[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, 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 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 (DCs), 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 via MHC-II-, IL-10 and Tgfß-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.

Evaluation of Non-Vector Transmission of Usutu Virus in Domestic Canaries (Serinus canaria).

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV "overwinters", the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>102 TCID50). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.

Differentiation of Bone Marrow Monocytes into Alveolar Macrophages-like Cells through Co-culture with Lung Epithelial Cells and Group 2 Innate Lymphoid Cells.

During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes. Key features • Ex vivo co-culture model of the alveolar niche. • Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.

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.

Dampening type 2 properties of group 2 innate lymphoid cells by a gammaherpesvirus infection reprograms alveolar macrophages.

Immunological dysregulation in asthma is associated with changes in exposure to microorganisms early in life. Gammaherpesviruses (γHVs), such as Epstein-Barr virus, are widespread human viruses that establish lifelong infection and profoundly shape host immunity. Using murid herpesvirus 4 (MuHV-4), a mouse γHV, we show that after infection, lung-resident and recruited group 2 innate lymphoid cells (ILC2s) exhibit a reduced ability to expand and produce type 2 cytokines in response to house dust mites, thereby contributing to protection against asthma. In contrast, MuHV-4 infection triggers GM-CSF production by those lung ILC2s, which orders the differentiation of monocytes (Mos) into alveolar macrophages (AMs) without promoting their type 2 functions. In the context of γHV infection, ILC2s are therefore essential cells within the pulmonary niche that imprint the tissue-specific identity of Mo-derived AMs and shape their function well beyond the initial acute infection.

Distinct blood protein profiles associated with the risk of short-term and mid/long-term clinical relapse in patients with Crohn's disease stopping infliximab: when the remission state hides different types of residual disease activity.

OBJECTIVE: Despite being in sustained and stable remission, patients with Crohn's disease (CD) stopping anti-tumour necrosis factor α (TNFα) show a high rate of relapse (~50% within 2 years). Characterising non-invasively the biological profiles of those patients is needed to better guide the decision of anti-TNFα withdrawal. DESIGN: Ninety-two immune-related proteins were measured by proximity extension assay in serum of patients with CD (n=102) in sustained steroid-free remission and stopping anti-TNFα (infliximab). As previously shown, a stratification based on time to clinical relapse was used to characterise the distinct biological profiles of relapsers (short-term relapsers: <6 months vs mid/long-term relapsers: >6 months). Associations between protein levels and time to clinical relapse were determined by univariable Cox model. RESULTS: The risk (HR) of mid/long-term clinical relapse was specifically associated with a high serum level of proteins mainly expressed in lymphocytes (LAG3, SH2B3, SIT1; HR: 2.2-4.5; p<0.05), a low serum level of anti-inflammatory effectors (IL-10, HSD11B1; HR: 0.2-0.3; p<0.05) and cellular junction proteins (CDSN, CNTNAP2, CXADR, ITGA11; HR: 0.4; p<0.05). The risk of short-term clinical relapse was specifically associated with a high serum level of pro-inflammatory effectors (IL-6, IL12RB1; HR: 3.5-3.6; p<0.05) and a low or high serum level of proteins mainly expressed in antigen presenting cells (CLEC4A, CLEC4C, CLEC7A, LAMP3; HR: 0.4-4.1; p<0.05). CONCLUSION: We identified distinct blood protein profiles associated with the risk of short-term and mid/long-term clinical relapse in patients with CD stopping infliximab. These findings constitute an advance for the development of non-invasive biomarkers guiding the decision of anti-TNFα withdrawal.

Enteric glial cells favor accumulation of anti-inflammatory macrophages during the resolution of muscularis inflammation.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206+ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206+ Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

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.

Airway Macrophages Encompass Transcriptionally and Functionally Distinct Subsets Altered by Smoking.

Alveolar macrophages (AMs) are functionally important innate cells involved in lung homeostasis and immunity and whose diversity in health and disease is a subject of intense investigations. Yet, it remains unclear to what extent conditions like smoking or chronic obstructive pulmonary disease (COPD) trigger changes in the AM compartment. Here, we aimed to explore heterogeneity of human AMs isolated from healthy nonsmokers, smokers without COPD, and smokers with COPD by analyzing BAL fluid cells by flow cytometry and bulk and single-cell RNA sequencing. We found that subpopulations of BAL fluid CD206+ macrophages could be distinguished based on their degree of autofluorescence in each subject analyzed. CD206+ autofluorescenthigh AMs were identified as classical, self-proliferative AM, whereas autofluorescentlow AMs were expressing both monocyte and classical AM-related genes, supportive of a monocytic origin. Of note, monocyte-derived autofluorescentlow AMs exhibited a functionally distinct immunoregulatory profile, including the ability to secrete the immunosuppressive cytokine IL-10. Interestingly, single-cell RNA-sequencing analyses showed that transcriptionally distinct clusters of classical and monocyte-derived AM were uniquely enriched in smokers with and without COPD as compared with healthy nonsmokers. Of note, such smoking-associated clusters exhibited gene signatures enriched in detoxification, oxidative stress, and proinflammatory responses. Our study independently confirms previous reports supporting that monocyte-derived macrophages coexist with classical AM in the airways of healthy subjects and patients with COPD and identifies smoking-associated changes in the AM compartment that may favor COPD initiation or progression.

IgE antibodies increase honeybee venom responsiveness and detoxification efficiency of mast cells.

BACKGROUND: In contrast to their clearly defined roles in allergic diseases, the physiologic functions of Immunoglobulin E antibodies (IgEs) and mast cells (MCs) remain enigmatic. Recent research supports the toxin hypothesis, showing that MCs and IgE-related type 2 immune responses can enhance host defense against certain noxious substances, including honeybee venom (BV). However, the mechanisms by which MCs can interfere with BV toxicity are unknown. In this study, we assessed the role of IgE and certain MC products in MC-mediated BV detoxification. METHODS: We applied in vitro and in vivo fluorescence microscopyimaging, and flow cytometry, fibroblast-based toxicity assays and mass spectrometry to investigate IgE-mediated detoxification of BV cytotoxicity by mouse and human MCs in vitro. Pharmacologic strategies to interfere with MC-derived heparin and proteases helped to define the importance of specific detoxification mechanisms. RESULTS: Venom-specific IgE increased the degranulation and cytokine responses of MCs to BV in vitro. Passive serum sensitization enhanced MC degranulation in vivo. IgE-activated mouse or human MCs exhibited enhanced potential for detoxifying BV by both proteolytic degradation and heparin-related interference with toxicity. Mediators released by IgE-activated human MCs efficiently degraded multiple BV toxins. CONCLUSIONS: Our results both reveal that IgE sensitization enhances the MC's ability to detoxify BV and also assign efficient toxin-neutralizing activity to MC-derived heparin and proteases. Our study thus highlights the potential importance of IgE, MCs, and particular MC products in defense against BV.

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.

Neutrophil-specific gain-of-function mutations in Nlrp3 promote development of cryopyrin-associated periodic syndrome.

Gain-of-function mutations in NLRP3 are responsible for a spectrum of autoinflammatory diseases collectively referred to as "cryopyrin-associated periodic syndromes" (CAPS). Treatment of CAPS patients with IL-1-targeted therapies is effective, confirming a central pathogenic role for IL-1ß. However, the specific myeloid cell population(s) exhibiting inflammasome activity and sustained IL-1ß production in CAPS remains elusive. Previous reports suggested an important role for mast cells (MCs) in this process. Here, we report that, in mice, gain-of-function mutations in Nlrp3 restricted to neutrophils, and to a lesser extent macrophages/dendritic cells, but not MCs, are sufficient to trigger severe CAPS. Furthermore, in patients with clinically established CAPS, we show that skin-infiltrating neutrophils represent a substantial biological source of IL-1ß. Together, our data indicate that neutrophils, rather than MCs, can represent the main cellular drivers of CAPS pathology.

Mast cells and IgE in defense against lethality of venoms: Possible "benefit" of allergy[].

Physicians think of mast cells and IgE primarily in the context of allergic disorders, including fatal anaphylaxis. This 'bad side' of mast cells and IgE is so well accepted that it can be difficult to think of them in other contexts, particularly those in which they may have beneficial functions. However, there is evidence that mast cells and IgE, as well as basophils (circulating granulocytes whose functions partially overlap with those of mast cells), can contribute to host defense as components of adaptive type 2 immune responses to helminths, ticks and certain other parasites. Accordingly, allergies often are conceptualized as "misdirected" type 2 immune responses, in which IgE antibodies are produced against any of a diverse group of apparently harmless antigens, and against components of animal venoms. Indeed, certain unfortunate patients who have become sensitized to venoms develop severe IgE-associated allergic reactions, including fatal anaphylaxis, upon subsequent venom exposure. In this review, we will describe evidence that mast cells can enhance innate resistance, and survival, to challenge with reptile or arthropod venoms during a first exposure to such venoms. We also will discuss findings indicating that, in mice surviving an initial encounter with venom, acquired type 2 immune responses, IgE antibodies, the high affinity IgE receptor (FcεRI), and mast cells can contribute to acquired resistance to the lethal effects of both honeybee venom and Russell's viper venom. These findings support the hypothesis that mast cells and IgE can help protect the host against venoms and perhaps other noxious substances.