Quentin Scanvion
email: quentin.scanvion@chru-lille.fr

Laurent Pascal
email: pascal.laurent@ghicl.net

Lidwine Stervinou-Wémeau

Anne-Laure Lejeune
email: anne-laure.lejeune@univ-lille.fr

Valérie Deken

Éric Hachulla
email: eric.hachulla@chru-lille.fr

Bruno Quesnel
email: bruno.quesnel@chru-lille.fr

Arsène Mékinian
email: arsene.mekinian@aphp.fr

David Launay
email: david.launay@chru-lille.fr

Louis Terriou
email: louis.terriou@chru-lille.fr

Systemic Pulmonary Events Associated with Myelodysplastic Syndromes: A Retrospective Multicentre Study

Keywords: interstitial lung disease, pneumonia, pleuritic effusion, pulmonary hypertension, pulmonary alveolar proteinosis, iatrogenic effects

Although pulmonary events are considered to be frequently associated with malignant haemopathies, they have been sparsely studied in the specific context of myelodysplastic syndromes (MDS). We aimed to describe their different types, their relative proportions and their relative effects on overall survival (OS). We conducted a multicentre retrospective cohort study. Patients with MDS (diagnosed according to the 2016 WHO classification) and pulmonary events were included. The inclusion period was 1 January 2007 to 31 December 2017 and patients were monitored until August 2019. Fifty-five hospitalized patients were included in the analysis. They had 113 separate pulmonary events. Thirteen patients (23.6%) had a systemic autoimmune disease associated with MDS. Median age at diagnosis of MDS was 77 years. Median time to onset of pulmonary events was 13 months. Pulmonary events comprised: 70 infectious diseases (62%); 27 interstitial lung diseases (23.9%), including 13 non-specific interstitial pneumonias and seven secondary organizing pneumonias or respiratory bronchiolitis-interstitial lung diseases; 10 pleural effusions (8.8%), including four cases of chronic organizing pleuritis with exudative effusion; and six pulmonary hypertensions (5.3%). The median OS of the cohort was 29 months after MDS diagnosis but OS was only 10 months after a pulmonary event. The OS was similar to that of the general myelodysplastic population. However, the occurrence of a pulmonary event appeared to be either an accelerating factor of death or an indicator for the worsening of the underlying MDS in our study. More than a third of pulmonary events were non-infectious and could be systemic manifestations of MDS.

Introduction

MDS are a heterogeneous clonal condition of pluripotent stem cells. They constitute the main haemopathy after 70 years of age. MDS are defined by chronic cytopaenia linked to qualitatively ineffective haematopoiesis [START_REF] Fozza | The Yin and Yang of Myelodysplastic Syndromes and Autoimmunity: The Paradox of Autoimmune Disorders Responding to Therapies Specific for MDS[END_REF][START_REF] Zeidan | Epidemiology of Myelodysplastic Syndromes: Why Characterizing the Beast Is a Prerequisite to Taming It[END_REF][START_REF] Arber | Revision to the World Health Organization Classification of Myeloid Neoplasms and Acute Leukemia[END_REF][START_REF] Montalban-Bravo | Myelodysplastic Syndromes: 2018 Update on Diagnosis, Risk-Stratification and Management[END_REF]. It has been shown that 15 to 33% of myelodysplastic patients have associated autoimmune or auto-inflammatory diseases (ADs) [START_REF] Fozza | The Yin and Yang of Myelodysplastic Syndromes and Autoimmunity: The Paradox of Autoimmune Disorders Responding to Therapies Specific for MDS[END_REF][START_REF] Mekinian | Biologics in Myelodysplastic Syndrome-Related Systemic Inflammatory and Autoimmune Diseases: French Multicenter Retrospective Study of 29 Patients[END_REF][START_REF] Komrokji | Autoimmune Diseases and Myelodysplastic Syndromes[END_REF]. Systemic vasculitis, connective tissue disease, inflammatory arthritis, neutrophilic pathologies [START_REF] Grignano | Autoimmune Manifestations Associated with Myelodysplastic Syndromes[END_REF] and, more rarely, autoimmune cytopaenia [START_REF] Saif | Autoimmune Phenomena in Patients with Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia[END_REF] have all been observed. Furthermore, there are systemic diseases that are incomplete and consequently more difficult to diagnose [START_REF] Grignano | Autoimmune Manifestations Associated with Myelodysplastic Syndromes[END_REF]. ADs isolated to a single organ have also been described [START_REF] Fozza | The Yin and Yang of Myelodysplastic Syndromes and Autoimmunity: The Paradox of Autoimmune Disorders Responding to Therapies Specific for MDS[END_REF][START_REF] Grignano | Autoimmune Manifestations Associated with Myelodysplastic Syndromes[END_REF][START_REF] Saif | Autoimmune Phenomena in Patients with Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia[END_REF]. MDS are now considered as complex disease and their clinical expression is not purely haematological.

Concerning pulmonary manifestations in MDS, data are scarce. Indeed, while the frequency of pulmonary events in the context of malignant haemopathies (all types combined) is commonly considered to be high and associated with a high morbidity and mortality [START_REF] Lamour | Non-Infectious Pulmonary Complications of Myelodysplastic Syndromes and Chronic Myeloproliferative Disorders[END_REF] especially in acute leukaemias, chronic myeloid leukaemias, and lymphomas [START_REF] Chagnon | Pulmonary Manifestations of Hematological Malignancies: Focus on Pulmonary Chronic Graft-Versus Host Disease[END_REF]. In the case of MDS, the data are less clear. Most lung diseases are attributed to infectious causes [START_REF] Chagnon | Pulmonary Manifestations of Hematological Malignancies: Focus on Pulmonary Chronic Graft-Versus Host Disease[END_REF]. However, the other pneumonias, especially interstitial pneumonia, have rarely been studied [START_REF] Lamour | Non-Infectious Pulmonary Complications of Myelodysplastic Syndromes and Chronic Myeloproliferative Disorders[END_REF][START_REF] Nanah | Deletion 5q Is Frequent in Myelodysplastic Syndrome (MDS) Patients Diagnosed with Interstitial Lung Diseases (ILD): Mayo Clinic Experience[END_REF]. The relative frequency of infectious versus non-infectious pneumonias in myelodysplastic patients has not been directly described. A better knowledge of the pulmonary events in these patients would allow a more effective and efficient diagnostic and therapeutic approach, by enabling the relevance of paraclinical examinations to be assessed.

The main purpose of our study was to describe all of the pulmonary events occurring in hospitalized patients with MDS, their type and their relative proportions. A secondary objective was to explore survival according to the type of event.

Materials and Methods

Patient Selection

We conducted a multicentre retrospective study. Patients with MDS (diagnosed according to the 2016 WHO classification) and pulmonary events were included at three centres in the north of France (Lille University Hospital, Lille Catholic University Hospitals, Armentières Hospital). The inclusion period was 1 January 2007 to 31 December 2017 and monitoring was carried out until August 2019. Patients were included if they had been hospitalized at least once for MDS and a pulmonary event (see online Supplementary Material S1). The exclusion criteria were cardiogenic acute oedema of the lungs, coding errors, lack of information and if MDS had worsened to acute myeloid leukaemia (AML) when the pulmonary event occurred.

Data Collection

Patients' medical records were individually searched for (i) demographic data; (ii) comorbidities (smoking status: active, absent or weaned off tobacco for more than 3 years; the updated age-adjusted Charlson comorbidity index [START_REF] Bannay | The Best Use of the Charlson Comorbidity Index With Electronic Health Care Database to Predict Mortality[END_REF]; the Adult Comorbidity Evaluation 27 score, the Cumulative Illness Rating Scale for Geriatrics); (iii) MDS characteristics; (iv) molecular biology data by next-generation sequencing (NGS) (which improved during the inclusion period; the most recent NGS included an analysis of 36 genes); (v) overall survival (OS); (vi) characteristics of pulmonary events. During data collection, if a non-infectious pulmonary event was highlighted, a potentially iatrogenic treatment was sought in the patient's history. The baseline considered was Pneumotox [START_REF] Camus | The Drug-Induced Respiratory Disease Website[END_REF]. The pulmonary events considered were those diagnosed during or after hospitalization or diagnosed a posteriori during the analysis of medical records for this study. These diagnoses were based on the synthesis of clinical data, pulmonary function test, bronchoscopy and biological (including bronchoalveolar lavage analysis) and radiological studies. The CT scans were reviewed by an independent radiologist (specialized in thoracic imaging). We adopted a "dynamic integrated approach" as in the Update of the International Multidisciplinary Classification of the Idiopathic Interstitial Pneumonias [START_REF] Travis | An Official American Thoracic Society/European Respiratory Society Statement: Update of the International Multidisciplinary Classification of the Idiopathic Interstitial Pneumonias[END_REF]. The process of achieving a multidisciplinary diagnosis was dynamic, requiring confrontations between data from clinicians, biologists, radiologists and, when available, pathologists. In order to be retained, cases of interstitial lung diseases associated with MDS did not have to have an identified secondary aetiology.

Statistical Analysis

Results are expressed as median (interquartile range) [minimum-maximum] for continuous variables and as frequencies and percentage for categorical variables. The normality of the distributions was assessed graphically and using the Shapiro-Wilk test. The factors associated with mortality were studied by Cox models, including factors that varied during follow-up as time-dependent variables. Ordinal qualitative variables were subjected to trend tests. For specific analyses of pulmonary events, Cox models with adjustment to the International Prognostic Scoring System-Revised (IPSS-R) score were developed. The effects of each factor on survival were evaluated using the hazard ratio (HR) and the 95% confidence interval (95% CI). The statistical tests were all carried out with a bilateral risk α of 5%. Statistical analyses were performed using SAS software (version 9.4, SAS Institute Inc. Cary, NC, USA). All the statistical analyses were discussed and validated by a group of statisticians, represented by Mrs Valérie Deken.

Ethical Considerations

In accordance with French law, the creation of the database necessary for this study was declared beforehand to the Commission Nationale de l'Informatique et des Libertés (CNIL) and was included in the Lille University Hospital's register of declared databases. The included patients had been informed beforehand in their letters of consultation or hospitalization that unless they expressed an objection, their medical data could be used in an anonymous form for medical research purposes. No objections were expressed by any of the included patients.

Results

Patients' Characteristics

Fifty-five myelodysplastic patients with a total of 113 pulmonary events were included in the study. A flow chart of patient inclusion is presented in Figure 1. The characteristics of the patients are detailed in Table 1.

Regarding smoking status, seven patients (16.3%) were active smokers, 19 (44.2%) had been weaned off smoking for more than three years at the time of diagnosis of MDS and 17 patients (39.5%) had never smoked; the remaining 12 patients had missing data. Among the 55 patients, the "NGS MDS" molecular biological status was known for 22 patients (40%). At least one pathogenic mutation was confirmed in 19 of these 22 patients (86%). The lack of sufficient molecular data prevented us from carrying out statistical analyses of these data. Thirteen patients (23.6%) had an AD: two had polyarteritis nodosa (one with myocardial involvement), one had granulomatosis with polyangiitis, two had giant cell arteritis, one had hypocomplementemic urticarial vasculitis, one had unclassifiable largevessel vasculitis, two had immune thrombocytopaenia (associated in one case with systemic lupus erythematosus), one had mixed connective tissue disease, one had psoriatic arthritis, one had unclassifiable auto-inflammatory disease and one had large granular lymphocyte leukaemia with systemic lupus erythematosus, anti-phospholipid syndrome and secondary Gougerot-Sjögren syndrome. Age at diagnosis of the various associated pulmonary events (all types combined) was 76 years (69-84) [

CPSS n = 4 Good 0 0 0 0 Intermediate 1 1 0 0 1 (25%) Intermediate 2 0 1 0 1 (25%) Poor 0 1 0 1 (25%) NA 0 1 0 1 (25%) Comorbidities Charlson index 1 5 (3-6) [0.0-8.0] & 6 (5-7) [2.0-9.0] 7 (4-7) [1.0-16.0] 6 (4-7) [0.0-16.0] & CIRS-G 9 (5-11) [0.0-18.0] & 8 (6-9.5) [3.0-12.0] 9 (7-10) [2.0-19.0] 9 (7-10) [0.0-19.0] & ACE-27 2 (2-3) [0.0-3.0] & 2.5 (2-3) [1.0-3.0] 3 (2-3) [0.0-3.0] 3 (2-3) [0.0-3.0] & ADs

Pulmonary Events

There were 113 pulmonary events, including 70 infectious pneumonias (61.9%), 27 interstitial lung diseases (ILDs) (23.9%), 10 pleural effusions (8.8%) and six pulmonary hypertensions (5.3%) (see Figure 2). The presence of ADs did not significantly modify the proportions of pulmonary events (Fisher's test, p = 0.88). In 36 of the 70 infectious pneumonias (51.4%) the pathogen was undetermined (see Figure 3). For five of them (7.1%), the infectious causes initially retained by clinicians appeared questionable with retrospective knowledge of the evolution of the cases (treatments undertaken, subsequent events, etc.). A diagnosis of secondary organizing pneumonia (SOP) was suspected for these cases, without sufficient retrospective arguments to be accepted. When the pathogen was known, it was mainly bacterial (n = 26, 37.1%). We also found six fungal infections (8.6%), one case of mycobacterial infection (1.4%) and one case of identified viral infection (1.4%). The pathogens are detailed in Table S1. Regarding ILD (see Figure 4), data synthesis revealed the following patterns: nonspecific interstitial pneumonias (NSIPs) (n = 13), some of which had a diffuse (n = 4) or focal (n = 3) fibrosing component; SOPs (n = 4); respiratory bronchiolitis-interstitial lung diseases in non-smoking patients (RB-ILD-ns) (n = 3); pulmonary alveolar proteinosis (PAP) (n = 1); diffuse alveolar damage (n = 1). There was also a case in which retrospective analysis of the data was unable to distinguish between pulmonary vasculitis and SOP and a case in which the CT scan and clinical history were suggestive of PAP but without confirmation by staining with periodic acid-Schiff with bronchoalveolar lavage. Among the seven cases of fibrosis, four patients had no autoantibodies, two had positive anti-nuclear antibodies (indirect immunofluorescence on HEp2 cells) but without specific antibodies, and one had a weakly positive myositis dot-blot assay, but without significant specific antibody. All six cases of pulmonary hypertension were precapillary and one also had a postcapillary component.

Figure 5 shows the monitoring of each patient with the different pulmonary events over time. Table S2 describe the treatments received for MDS. Our study was not designed to assess the impact of treatment on outcome and the heterogeneity of treatment precludes any firm conclusion. The secondary aim of the study was to study survival according to the different types of pulmonary events. For this purpose, we formed the following groups of analyses: Group A ("Infectious"): comprising the infectious pneumonias (excluding the five doubtful cases) and the bacterial pleurisy case (66 cumulative pulmonary events (58%)); Group B ("Organizing"): comprising the SOPs, the RB-ILD-ns cases and the COPE cases (12 cumulative pulmonary events (11%)); Group C ("NSIP"): comprising the NSIP cases (13 cumulative pulmonary events (11%)).

For the myelodysplastic patients in our cohort, according to a Cox model the diagnosis of a first infectious pneumonia (Group A) did not influence survival any differently from the diagnosis of Group B impairment or the diagnosis of NSIP (i.e., Group C) (see Table 2). The recurrence of at least one infectious pneumonia increased the hazard ratio versus the first infectious pneumonia. HR: hazard ratio adjusted for IPSS-R score; HR 95% CI: 95% confidence interval of the hazard ratio.

Iatrogenic Data

The information collected shows that one of the NSIP cases with a focal fibrosing component occurred in a patient who had been treated for one year with methotrexate. A patient with chronic myelomonocytic leukaemia (CMML) had been treated with hydroxyurea; during his follow-up, one SOP and one COPE occurred. Finally, one case of NSIP, one case of SOP and the case of pulmonary vasculitis or SOP occurred during treatment with azacitidine.

Discussion

To our knowledge, this retrospective multicentre study is the first to describe the proportions of pulmonary events (both infectious and non-infectious) occurring in the myelodysplastic population and their relative effects on patient OS. The general characteristics of our cohort are close to those of the general myelodysplastic population [START_REF] Zeidan | Epidemiology of Myelodysplastic Syndromes: Why Characterizing the Beast Is a Prerequisite to Taming It[END_REF][START_REF] Duchmann | Prise En Charge Des Myélodysplasies[END_REF]. Thus, myelodysplastic patients with pulmonary events do not seem to form a particular clinical population of MDS [START_REF] Nanah | Deletion 5q Is Frequent in Myelodysplastic Syndrome (MDS) Patients Diagnosed with Interstitial Lung Diseases (ILD): Mayo Clinic Experience[END_REF]. The distribution of different types of MDS was broadly consistent with that of previous international cohorts. However, we noted a tendency for an over-representation of multilineage dysplasia (as opposed to single lineage) and MDS with excess blasts. This finding is also described in MDS with associated systemic inflammatory and autoimmune manifestations [START_REF] Mekinian | Systemic Inflammatory and Autoimmune Manifestations Associated with Myelodysplastic Syndromes and Chronic Myelomonocytic Leukaemia: A French Multicentre Retrospective Study[END_REF], which represent 15% to 33% of MDS cases [START_REF] Fozza | The Yin and Yang of Myelodysplastic Syndromes and Autoimmunity: The Paradox of Autoimmune Disorders Responding to Therapies Specific for MDS[END_REF][START_REF] Mekinian | Biologics in Myelodysplastic Syndrome-Related Systemic Inflammatory and Autoimmune Diseases: French Multicenter Retrospective Study of 29 Patients[END_REF][START_REF] Komrokji | Autoimmune Diseases and Myelodysplastic Syndromes[END_REF]. The representativeness of our cohort was strengthened by multicentre inclusion. Indeed, the three inclusion centres have a varied patient base. Lille University Hospital is a national reference centre for systemic diseases. Armentières Hospital is a rural hospital. Lille Catholic University Hospitals are city hospitals. However, the retrospective nature of the data did not allow us to overcome all selection and information biases.

MDS are chronic pathologies that usually induce an immunocompromised status. Up to 60% of patients with neutropaenia (whatever the cause) may develop a pulmonary infiltrate during follow-up [START_REF] Nováková | Potential Sites of Infection That Develop in Febrile Neutropenic Patients[END_REF]. In practice, most MDS lung diseases are therefore attributed to infectious causes [START_REF] Lamour | Non-Infectious Pulmonary Complications of Myelodysplastic Syndromes and Chronic Myeloproliferative Disorders[END_REF]. However, in immunocompromised patients or patients with cancer (not specifically MDS), some authors estimate that a quarter to a half of infiltrates would be non-infectious [START_REF] Vento | Lung Infections after Cancer Chemotherapy[END_REF]. Our results support this since infectious pneumonias represented at most only 61.9% (i.e., 70/113) of pulmonary events. Furthermore, this proportion may have been overestimated because it is common for pneumonia to be diagnosed, even in the absence of an identified pathogen. However, we noticed that there were some patients whose pneumonias were not improved by several lines of antibiotic therapy, except when it was associated with corticosteroids. These cases could therefore be unrecognized SOP. However, we only included hospitalized patients; consequently, less severe pneumonias occurring at home were not counted. Our results are therefore not applicable to outpatient MDS patients Twenty-four percent of pulmonary events had patterns of ILD. These pulmonary manifestations have been little evaluated apart from a few case reports or case series. A retrospective study of 827 myelodysplastic patients at the Mayo Clinic found 18 (2%) ILDs, including 44% NSIP (half of which had a fibrosing component) and 22% organizing pneumonias [START_REF] Nanah | Deletion 5q Is Frequent in Myelodysplastic Syndrome (MDS) Patients Diagnosed with Interstitial Lung Diseases (ILD): Mayo Clinic Experience[END_REF]. Our study confirmed this relative distribution of patterns, with 48% NSIP and 30% SOP and RB-ILD-ns. However, we did not confirm the previously reported high prevalence of 5q-abnormalities in MDS patients [START_REF] Nanah | Deletion 5q Is Frequent in Myelodysplastic Syndrome (MDS) Patients Diagnosed with Interstitial Lung Diseases (ILD): Mayo Clinic Experience[END_REF], with only two cases in our study. It should be noted that the presence of ILD predisposes to infections, especially fungal infections. In our cohort, an NSIP pre-existed for four out of six fungal infections. On the other hand, for 32 clinical and radiological suspicions of invasive pulmonary aspergillosis, Kim et al. showed that a lung biopsy corrected the diagnosis in favour of SOP in seven cases (22%) [START_REF] Kim | Importance of Open Lung Biopsy in the Diagnosis of Invasive Pulmonary Aspergillosis in Patients with Hematologic Malignancies[END_REF]. This supports our suspicion of underdiagnosis of SOP. Between the hypotheses of a fortuitous association or systemic pulmonary manifestations associated with MDS, the literature provides several arguments in favour of the latter. Organizing pneumonia (OP) is an inflammatory pathology of the lower respiratory tract. A distinction is made between cryptogenic and secondary forms. Secondary aetiologies are multiple. Among them we found malignant haemopathies such as acute lymphomas and leukaemias, but MDS are not often cited [START_REF] Daniels | Organizing Pneumonia in Patients with Hematologic Malignancies: A Steroid-Responsive Lesion[END_REF][START_REF] Cordier | Cryptogenic Organising Pneumonia[END_REF][START_REF] Oymak | Bronchiolitis Obliterans Organizing Pneumonia. Clinical and Roentgenological Features in 26 Cases[END_REF][START_REF] Cazzato | Bronchiolitis Obliterans-Organizing Pneumonia: An Italian Experience[END_REF]. However, in line with our results, case series of OP induced by MDS have been described [START_REF] Kataoka | Interstitial Pneumonia Associated with Progression of Myelodysplastic Syndrome[END_REF][START_REF] Kamiya | Disseminated Cryptococcosis Complicated with Bilateral Pleural Effusion and Ascites during Corticosteroid Therapy for Organizing Pneumonia with Myelodysplastic Syndrome[END_REF][START_REF] Mobeireek | Obliterative Bronchiolitis in a Patient with Myelodysplastic Syndrome before Bone Marrow Transplantation[END_REF][START_REF] Hosono | An autopsy case of myelodysplastic syndrome complicated by recurrent organizing pneumonia[END_REF][START_REF] Yamakawa | A case of myelodysplastic syndrome complicated with organizing pneumonia[END_REF][START_REF] Patel | Bronchiolitis Obliterans Organizing Pneumonia in a Patient with Chronic Myelogenous Leukemia Developing after Initiation of Interferon and Cytosine Arabinoside[END_REF][START_REF] Wohlrab | A Patient with Myelodyplastic Syndrome, Pulmonary Nodules, and Worsening Infiltrates[END_REF][START_REF] Kobara | Clinical study of three myelodysplastic syndrome (MDS) patients with BOOP-like pulmonary disease[END_REF][START_REF] Mito | A case of pulmonary manifestation associated with myelodysplastic syndrome[END_REF][START_REF] Drent | Pulmonary Infiltration Associated with Myelodysplasia[END_REF][START_REF] Stemmelin | Bronchiolitis with Leukemia[END_REF]. The first case was published in 1990 [START_REF] Tenholder | The Myelodysplastic Syndrome and Bronchiolitis Obliterans[END_REF]. From a pathophysiological point of view, in this pulmonary involvement, inflammatory cells infiltrate the alveolar interstitial tissue after any primary endothelial lesion [START_REF] Cordier | Cryptogenic Organising Pneumonia[END_REF]. Since the neutrophils and eosinophils are dysplastic in MDS, these appear to be a probable aetiology of OP. Furthermore, Sweet's syndrome (SS) is another strong argument for a causal association between MDS and OP. Indeed, the malignant form (as opposed to the idiopathic form) of SS is a paraneoplastic syndrome and is particularly induced by myeloid haemopathies (dermatosis can happen before, during or following the haemopathy) [START_REF] Drent | Pulmonary Infiltration Associated with Myelodysplasia[END_REF][START_REF]Orphanet-Le Portail Des Maladies Rares et Des Médicaments Orphelins[END_REF][START_REF] Lepelletier | Neutrophilic Dermatoses Associated with Myeloid Malignancies[END_REF][START_REF] Chan | Subcutaneous Sweet Syndrome in the Setting of Myeloid Disorders: A Case Series and Review of the Literature[END_REF]. It is a neutrophilic dermatosis. However, SS frequently associates with cutaneous involvement, a secondary OP [START_REF] Lepelletier | Neutrophilic Dermatoses Associated with Myeloid Malignancies[END_REF][START_REF] Chan | Subcutaneous Sweet Syndrome in the Setting of Myeloid Disorders: A Case Series and Review of the Literature[END_REF][START_REF] Tzelepis | Cryptogenic Organizing Pneumonia in Sweet's Syndrome: Case Report and Review of the Literature[END_REF][START_REF] Angeline | Pulmonary Involvement in Sweet's Syndrome (Acute Febrile Neutrophilic Dermatosis)[END_REF]. Its pulmonary involvement is one of the most frequent and most studied systemic neutrophilic infiltrates. It occurs during dermatosis, but can be inaugural and therefore occurs alone [41]. On the other hand, a particular histological form, called histiocytoid Sweet syndrome by some authors [START_REF] Requena | Histiocytoid Sweet Syndrome: A Dermal Infiltration of Immature Neutrophilic Granulocytes[END_REF] myelodysplasia cutis by others [START_REF] Osio | Myelodysplasia Cutis Versus Leukemia Cutis[END_REF] is described. In this form, the tumour cells have a combined myeloid and monocytic phenotype and CD3+ T cells are also present. This form of SS is said to be a cutaneous MDS. Given the existence of only cutaneous SS associated with MDS and myelodysplasia cutis, it is possible to consider only pulmonary SS associated with MDS or "myelodysplastic organizing pneumonia". In addition, the signature profiles of cytokines in myelodysplastic patients confirm higher levels of TNF-α, IFN-γ, TGF-β, IL-6 and IL-18 [START_REF] Gañán-Gómez | Deregulation of Innate Immune and Inflammatory Signaling in Myelodysplastic Syndromes[END_REF]. Similarly, subjects with interstitial lung disease were found to have higher levels of IL-6 [START_REF] Malaviya | Anti-TNFα Therapy in Inflammatory Lung Diseases[END_REF], TNF-α [START_REF] Hou | TNF-α-Induced NF-KB Activation Promotes Myofibroblast Differentiation of LR-MSCs and Exacerbates Bleomycin-Induced Pulmonary Fibrosis[END_REF], TGF-β [START_REF] Bao | Expression of Suppressor of Cytokine Signaling 1 in the Peripheral Blood of Patients with Idiopathic Pulmonary Fibrosis[END_REF], IFN-γ [START_REF] Ishikawa | Relevance of Interferon-Gamma in Pathogenesis of Life-Threatening Rapidly Progressive Interstitial Lung Disease in Patients with Dermatomyositis[END_REF] and other inflammatory cytokines. Thus the link of interstitial lung disease with MDS could be due to immune deregulation [START_REF] Nanah | Deletion 5q Is Frequent in Myelodysplastic Syndrome (MDS) Patients Diagnosed with Interstitial Lung Diseases (ILD): Mayo Clinic Experience[END_REF]. In addition, myelodysplastic patients have higher G-CSF levels [START_REF] Gañán-Gómez | Deregulation of Innate Immune and Inflammatory Signaling in Myelodysplastic Syndromes[END_REF] and drug-induced SS with OP occurs most often after treatment with G-CSF [START_REF]Orphanet-Le Portail Des Maladies Rares et Des Médicaments Orphelins[END_REF]. We note another element of link. Indeed, in our patients (as in other case reports) a close linked evolution between interstitial lung diseases and MDS was observed. In view of all the above arguments, we propose to classify and consider OP in myelodysplastic patients as secondary OP (and not cryptogenic). It is also sometimes proposed that interstitial lung diseases are caused by pulmonary leukocytoclastic vasculitis [START_REF] Giannouli | Autoimmune Phenomena in Myelodysplastic Syndromes: A 4-Yr Prospective Study[END_REF][START_REF] Farris | Diffuse Cellular and Fibrosing Interstitial Pneumonitis with Desquamative Interstitial Pneumonitis-like Features Associated with Myeloid Neoplasia[END_REF][START_REF] Hamidou | Systemic Medium-Sized Vessel Vasculitis Associated with Chronic Myelomonocytic Leukemia[END_REF][START_REF] Smail | Wegener's disease and myelomonocytic leukemia: A fortuitous association?[END_REF], associated with systemic autoimmune or auto-inflammatory diseases [START_REF] Cordier | Cryptogenic Organising Pneumonia[END_REF] The pulmonary involvement then constitutes only a part of systemic vasculitis, itself associated or not with MDS. In our cohort, this pulmonary vasculitis attack was strongly suspected for a pulmonary manifestation occurring in patient No. 41, who also had a severe hypocomplementemic urticarial vasculitis (with amputation of the lower limbs).

Pulmonary alveolar proteinosis (PAP) is a rare condition characterized by the alveolar accumulation of components of the surfactant. Autoimmune PAP (anti-G-CSF serum antibodies) represents 90% of PAP cases [START_REF] Suzuki | Pulmonary Alveolar Proteinosis Syndrome[END_REF][START_REF] Jouneau | La Protéinose Alvéolaire Pulmonaire[END_REF]; the remaining cases are secondary (8%) or hereditary (2%). Among secondary PAPs, more than 3/4 are caused by MDS, and therefore 6% of PAPs in total [START_REF] Suzuki | Pulmonary Alveolar Proteinosis Syndrome[END_REF][START_REF] Jouneau | La Protéinose Alvéolaire Pulmonaire[END_REF]. They are due to a qualitative or quantitative macrophages dysfunction for the clearance of the surfactant [START_REF] Suzuki | Pulmonary Alveolar Proteinosis Syndrome[END_REF][START_REF] Jouneau | La Protéinose Alvéolaire Pulmonaire[END_REF][START_REF] Nagai | Cytogenetic Evidence for the Clonal Hematopoietic Cell Origin of Alveolar Macrophages in Myelodysplastic Syndrome-Associated Pulmonary Alveolar Proteinosis[END_REF]. GATA2 haploinsufficiency has been described as a possible aetiology of secondary MDS and PAP [START_REF] Griese | GATA2 Deficiency in Children and Adults with Severe Pulmonary Alveolar Proteinosis and Hematologic Disorders[END_REF][START_REF] Spinner | GATA2 Deficiency: A Protean Disorder of Hematopoiesis, Lymphatics, and Immunity[END_REF]. When MDS ± PAP are associated with mycobacterial infections, disseminated HPV (human papilloma virus) or opportunistic fungal infections, and with lack of monocytes, B cells and NK cells, they constitute the MonoMAC syndrome [START_REF] Vinh | Autosomal Dominant and Sporadic Monocytopenia with Susceptibility to Mycobacteria, Fungi, Papillomaviruses, and Myelodysplasia[END_REF]. This is the case in our patient No. 55: multilineage MDS + PAP + pneumonia caused by Mycobacterium kansasii + NSIP. GATA2 haploinsufficiency is also associated with pulmonary hypertension [START_REF] Sanges | Haemodynamically Proven Pulmonary Hypertension in a Patient with GATA2 Deficiency-Associated Pulmonary Alveolar Proteinosis and Fibrosis[END_REF].

Concerning the iatrogenic effects, apart from the possible involvement of methotrexate in a case of fibrosing NSIP, there is no strong argument in favour of other iatrogenic treatment effects. Indeed, the toxic hypothesis of hydroxyurea must be put into perspective. Firstly, in our cohort, pleurisy and SOP occurred, respectively 23 and 29 months after the end of treatment. Secondly, in the literature, only two cases (one per complication [START_REF] Kamal | Hydroxyurea-Induced Interstitial Pneumonitis: A Rare Clinical Entity[END_REF][START_REF] Kalambokis | Fulminant Bronchiolitis Obliterans Organizing Pneumonia Following 2 d of Treatment with Hydroxyurea, Interferon-α and Oral Cytarabine Ocfosfate for Chronic Myelogenous Leukemia[END_REF]) have been reported and they clearly lack arguments of accountability or suffer from confusion bias. For example, hydroxyurea was concomitant with IFN-γ and cytarabine, which are themselves listed as possible causes of SOP [START_REF] Camus | The Drug-Induced Respiratory Disease Website[END_REF]. Furthermore, azacitidine was suspected in about ten case reports describing different pulmonary events [START_REF] Camus | The Drug-Induced Respiratory Disease Website[END_REF]. However, this treatment is indicated for CMML and AML, and for MDS with an intermediate to poor prognosis. Independently of azacitidine, all these pathologies can themselves be associated with ILD. Prior MDS treatment and ADs seemed to play no significant role in ILD development.

Nine non-purulent pleural effusions occurred, accounting for 8% of pulmonary events in the cohort. This relatively large proportion opens a new perspective on the pleural complications of MDS, which have been very little studied. Indeed, although pleural effusions are frequently associated with lymphomas, they are only rarely discussed for MDS [START_REF] Afessa | Effusions in immunocompromised hosts[END_REF][START_REF] Alexandrakis | Pleural Effusions in Hematologic Malignancies[END_REF]. Four cases of COPE were observed, in 4 out of 55 patients. COPE is a benign mesothelial proliferation, with varying degrees of more or less fibrous reactive hyperplasia, when the pleura is injured [START_REF] Churg | The Separation of Benign and Malignant Mesothelial Proliferations[END_REF]. This is the first time that the MDS and COPE association has been described. Two patients had at least one severe respiratory infection in their history. However, for the other two patients, no aetiology was highlighted.

Pulmonary hypertension is an initially insidious and paucisymptomatic pathology, and this is especially problematic when associated with MDS, as dyspnoea can be attributed to anaemia. Unlike myeloproliferative syndromes, there are few data for MDS (the pathophysiological link has not yet been clearly established). There are three case reports [START_REF] Dingli | Unexplained Pulmonary Hypertension in Chronic Myeloproliferative Disorders[END_REF][START_REF] Gámez-López | Effectiveness of Bosentan in a Case of Pulmonary Arterial Hypertension Associated with Myelodysplastic Syndrome[END_REF], and a recent prospective study found three cases of pulmonary hypertension in 36 myelodysplastic patients (8%) [START_REF] Sill | Pulmonary Arterial Pressure in Patients with Myelodysplastic Syndromes[END_REF].

The median OS was similar to that of the general myelodysplastic population [START_REF] Zeidan | Epidemiology of Myelodysplastic Syndromes: Why Characterizing the Beast Is a Prerequisite to Taming It[END_REF]. MDS with pulmonary events does not appear to be a distinct subgroup of MDS in terms of survival. However, pulmonary events are significant events in the history of the disease. In our study, pulmonary events occurred towards the end of the disease history (median 10 months before death). Thus, the occurrence of a pulmonary event appears to be a marker of severity during the natural history of MDS. However, the retrospective nature of the study must be kept in mind as a source of potential bias if the start or end of follow-up was not carried out in our inclusion centres. This study did not allow us to determine a relative risk of death for MDS patients with pulmonary events versus those without. However, it is interesting to note that one type of pulmonary event did not decrease survival more significantly than another (i.e., similar HR CI 95% in the three groups, A, B and C). Nevertheless, it should be borne in mind that the effect of the complications in Groups B and C may have been underestimated by our model because of a smaller number of cases (a lack of power cannot be excluded). We also noted a logical cumulative effect, since the recurrence of at least one case of infectious pneumonia in a given patient increased the HR. In addition, it should be remembered that our study focused on information from hospital medical records. Thus, the known infectious complications were more severe than ambulatory pneumonia. The presence of ADs had no significant effect on survival. The effect of this parameter on the OS of MDS patients is discordant with various other studies [START_REF] Fozza | The Yin and Yang of Myelodysplastic Syndromes and Autoimmunity: The Paradox of Autoimmune Disorders Responding to Therapies Specific for MDS[END_REF]. Nevertheless, in a series of 123 myelodysplastic patients with an associated autoimmune or auto-inflammatory pathology versus 665 patients without, the survival did not differ [START_REF] Mekinian | Systemic Inflammatory and Autoimmune Manifestations Associated with Myelodysplastic Syndromes and Chronic Myelomonocytic Leukaemia: A French Multicentre Retrospective Study[END_REF].

Conclusions

In conclusion, a wide variety of pulmonary complications can occur in patients with MDS. More than a third of them could correspond to systemic pulmonary manifestations, comprising several ILDs, pleural effusions and pulmonary hypertension. Future prospective studies will be necessary to assess the risk of developing a pulmonary complication and to explore appropriate therapeutic strategies (corticosteroid therapy, hypomethylating treatments, etc.).

Supplementary Materials:

The following are available online at https://www.mdpi.com/2077-0 383/10/6/1162/s1, Online supplementary material S1: Inclusion procedure, Table S1: Details of pathogens identified during infectious pneumonias in myelodysplastic patients, Table S2 Informed Consent Statement: Study was retrospective. The included patients had been informed beforehand in their letters of consultation or hospitalization that unless they expressed an objection, their medical data could be used in an anonymous form for medical research purposes. No objections were expressed by any of the included patients.

  29.0-92.0]. The time from diagnosis of MDS to each pulmonary event was 13 months (1-40) [-93.0-139.0]. Age at diagnosis of ADs was 69 years (42-71) [6.0-79.0]. This corresponded to a delay of -39•months (-139-0) [-361.0-16.0] between the diagnosis of MDS and ADs (which therefore mostly predated MDS diagnosis).

Figure 1 .

 1 Figure 1. Patient inclusion flow chart. (AML: MDS had worsened to acute myeloid leukaemia when the pulmonary event occurred).

Figure 2 .

 2 Figure 2. Pie charts of the different types of pulmonary events occurring in myelodysplastic patients. (A): Overall cohort. (B): myelodysplastic syndrome with associated autoimmune or auto-inflammatory diseases (ADs +) versus without (ADs -).

Figure 3 .

 3 Figure 3. Pie chart of the causes of infectious pneumonias occurring in myelodysplastic patients.

Figure 4 .

 4 Figure 4. Pie chart of the patterns of ILDs in myelodysplastic patients. (* in non-smoking patients). The 10 cases of pleural effusion comprised chronic organizing pleuritis with exudative effusion (COPE, n = 4), pleural calcification with exudate (n = 2), lymphocytic exudate

Figure 5 .

 5 Figure 5. Historiogram of the cohort: chronology of pulmonary events in myelodysplastic patients. (T 0 : diagnosis of myelodysplastic syndrome).

3. 3 .

 3 Overall Survival Thirty-five patients (64%) died during the study period (i.e., 12 years and 8 months). Median age at death was 79 years (72-85) [32.0-93.0]. The Kaplan-Meier survival function is presented in Figure 6. According to this, the median OS was 29 months (16-127) [1.0-184.0] after the diagnosis of MDS. The OS after pulmonary events (all types) was 10 months (1-22) [0.0-198.0]. AML occurred in six patients (11%) after 20 months (11-45) [11.0-52.0]. It was significantly predictive of death (p < 0.001). Five of the six patients died during the study period. Their post-AML survival was 5 months (2-7) [<1.0-8.0]. One patient was still alive 2 months after AML diagnosis (i.e., at the end of the study). The results of the univariate analyses of the predictive factors of post-MDS survival are available in Figure S1).

Figure 6 .

 6 Figure 6. Kaplan-Meier survival curve for our cohort.

  : Details of treatments received for myelodysplastic syndromes, Figure S1: Univariate analyses of the predictive factors of post-MDS survival. Author Contributions: Q.S. and L.T. designed the study. Q.S. collected the data with the contribution of L.P. and T.S. A.-L.L. reviewed the CT-scans. Statistical analyses were done by V.D. and Q.S. Q.S. wrote the first draft with input from L.S.-W., É.H., D.L. and L.T.; B.Q. and A.M.: Validation; Writingreview and editing. All authors revised the manuscript and approved the final form. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the study are appropriately investigated and resolved.Funding: This research received no external funding.Institutional Review Board Statement:In accordance with French law, the creation of the database necessary for this study was declared beforehand to the Commission Nationale de l'Informatique et des Libertés (CNIL) and was included in the Lille University Hospital's register of declared databases.

Table 1 .

 1 General characteristics of patients.

		Lille University Hospital (n = 18)	Armentières Hospital (n = 12)	Lille Catholic University Hospitals (n = 25)		Total (n = 55)
	Gender ratio (M/F)	13/5 = 2.6	7/5 = 1.4	23/2 = 11.5		43/12 = 3.6
	Median age at diagnosis of MDS	69 (50-73) [29.0-87.0]	80 (78-82) [64.0-88.0]	78 (69-83) [56.0-92.0]	77 (66-83) [29.0-92.0]
	MDS or MDS/MPN (WHO
	2016)
	MDS-SLD MDS-MLD	1 10	2 2 ¤	4 § 10	7 22	(52.7%)
	MDS-EB-1 MDS-EB-2	0 3	3 1	3 ¤ 8 ¤	6 12	(32.7%)
	MDS-U	2	0	0	2	(3.6%)
	CMML	1	3	0	4	(7.3%)
	MDS-EO-PDGFRA	1	0	0	1	(1.8%)
	Primary myelofibrosis	0	1	0	1	(1.8%)
	MDS
	Primary	15	11	20	46	(83.6%)
	Secondary
	Cancer chemotherapy Exposure ¶	1 2	1 0	4 1	6 3	(16.3%)
	IPSS					N = 50
	Good	4	1	4	9	(18%)
	Intermediate 1	6	3	6	15	(30%)
	Intermediate 2	5	1	11	17	(34%)
	Poor	1	0	2	3	(6%)
	NA	1	3	2	6	(12%)
	IPSS-R					N = 50
	Very good	3	1	2	6	(12%)
	Good	4	0	5	9	(18%)
	Intermediate	3	3	7	13	(26%)
	Poor	2	0	3	5	(10%)
	Very poor	4	1	6	11	(22%)
	NA	1	3	2	6	(12%)

Table 2 .

 2 Comparison of predictive effects of the three main groups of pulmonary events (Infectious, SOP, NSIP) on survival, according to a multivariate time-dependent Cox model.

	Pulmonary Events	Patient Months	Deaths	HR	[HR 95% CI]
	Group A ("Infectious"):
	1 event		14,644	15	5.6	[2.2-14.3]
	≥2 events		5471	11	8.6	[2.8-26.3]
	No event		43,421	9
	Group B	Yes	2991	6	4.5	[1.7-12.0]
	("Organizing"):	No	60,545	29
	Group C ("NSIP"):	Yes No	6655 56,881	7 28	5.8	[1.6-20.9]

J. Clin. Med. 2021, 10, 1162. https://doi.org/10.3390/jcm10061162 https://www.mdpi.com/journal/jcm

Acknowledgments:

The authors would like to thank the collaborators and patients at the investigative sites for their support of this study. We thank Nick Barton for language editing.

Data Availability Statement:

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the included patients have consented to the use of their medical data in anonymous form for medical research purposes, but not to its access in a publicly accessible repository.

Conflicts of Interest: STERVINOU-WEMEAU reports personal fees and non-financial support from Roche, personal fees and non-financial support from Boehringer-Ingelheim, personal fees from BMS, personal fees from Sanofi Genzyme, and personal fees from GSK, outside the submitted work. Prof. LAUNAY reports grants from CSL Behring, personal fees from Biocryst, grants and personal fees from Takeda, outside the submitted work. The other authors have nothing to disclose.


Benoît Geslin

Sophie Gachet

Magali Deschamps-Cottin

Floriane Flacher

Benjamin Ignace

Corentin Knoploch

Éric Meineri

Christine Robles

Lise Ropars

Lucie Schurr

Violette Le Féon

Bee hotels host a high abundance of exotic bees in an urban context

Keywords: Bee hotels, native and exotic bees, Megachile sculpturalis

Bee hotels are increasingly set up by land managers in public parks to promote wild bee populations. However, we have very little evidence of the usefulness of bee hotels as tools to help the conservation of wild bees within cities. In this study, we installed 96 bee hotels in public parks of Marseille (France) for a year and followed their use as a nesting substrate by the local fauna. The most abundant species that emerged from bee hotels was the exotic bee species Megachile sculpturalis, representing 40% of all individuals. Moreover, we only detected four native bee species all belonging to the Osmia genus. More worryingly, we found a negative correlation between the occurrence of M. sculpturalis in bee hotels and the presence of native bees. One hypothesis to explain this result might be linked to the described territorial and aggressive behaviour of M. sculpturalis toward the nests built by the native fauna. This study raises the question about the usefulness of bee hotels for the conservation of native bees especially within cities harbouring high abundance of exotic bees. We provide here concrete advices to land managers to build bee hotels that can both host native bees and prevent the installation of M. sculpturalis.

INTRODUCTION

Megachile sculpturalis Smith, 1853 (Hymenoptera, Apoidea, Megachilidae) is the first exotic bee species that has been detected in continental Europe. This large bee native to East Asia and easily recognisable by its infuscate wings and its orange hair on the thorax was first detected in 2008, near Marseille [START_REF] Vereecken | Premières données sur la présence de l'abeille asiatique Megachile (Callomegachile) sculpturalis SMITH (Hymenoptera, Megachilidae) en Europe[END_REF]. Since this first detection, M. sculpturalis now occupies the whole southeast of France (Le Féon et al. 2018;Le Féon & Geslin 2018) and has also been recorded in several other European countries: Germany [START_REF] Westrich | Megachile sculpturalis Smith 1853 (Hymenoptera, Apidae), a new species for the bee fauna of Germany, now north of the Alps[END_REF], Hungary [START_REF] Kov | Megachile sculpturalis Smith, 1853 in Hungary (Hymenoptera , Megachilidae)[END_REF], Italy [START_REF] Quaranta | A new species for the bee fauna of italy: Megachile sculpturalis continues its colonization of europe[END_REF], Slovenia (Gogala & Zadravek 2018), Spain [START_REF] Aguado | Megachile (Callomegachile) sculpturalis Smith, 1853 (Apoidea: Megachilidae): A new exotic species in the Iberian Peninsula, and some notes about its biology[END_REF], and Switzerland [START_REF] Amiet | Die Blattschneiderbiene Megachile sculpturalis Smith, 1853 (Hymenoptera, Apidae) nun auch in der Schweiz[END_REF].

Regarding its ecology and its interactions with the local flora and fauna, the little evidence we have suggests that M. sculpturalis tends to seek pollen preferentially from exotic plants, including Sophora japonica L. (= Styphnolobium japonicum Schott, Fabaceae; [START_REF] Andrieu-Ponel | A propos du comportement de butinage de Megachile sculpturalis Smith, 1853, en France méditerranéenne (Nîmes et Montpellier) (Hymenoptera , Megachilidae)[END_REF] or Ligustrum sp. (Olaceae; Quaranta et al. 2012). This species nests in hollow stems or pre-existing cavities in dead wood. More worryingly, several events of aggressive eviction of wild bees from their nest by M. sculpturalis (mostly Osmia spp. and Xylocopa spp.) has been previous documented [START_REF] Laport | Occupation of Active Xylocopa virginica Nests by the Recently Invasive Megachile sculpturalis in Upstate New York[END_REF][START_REF] Roulston | Aggressive Eviction of the Eastern Carpenter Bee (Xylocopa virginica (Linnaeus)) from its Nest by the Giant Resin Bee (Megachile sculpturalis Smith)[END_REF], Le Féon et al. 2018), showing potential negative interactions between the local bee fauna and this exotic species. [START_REF] Quaranta | A new species for the bee fauna of italy: Megachile sculpturalis continues its colonization of europe[END_REF] suggested the use of trap-nests (i.e., bee hotels) to monitor the expansion of M. sculpturalis. The trap-nests used in that study were composed of giant reeds and dead trunks previously drilled to create holes. Such trap-nests are widely used and study in the literature either to sample local bee diversity (e.g. [START_REF] Tscharntke | Bioindication using trap-nesting bees and wasps and their natural enemies: community structure and interactions[END_REF][START_REF] Loyola | Habitat structure components are effective predictors of trapnesting Hymenoptera diversity[END_REF][START_REF] Westphal | Measuring bee diversity in different European habitats and biogeographical regions[END_REF][START_REF] Nielsen | Assessing bee species richness in two Mediterranean communities: Importance of habitat type and sampling techniques[END_REF][START_REF] Fabian | Plant diversity in a nutshell: testing for small-scale effects on trap nesting wild bees and wasps[END_REF][START_REF] Da Rocha-Filho | Cavity-nesting bees and wasps (Hymenoptera: Aculeata) in a semi-deciduous Atlantic forest fragment immersed in a matrix of agricultural land[END_REF], to improve pollination service for crops (e.g. [START_REF] Sheffield | Diversity of cavity-nesting bees (Hymenoptera : Apoidea ) within apple orchards and wild habitats in the Annapolis Valley, Nova Scotia, Canada Diversity of cavity-nesting bees ( Hymenoptera : Apoidea ) within apple orchards and wild habitats in the Ann[END_REF][START_REF] Dainese | Managing trap-nesting bees as crop pollinators: Spatiotemporal effects of floral resources and antagonists[END_REF], to study host-parasitoides relationships (e.g. [START_REF] Tscharntke | Bioindication using trap-nesting bees and wasps and their natural enemies: community structure and interactions[END_REF][START_REF] Pitts-Singer | The Alfalfa Leafcutting Bee, Megachile rotundata: The World's Most Intensively Managed Solitary Bee[END_REF][START_REF] Groulx | Nesting aggregation as a predictor of brood parasitism in mason bees (Osmia spp.)[END_REF][START_REF] Happe | Small-scale agricultural landscapes and organic management support wild bee communities of cereal field boundaries[END_REF], food preferences of bees (e.g. Jauker et al. 2012;[START_REF] Macivor | Pollen specialization by solitary bees in an urban landscape[END_REF], or even in a context of biological invasions [START_REF] Barthell | Invader effects in a community of cavity nesting megachilid bees (Hymenoptera : Megachilidae)[END_REF]. Trap-nests also called "bee hotels" (hereafter) are also increasingly set up by land managers in public parks to promote wild bee populations. The body of literature regarding the usefulness of bee hotels to enhance or protect bee populations within cities and urban parks has starting to grow past few years (e.g. [START_REF] Gaston | Urban domestic gardens (II): Experimental tests of methods for increasing biodiversity[END_REF][START_REF] Pereira-Peixoto | Spillover of trap-nesting bees and wasps in an urban-rural interface[END_REF][START_REF] Von Königslöw | Benchmarking nesting aids for cavity-nesting bees and wasps[END_REF]. As an example, in France (Lyon), [START_REF] Fortel | Use of human-made nesting structures by wild bees in an urban environment[END_REF] collected 21 species in a two-year survey of complex bee hotels composed of multiples substrates for below-ground nesting bees and several nesting materials for above-ground nesting bees (logs of several tree species drilled with holes, hollow or pithy stems). All species were native except M. sculpturalis (16 out of 3102 bees in total). In Canada, in Toronto, a city known to harbour many exotic bee species [START_REF] Sheffield | Hylaeus punctatus (Hymenoptera: Colletidae), a bee species new to Canada, with notes on other non-native species[END_REF][START_REF] Macivor | Bee hotels" as tools for native pollinator conservation: A premature verdict?[END_REF] found 31 species in a three years survey of 600 bee hotels in Toronto (Canada), with 47% of individuals being non-native bees.

In this study, we had two main aims. Firstly, we explored the role of bee hotels in hosting a diverse community of wild bees (above-ground nesting species) in a Mediterranean city (Marseille). The Mediterranean area is considered as a bee diversity hotspot at the European scale [START_REF] Nieto | European Red List of Bees[END_REF]) and we wanted to know whether simple bee hotels could provide nesting opportunities for above-ground nesting bee species. Secondly, we evaluated the use of bee hotels by the exotic bee M. sculpturalis. As previously mentioned, this species has rapidly extended its geographical distribution within a few years in Europe, and we hypothesise here that M. sculpturalis might widely use bee hotels to nest. Moreover, as reported above, M. sculpturalis may exhibit aggressive behaviour towards the local native fauna. Its presence in bee hotels might therefore be detrimental for the nesting of native bees, and we also sought to acquire some insight concerning the potential of native and exotic bees to coexist in the same bee hotels.

For this purpose, we installed 96 bee hotels in 12 parks in the city of Marseille and we monitored their use as a nesting substrate by the local bee fauna. We specifically wanted i) to monitor the occupation of bee hotels by M. sculpturalis and by the local native bee fauna, ii) to estimate the potential coexistence of both native and exotic bees in the same bee hotels and iii) to provide information on their nesting preferences in terms of size of the nest entrance. The ultimate goal of this study is to provide management recommendations to stakeholders when installing bee hotels.

METHODS

Study sites

The city of Marseille is the second biggest town in France after Paris with nearly 870 000 inhabitants (INSEE, 2015). Marseille is located in the south of France on the Mediterranean coast (43.3N, 5.4E). The climate is typical of the Mediterranean basin, with hot, dry summers, a mean annual temperature of 14.6 °C and mean annual rainfall of 552 mm. In this city, we selected 12 public parks of with a minimum area of 1 ha (for precise location see Fig. 1, see also Table 1 for further information).

Bee hotels

In February 2016, we installed the 96 bee hotels in the twelve public parks (8 per parks). Bee hotels were homogeneously distributed throughout the parks. Each hotel was placed at a minimum of 1.5 m from the ground, carefully tied to a tree and oriented southwards. Each bee hotel measured 45 × 35 × 30 cm and was composed of two substrates: trunk and bamboo or reed stems. Trunks (Pinus spp.) were drilled with holes of 6, 8, 10 and 12 mm on a single end (8-10 holes per trunk) with a depth of 0.2 m. The diameter of the holes are representative of the bee's preferences for nesting sites [START_REF] Quaranta | A new species for the bee fauna of italy: Megachile sculpturalis continues its colonization of europe[END_REF][START_REF] Fortel | Use of human-made nesting structures by wild bees in an urban environment[END_REF]. Then, hotels were entirely filled with reed or bamboo stems (Fig. 2). The diameter of stems ranged from 3 to 20 mm (mean 7.5 mm; sd = 3.5).

Bee sampling

After a year, in February 2017, 71 of the 96 bee hotels were still in place, the rest having undergone severe damages. We collected each stem and trunk used by the local fauna to nest (entrance filled with mud or resin). Each nesting substrate was individually placed in plastic bags and stems and trunks from the same hotel were placed in the same box when possible. We tried to prevent as far as possible emerging bees from escaping from the plastic bags, but some individuals still managed to escape by chewing the plastic of the bags. From February 10 th to June 1 st , each bag was monitored to look for emerging bees. Each bee was then collected and frozen for further identification to species level by expert-taxonomists (David Genoud & Matthieu Aubert).

To assess the relationship between the adult body size and the nest entrances (holes), we first measured this entrance with a digital calliper (Digit-Cal MK IV, Brown & Sharpe). The intertegular distance (ITD) of a subset of individuals was then measured (238 individuals were measured, 129 Megachile sculpturalis and 109 native Osmia).For this purpose, we used a binocular magnifier, a digital camera (Motic -1.3 MP) and an image processing software (Motic Images Plus 2.0 ML).

Statistical analysis

The link between the presence of the native bees and the abundance M. sculpturalis was assessed using the two-step Hurdle modelling process. This approach combines logistic models with log-linear zero-truncated models in order to analyse account zero-inflated Poisson data [START_REF] Zuur | Mixed Effects Models and Extensions in Ecology with R[END_REF]. Firstly, we fitted a binomial generalized linear mixed model to test whether the presence (or absence) of native bees was influenced by the abundance of M. sculpturalis in each bee hotels. Secondly, we fitted a Poisson generalized linear mixed model linking the abundance of native bees as a response variable and the abundance of M. sculpturalis as explanatory variable (see Meineri et al., 2013 for examples of similar analyses). The two models described above included the park as a random effect to account for repeated measurement within each of the height parks. The abundance of M. sculpturalis was log transformed in both models to obtain a variable closer to a normal distribution. Size of nest entrances were compared between emerging adults of M. sculpturalis and emerging native bees using mixed effect linear models nested on both parks and hotels. The size of nest entrances of M. sculpturalis males and females such as the size of emerging adult males and females were compared using mixed effect linear models nested on parks.

Finally, to explore potential competitive interactions between native and exotic bees, we linked the body size of mature individuals of native bees with the presence/absence of M. sculpturalis individuals using linear mixed model using parc as random effect.

All statistical analyses were performed with R 3.5.2 (R Core Team, 2018). Mixed effect models were run within the libraries lme4 [START_REF] Bates | lme4: linear mixed-effect models using Eigen and S4. R package version 1[END_REF]. The library lmerTest was used for the estimations of P-values for the coefficients [START_REF] Kuznetsova | lmerTest Package: Tests in Linear Mixed Effects Models[END_REF].

RESULTS

Bees emerged from 41 of the 71 remaining bee hotels. No bee was found in 30 of the hotels after a single year. In total, 889 individuals emerged. Megachile sculpturalis was the most abundant species with 356 individuals (~40% of the total abundance). The sex-ratio of this species was highly skewed in favor of males (296 males i.e. 83% of the specimens). We only found four native bee species, all belonging to the genus Osmia: Osmia niveata (Fabricius, 1804), O. caerulescens L., O. bicornis L. and O. cornuta (Latreille, 1805). The most abundant native species was O. bicornis with 308 individuals (~34%) followed by O. cornuta with 143 individuals (~16%), O. niveata, 59 individuals (~7%) and O. caerulescens, 19 individuals (~2%). The remaining individuals belonged to Osmia but could not be determined to the species level due to their conservation state (<1%). Unfortunately, some bees chewed their way out of the plastic bags used for rearing them, as a result, we could only rely 716 of those individuals to a particular park (see Table 1) and 699 individuals to a particular bee hotel (supplementary material S1).

Link between native and exotic bees

The probability of presence of a native bee within a bee hotel significantly decreased as the abundance of M. sculpturalis increased (P=0.002). According to the model, the probability of finding native bees drop by 51% (from 0.51 to 0.25) after a single individual of M. sculpturalis was observed (Fig. 3). Only ten hotels harbour both native and exotic bees.

The abundance of M. sculpturalis had however not significantly affected the abundance of native bees when focussing only on the hotels that were colonised by both exotic and native bees.

Body and nest entrance size.

As expected, individuals of M. sculpturalis were significantly larger than native bees (P <0.001; M. sculpturalis mean ITD = 0.42 mm, native bees mean ITD = 0.30 mm) and nested in larger cavities (P =0.032; M. sculpturalis mean nest entrance's size = 9.57 mm, native bees mean nest entrance's size = 7.76, Fig. 4). Emerging males of M. sculpturalis were smaller than emerging females (P <0.001; males mean ITD = 0.41 mm, female mean ITD = 0.55), and emerged from smaller stems (P<0.001, males mean stem's size = 9.46 mm; female mean stem's size = 12.03 mm).

We also asked whether native bees tended to nest in larger cavities in the absence of M. sculpturalis. However, we did not find any effect of the presence/absence of M. sculpturalis on the mean size of nest entrance of native bees.

DISCUSSION

The first insight from our study concerns the limited species richness we observed in our bee hotels. Only five species were detected in a one-year survey. In comparison with the study of [START_REF] Fortel | Use of human-made nesting structures by wild bees in an urban environment[END_REF], which detected 21 species (3,102 specimens) in a comparable city in terms of population numbers (Lyon -300 km north of Marseille), this richness appeared very weak.

In 2016, we surveyed the bee populations of urban parks of Marseille (Geslin et al. unpublished data) and found 114 wild bee species. Among these, we might have expected to find some species nesting in our bee hotels such as Megachile rotundata (Fabricius, 1787), Xylocopa violacea L. or Anthidium manicatum L., but we did not find any. Several factors might explain this result. Firstly, our bee hotels were much simpler than those of [START_REF] Fortel | Use of human-made nesting structures by wild bees in an urban environment[END_REF]. We used only two types of nesting substrates to build our hotels, bamboo/reed stems and holes drilled in trunks of Pinus spp. [START_REF] Fortel | Use of human-made nesting structures by wild bees in an urban environment[END_REF] used a wide diversity of logs (e.g. Acer sp., Ailanthus sp., Fraxinus sp., Platanus sp., Populus sp., Prunus sp., Robinia sp., and Tilia sp.), and stems (e.g. Arundo sp., Phragmites sp, and Phyllostachys sp.) and built larger bee hotels. Furthermore, in the latter study, the authors showed an increase in the species richness of bees that nest in man-made structures between the two years of their experiments. Here, we installed bee hotels for a single year, and we can hypothesise that the richness might be higher in the longer term.

Despite this, the bee richness we did find nevertheless appeared very low in comparison to the richness of urban park we found. In addition to this low richness, we also found a very high abundance of the exotic bee Megachile sculpturalis. The proportion we did found is quite similar to that reported in the study of [START_REF] Macivor | Bee hotels" as tools for native pollinator conservation: A premature verdict?[END_REF], who also found a very high proportion of exotic bees in their bee hotels (47%). The context is however slightly different, because many exotic bee species have been introduced in Canada (the study of MacIvor & Packer was carried out in Toronto, [START_REF] Sheffield | Hylaeus punctatus (Hymenoptera: Colletidae), a bee species new to Canada, with notes on other non-native species[END_REF]) and only one (to date) in France, but it highlights that bee hotel might be widely use by exotic bee species. [START_REF] Barthell | Invader effects in a community of cavity nesting megachilid bees (Hymenoptera : Megachilidae)[END_REF] in California, already found that some exotic bee species (two Megachile species) could be frequent in artificial nesting cavities. In another study relative to trap nests in Europe, the exotic wasp Isodontia mexicana has also been previously captured (e.g. [START_REF] Fabian | Plant diversity in a nutshell: testing for small-scale effects on trap nesting wild bees and wasps[END_REF], and [START_REF] Quaranta | A new species for the bee fauna of italy: Megachile sculpturalis continues its colonization of europe[END_REF] have already used trap nests to capture M. sculpturalis in Italy. Finally, in a previous study we published relative to citizen monitoring of M. sculpturalis, we already mentioned several events of M. sculpturalis individuals nesting in bee hotels (Le Féon et al., 2018 -26 cases among 39 reported nesting events). In the present study, M. sculpturalis was the most abundant species and its use of bee hotels might facilitate the extending of its geographical distribution.

More worryingly, we found a strong negative correlation between the presence of M. sculpturalis individuals and the presence of native bee species in our bee hotels. In other words, the probability of an Osmia to emerge from an hotel also occupied by M. sculpturalis is light.

At this stage, it might be too straightforward to directly explain this negative correlation by the reported aggressive behaviour of M. sculpturalis toward other bee species (aggressive nest eviction -Laport & Minckley 2012, [START_REF] Roulston | Aggressive Eviction of the Eastern Carpenter Bee (Xylocopa virginica (Linnaeus)) from its Nest by the Giant Resin Bee (Megachile sculpturalis Smith)[END_REF], Le Féon & Geslin, 2018) for several reasons. Firstly, as we have shown, M. sculpturalis and native Osmia seemed not to share the same nesting preferences, and M. sculpturalis tends to nest in larger cavities than the native fauna. Competition for nesting sites is therefore unlikely. Such competition might however occur with the larger bee species we previously sampled in Marseille urban parks (Geslin et al., unpublished data) such as Xylocopa spp. or Anthidium spp. Secondly, the two most abundant Osmia species we found (O. bicornis and O. cornuta) and M. sculpturalis do not share the same phenology. These two Osmia species are spring species whereas M. sculpturalis is active in summer (Le Féon et al., 2018). Direct competition (aggression or eviction) similar to aggressive events reported by Roulson & Malfi (2012) between active adults is therefore unlikely. Furthermore, we cannot exclude that the negative correlation we found between the presence of Osmia and that of M. sculpturalis in bee hotels could be due to other environmental factors and that environmental conditions (feeding resources, proximity to suitable habitats) could be highly favourable for M. sculpturalis and not for native bee species. However, we believe that the most likely hypothesis to explain the negative correlation we observed is probably linked to the territorial behaviour of M. sculpturalis toward the native fauna. Our main explanation lies in an exclusion of larvae of native bees by adults of M. sculpturalis. This behaviour has been previously documented on occasion (see Le Féon & Geslin 2018 for an example with Isodontia Mexicana) and may be more widespread. If this is the case, the negative impact of M. sculpturalis on the wild fauna could be greater than previously thought. This might also potentially affect the large bee species of the French Fauna such as Xylocopa spp. and some Anthidium and Megachile spp.

Taken together and given the widespread use of insect hotels by stakeholders, landowners and by the general public, our findings raise the question of the usefulness of simple bee hotels for the conservation of native wild bee species in urban park facing high abundance of above ground nesting exotic bees. Rather, in some cases, it appears that those simple hotels might be particularly hospitable for exotic species. M. sculpturalis extensively use bee hotels to nest, and these hotels might help this species to extend its geographical range. This could be worrying for the native bee fauna as M. sculpturalis might develop aggressive behaviours towards a large diversity of above ground nesting native bees by evicting their larvae and thus preclude their emergence. This assertion needs however to be more widely explored and we advocate here future studies to investigate the value of bee hotels as a conservation tool. More complex bee hotels harbouring several nesting substrates might for example be more efficient to support urban bee biodiversity (see von Königsloöw et al. 2019 as an example).

Recommendations for land-managers

Megachile sculpturalis is a large species. As we show, this species nests in larger cavities than most species of the native fauna that is commonly found in bee hotels. Female in particular are laid in large cavities of about 12 mm. To discourage the spread of this species, one should bring attention to the diameter of the holes of the substrate use to build the bee hotels. At this stage, we advise land-managers to use nesting substrates designed to favour the reproduction of the native fauna (cavities of 4 to 8 mm) and to banish larger cavities (above 8 to 10 mm) in their bee hotels to prevent the installation of M. sculpturalis. TABLE Table 1: Details of public parks' names, superficies and the abundance of each bee species.

FIGURES

Figure 1 :Figure 2 :

 12 Figure 1: Location of public parks in the city of Marseille (France)

Figure 3 :

 3 Figure 3: Probability of observing at least an individual of native bee emerging from a bee

Figure 4 :

 4 Figure 4: Boxplots of the mean sizes of the nest's entrances between M. sculpturalis and native

Figure 1 :Figure 2 :

 12 Figure 1: Location of public parks in the city of Marseille (France)

Figure 3 :

 3 Figure 3: Probability of observing at least an individual of native bee emerging from a bee

Figure 4 :

 4 Figure 4: Boxplots of the mean sizes of the nest's entrances between M. sculpturalis and native

ACKNOWLEDGEMENTS

We would like to thank the city of Marseille for its help for setting up bee hotels in public parks.

We particularly appreciated the help of Sarah Bourdon, Thomas Muti, Patrick Bayle, Catherine Steunou and Josette Sakakini and the other students in civic services. We thank David Genoud and Matthieu Aubert for bee identifications. This work has been partly funded by the OHM (Observatoire Hommes-Milieux, Labex DRIIHM): Bassin Minier de Provence.

Authors' contributions :

Benoît Geslin conceived the study, achieved the field work and wrote the paper.

Violette Le Féon wrote the paper