Low exposure to microbial products, respiratory viral infections and air pollution are major risk factors for allergic asthma, yet the mechanistic links between such conditions and host susceptibility to type 2 allergic disorders remain unclear. Through the use of single-cell RNA sequencing, we characterized lung neutrophils in mice exposed to a pro-allergic low dose of lipopolysaccharide (LPS) or a protective high dose of LPS before exposure to house dust mites. Unlike exposure to a high dose of LPS, exposure to a low dose of LPS instructed recruited neutrophils to upregulate their expression of the chemokine receptor CXCR4 and to release neutrophil extracellular traps. Low-dose LPS-induced neutrophils and neutrophil extracellular traps potentiated the uptake of house dust mites by CD11b+Ly-6C+ dendritic cells and type 2 allergic airway inflammation in response to house dust mites. Neutrophil extracellular traps derived from CXCR4hi neutrophils were also needed to mediate allergic asthma triggered by infection with influenza virus or exposure to ozone. Our study indicates that apparently unrelated environmental risk factors can shape recruited lung neutrophils to promote the initiation of allergic asthma.

Non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung.

Resident tissue macrophages (RTM) can fulfill various tasks during development, homeostasis, inflammation and repair. In the lung, non-alveolar RTM, called interstitial macrophages (IM), importantly contribute to tissue homeostasis but remain little characterized. Here we show, using single-cell RNA-sequencing (scRNA-seq), two phenotypically distinct subpopulations of long-lived monocyte-derived IM, i.e. CD206+ and CD206-IM, as well as a discrete population of extravasating CD64+CD16.2+ monocytes. CD206+ IM are peribronchial self-maintaining RTM that constitutively produce high levels of chemokines and immunosuppressive cytokines. Conversely, CD206-IM preferentially populate the alveolar interstitium and exhibit features of antigen-presenting cells. In addition, our data support that CD64+CD16.2+ monocytes arise from intravascular Ly-6Clo patrolling monocytes that enter the tissue at steady-state to become putative precursors of CD206-IM. This study expands our knowledge about the complexity of lung IM and reveals an ontogenic pathway for one IM subset, an important step for elaborating future macrophage-targeted therapies.

Ozone-primed neutrophils promote early steps of tumour cell metastasis to lungs by enhancing their NET production.

BACKGROUND: Air pollution, including particulates and gazes such as ozone (O3), is detrimental for patient's health and has repeatedly been correlated to increased morbidity and mortality in industrialised countries. Although studies have described a link between ambient particulate matter and increased lung cancer morbidity, no direct relation has yet been established between O3 exposure and metastatic dissemination to lungs. OBJECTIVES: To outline the mechanisms through which pulmonary O3 exposure modulates metastasis kinetics in an experimental mouse model of O3 exposure. METHODS: Metastatic responses to pulmonary O3 exposure were assessed using a reliable experimental mouse model of concomitant pulmonary O3 exposure and tumour cell injection. Roles of neutrophils in O3-induced lung metastasis were highlighted using blocking anti-Ly6G antibodies; moreover, the implication of neutrophil extracellular traps (NETs) in metastatic processes was evaluated using MRP8cre-Pad4lox/lox mice or by treating mice with DNase I. RESULTS: Pulmonary O3 exposure strongly facilitates the establishment of lung metastasis by (1) Inducing a pulmonary injury and neutrophilic inflammation, (2) Influencing very early steps of metastasis, (3) Priming neutrophils' phenotype to release NETs that favour tumour cell colonisation in lungs. The ability of O3-primed neutrophils to enhance lung colonisation by tumour cells was proven after their adoptive transfer in Balb/c mice unexposed to O3. CONCLUSIONS: Pulmonary neutrophils induced by O3 promote metastatic dissemination to lungs by producing NETs. These findings open new perspectives to improve treatment and prevention strategies in patients affected by metastatic diseases.

Role of neutrophils in allergic asthma.

The contribution of neutrophils to asthma pathogenesis has been mainly studied in the context of non-allergic neutrophilic asthma. However, neutrophils can also be rapidly recruited and are largely present in the airways of allergic eosinophilic asthmatic patients. Under these circumstances, they possess specific phenotypic features distinguishing them from resting blood neutrophils and are endowed with particular functions. The exact contribution of neutrophils to allergic asthma pathogenesis is still unclear, but growing experimental evidence supports the ability of neutrophils or neutrophil-derived products to influence the underlying allergic type 2 immune response and cardinal features of allergic asthma, thus shedding new light on neutrophil biology and functions in an allergic context.

Corrigendum: Host DNA released by NETosis promotes rhinovirus-induced type-2 allergic asthma exacerbation.

This corrects the article DOI: 10.1038/nm.4332.

Host DNA released by NETosis promotes rhinovirus-induced type-2 allergic asthma exacerbation.

Respiratory viral infections represent the most common cause of allergic asthma exacerbations. Amplification of the type-2 immune response is strongly implicated in asthma exacerbation, but how virus infection boosts type-2 responses is poorly understood. We report a significant correlation between the release of host double-stranded DNA (dsDNA) following rhinovirus infection and the exacerbation of type-2 allergic inflammation in humans. In a mouse model of allergic airway hypersensitivity, we show that rhinovirus infection triggers dsDNA release associated with the formation of neutrophil extracellular traps (NETs), known as NETosis. We further demonstrate that inhibiting NETosis by blocking neutrophil elastase or by degrading NETs with DNase protects mice from type-2 immunopathology. Furthermore, the injection of mouse genomic DNA alone is sufficient to recapitulate many features of rhinovirus-induced type-2 immune responses and asthma pathology. Thus, NETosis and its associated extracellular dsDNA contribute to the pathogenesis and may represent potential therapeutic targets of rhinovirus-induced asthma exacerbations.

Exposure to Bacterial CpG DNA Protects from Airway Allergic Inflammation by Expanding Regulatory Lung Interstitial Macrophages.

Living in a microbe-rich environment reduces the risk of developing asthma. Exposure of humans or mice to unmethylated CpG DNA (CpG) from bacteria reproduces these protective effects, suggesting a major contribution of CpG to microbe-induced asthma resistance. However, how CpG confers protection remains elusive. We found that exposure to CpG expanded regulatory lung interstitial macrophages (IMs) from monocytes infiltrating the lung or mobilized from the spleen. Trafficking of IM precursors to the lung was independent of CCR2, a chemokine receptor required for monocyte mobilization from the bone marrow. Using a mouse model of allergic airway inflammation, we found that adoptive transfer of IMs isolated from CpG-treated mice recapitulated the protective effects of CpG when administered before allergen sensitization or challenge. IM-mediated protection was dependent on IL-10, given that Il10-/- CpG-induced IMs lacked regulatory effects. Thus, the expansion of regulatory lung IMs upon exposure to CpG might underlie the reduced risk of asthma development associated with a microbe-rich environment.