Sea cucumbers (Holothuroidea, Echinodermata) are marine deuterostomes possessing a complex innate immune system composed of a wide diversity of immune cells—coelomocytes, making them compelling models for exploring the evolution of immunity. This study investigates the functional specialisation of coelomocytes within the two main echinoderm body fluids, namely the perivisceral fluid (PF) from the perivisceral cavity and the hydrovascular fluid (HF) from the hydrovascular— ambulacral system. Given their specific distribution restricted to the HF, hemocyte-like cells (HELs) are particularly investigated. In echinoderms, hemocytes have been described as reddish cells containing haemoglobin and thus presenting a function in oxygen transport. Using an integrative approach combining cell morphological analyses, pigment profiling and multi-omics technologies, we demonstrate, in the sea cucumber Holothuria forskali , that HELs harbour exceptionally high concentrations of carotenoids, primarily canthaxanthin and astaxanthin—potent antioxidant molecules responsible for their pigmentation. Transcriptomics and proteomics analyses reveal that HELs express candidate genes involved in the carotenoid metabolism pathway as well as catalase, an antioxidant enzyme. Additionally, spectral flow cytometry assays reveal that HELs do not produce reactive oxygen species in contrast to most coelomocyte types, reinforcing the hypothesis of their antioxidant function. HELs also contribute to the formation of large red bodies (i.e., coelomocyte aggregates) and increase in concentration following lipopolysaccharide injections, indicating an active role in immune defence. Given these results, we hypothesise that these cells act after the culmination of the immune response, forming an antioxidant shell around the cellular aggregates to mitigate oxidative stress from reactive oxygen species (ROS) produced within the aggregate while encapsulating pathogens, thus protecting the host tissues. The discovery of carotenoid-carrying coelomocytes constitutes the first report of pigmented coelomocytes in sea cucumbers (except respiratory pigments), challenging the long-standing assumption that these cells contain haemoglobin. Therefore, we propose renaming hemocytes into carotenocytes, at least in this species. However, we think that this newly described coelomocyte type has been wrongly identified as haemoglobin-containing cells in many previous studies and could be present in many other holothuroid species. Our findings thus establish a new paradigm in the study of coelomocytes in echinoderms as well as the function of the hydrovascular system, unique to this phylum. ### Competing Interest Statement The authors have declared no competing interest. The SRA files from transcriptomics analysis were submitted to NCBI and are available under the BioProject ID PRJNA1259574. TSA files (including the merged transcriptome and translated proteome) and gene annotation, expression files are publicly available on the figshare repository following this link: . Additional raw and partially processed data regarding the results of this study will be provided under reasonable request. FRIA F.R.S.-FNRS, 47487, 57909 PDR F.R.S.-FNRS, 40013965

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.

Background Mechanisms that preclude lung metastasis are still barely understood. The possible consequences of allergic airways inflammation on cancer dissemination were studied in a mouse model of breast cancer. Methods Balb/c mice were immunized and daily exposed to ovalbumin (OVA) from day 21. They were subcutaneously injected with 4T1 mammary tumor cells on day 45 and sacrificed on day 67. Lung metastases were measured by biophotonic imaging (IVIS® 200 Imaging System) and histological measurement of tumor area (Cytomine software). Effects of CCL11 were assessed in vivo by intratracheal instillations of recCCL11 and in vitro using Boyden chambers. CCR3 expression on cell surface was assessed by flow cytometry. Results The extent of tumor metastases was significantly higher in lungs of OVA-exposed mice and increased levels of CCL11 expression were measured after OVA exposure. Migration of 4T1 cells and neutrophils was stimulated in vitro and in vivo by recCCL11. 4T1 cells and neutrophils express CCR3 as shown by flow cytometry and a selective CCR3 antagonist (SB-297006) inhibited the induction of 4T1 cells migration and proliferation in response to recCCL11 . Conclusions Allergic inflammation generated by exposure to allergens triggers the implantation of metastatic cells from primary breast tumor into lung tissues plausibly in a CCL11–CCR3-dependent manner. This indicates that asthma related inflammation in lungs might be a risk factor for lung metastasis in breast cancer patients.

Malignant pleural mesothelioma (MPM) standard therapy is a cisplatin-pemetrexed combination. Unfortunately, MPM cells become very rapidly therapy-resistant in most of cases. Numerous ADAMs (A Disintegrin And Metalloprotease) have been recognized as key contributors to resistance to chemotherapy. The aim of the study was to unveil mechanisms of MPM resistance to treatment and assess the possible implication of ADAM proteases. A measurement of ADAMs expression showed that ADAM8 mRNA is overexpressed as compared to control pleura in human MPM samples and in a mouse model of MPM. In vitro, the mesothelioma cell line AB12 transfected with shRNAs targeting ADAM8 and subsequently exposed to cisplatin displayed a decreased survival as compared to control-ShRNA treated counterparts. Several cisplatin-resistant mesothelioma cell lines were generated by exposing different MPM cell lines to increasing concentrations of cisplatin. A RNA sequencing was performed in order to compare cisplatin-resistant cells to matching parental cells. Surprisingly, a striking overexpression of ADAM8 was observed in cell lines rendered resistant to cisplatin. Moreover, in vivo experiments showed that tumors developed when these cisplatin-resistant cells were injected into the flanks of mice were growing faster and displayed a significant overexpression of ADAM8 mRNA as compared to tumors developed after parental cells injection. In conclusion, our results unveil ADAM8 as a key factor of cisplatin resistance in mesothelioma. Our future researches will focus on the downstream molecular pathways implicating ADAM8 in order to understand how it might affect MPM cisplatin resistance.

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.

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.

Air pollution is becoming a major health problem since it is responsible for millions of deaths related to cardiovascular and lung diseases. Particulate matter and gazes such as ozone produced have been shown to increase lung cancer morbidity in industrialized countries. However, no clear link between air quality levels and cancer cell dissemination to lung tissue has yet been established. A reliable murine model of concomitant pulmonary O3 exposure and tumor cell injection was used to evaluate metastatic burden in the lungs after pulmonary ozone exposure. The implication of neutrophils in this process was studied by using anti-Ly6G antibodies to prevent recruitment of neutrophils to the lungs. Additionally, the implication of neutrophil extracellular traps (NETs) in metastatic processes was evaluated using MRP8cre-Pad4lox/lox mice or by treating mice with DNase I. Pulmonary ozone exposure induces 1) a strong inflammatory response in lung tissues characterized by the recruitment of neutrophils and, 2) colonization of lung tissues by cancer cells and this already at early steps of the metastatic dissemination process. Additionally, an increased production of NETs by ozone-primed neutrophils was observed. Interestingly, neutrophil depletion and inhibition of NET formation greatly diminished the metastatic burden in lungs of mice exposed to ozone. The ability of O3-primed neutrophils to enhance lung colonization by tumor cells was further confirmed after their adoptive transfer in Balb/c mice unexposed to O3. 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.

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.