# Functional insights into human macrophage response against Scedosporium apiospermum and Scedosporium dehoogii

Dorra Elhaj Mahmoud, Mariem Hanachi, Hajar Yaakoub, Simon Blanchard, Pascale Pignon, Oussama Souiai, Yves Delneste, Jean-Philippe Bouchara, Nicolas Papon, Anaïs Hérivaux

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## To cite this version:

Dorra Elhaj Mahmoud, Mariem Hanachi, Hajar Yaakoub, Simon Blanchard, Pascale Pignon, et al. Functional insights into human macrophage response against Scedosporium apiospermum and Scedosporium dehoogii. Cytokine, 2023, 172, pp.156384. 10.1016/j.cytokine.2023.156384. hal-04269253

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1 Functional insights into human macrophage response against *Scedosporium*

2 *apiospermum* and *Scedosporium dehoogii*

3 Dorra Elhaj Mahmoudª, Mariem Hanachiᵇ, Hajar Yaakoubª, Simon Blanchardᶜ, Pascale

4 Pignonᶜ, Oussama Souiaiᵇ, Yves Delnesteᶜ,>d, Jean-Philippe Boucharaª, Nicolas Paponª, Anaïs

5 Hérivauxª

6 ª Univ Angers, Univ Brest, IRF, SFR ICAT, 49000, Angers, France.

7 ᵇ Laboratory of Bioinformatics, Biomathematics and Biostatistics-LR16IPT09, Institut Pasteur de

8 Tunis, Tunis, Tunisia.

9 ᶜ Univ Angers, Nantes Université, Inserm, CNRS, CRCI²NA, SFR ICAT, F-49000 Angers, France.

10 ᵈ Immunology and Allergology laboratory, University Hospital of Angers, Angers, France.

11 **Corresponding author**

12 Dr. Anaïs Hérivaux (PharmD, PhD)

13 Fungal Respiratory Infections Unit

14 University of Angers, IRIS-IBS

15 4, rue Larrey – 49933 Angers

16 France

17 +33 (0) 244 688 363

18 anais.herivaux@univ-angers.fr

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**Abstract**

Fungal infections caused by *Scedosporium* species are rising among immunocompromised and immunocompetent patients. Within the immunocompetent group, patients with cystic fibrosis (pwCF) are at high risk of developing a chronic airway colonization by these molds. While *S. apiospermum* is one of the major species encountered in the lungs of pwCF, *S. dehoogii* has rarely been reported. The innate immune response is believed to be critical for host defense against fungal infections. However, its role has only recently been elucidated and the immune mechanisms against *Scedosporium* species are currently unknown. In this context, we undertook a comparative investigation of macrophage-mediated immune responses toward *S. apiospermum* and *S. dehoogii* conidia. Our data showed that *S. apiospermum* and *S. dehoogii* conidia strongly stimulated the expression of a set of pro-inflammatory cytokines and chemokines such as IL-1β, IL-8, IL-6 and TNFα. We demonstrated that *S. dehoogii* was more potent in stimulating the early release of pro-inflammatory cytokines and chemokines while *S. apiospermum* induced a late inflammatory response at a higher level. Flow cytometry analysis showed that M1-like macrophages were able to internalize both *S. apiospermum* and *S. dehoogii* conidia, with a similar intracellular killing rate for both species. In conclusion, these results suggest that M1-like macrophages can rapidly initiate a strong immune response against both *S. apiospermum* and *S. dehoogii*. This response is characterized by a similar killing of internalized conidia, but a different time course of cytokine production.

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**Keywords:** *Scedosporium*, Macrophages, Inflammation, Immune response, Cytokines, Gene expression

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## 1 Introduction

Scedosporium species are environmental filamentous fungi that usually live as saprophytes in highly polluted soils and contaminated water [1]. They are considered as emerging fungal pathogens in both immunocompetent and immunocompromised individuals. Scedosporium infections result in a wide range of clinical manifestations, from colonization of the respiratory tract in patients with cystic fibrosis (pwCF) and subcutaneous mycetomas to severe and often fatal disseminated infections in immunocompromised patients [2,3]. Within the immunocompetent group, pwCF are among those at the highest risk of suffering from these mycoses [4,5]. In fact, after Aspergillus fumigatus, Scedosporium species are the second most frequent molds involved in the colonization of the airways of these patients with a frequency ranging from 2 to 17.4% depending on the geographical area and the group of pwCF studied [4,6,7]. Generally, the distribution of Scedosporium species in the CF lung follows their respective frequency in the environment with the exception of Scedosporium dehoogii [8]. Conversely to Scedosporium apiospermum and some other Scedosporium species, Scedosporium dehoogii has only been isolated once from respiratory secretions, whereas it is common in soil, thus questioning its ability to colonize the respiratory tract of pwCF [9]. A comparative genomic analysis of the main Scedosporium species revealed a number of candidate genes potentially involved in the adaptive mechanisms of pathogenic Scedosporium species within the respiratory tract of pwCF. Interestingly, more than a hundred genes exhibit point mutations that potentially lead to a loss of function in S. dehoogii, including genes encoding some transcription factors, as well as some GPI-anchored proteins and two thioredoxin-like proteins [10].

The airways are considered to be the primary route used by Scedosporium to colonize or infect the host. In order to colonize the respiratory tract, fungi must evade the host immune response. In the lungs, macrophages are one of the first lines of antifungal defense [11,12]. According to their activation state and function, macrophages can be broadly divided into M1-type (pro-inflammatory phenotype) and M2-type (wound healing phenotype) [13,14]. M1 macrophages are characterized by CD80, CD86, CD40 and major histocompatibility complex class II (MHC-II) surface molecules, whereas M2 macrophages express M2-specific markers such as CD163 and CD206 [14]. It is now recognized that the M2 type comprises a diverse group of macrophages rather than a single activation state. Accordingly, M2 macrophages can be further-categorized into distinct subsets: M2a, M2b, M2c and M2d, based on their distinct transcriptional profiles and biological functions [15]. Unlike M2 macrophages, M1
macrophages are typically fungicidal cells and their activation provides a host protection against a myriad of fungal infections [16]. The common response of M1 macrophages to fungal infections involves variety of pattern recognition receptors (PRRs) which may act individually or in concert [13,17]. *Scedosporium* cell wall components, including peptidorhamnomannans (PRMs), α-glucans, and glucosylceramides, are important activators of immune cells following their recognition by Toll-like receptor 2 (TLR2), TLR4, and Dectin-1 and through receptors that are yet unknown [18]. Following fungal recognition, M1 macrophages produce pro-inflammatory cytokines (dominated by interleukin (IL)-1β, IL-6, IL-12, IL-23 and Tumor Necrosis Factor (TNF) α) and chemokines that can recruit other immune cells that contribute to fungal clearance [19,20]. Phagocytosis and the intracellular killing of fungal elements constitute other key elements of the M1 macrophage-mediated immune response [21,22].

Remarkably, very little research has focused on understanding the immune response against *Scedosporium* species [23,24]. Among the unknowns that remain to be elucidated, the most important is the complexity of the mechanisms developed by these fungi to evade the host immune response and the differences in the inflammatory response induced by each species. This may explain, at least in part, the inability of *S. dehoogii* to develop in the respiratory tract and to cause a chronic colonization of the airways. In this context, we undertook a comparative investigation of the M1-like macrophage-mediated immune response upon conidia of *S. apiospermum* and *S. dehoogii*.

## 2 Materials and Methods

### 2.1 *Scedosporium* strains and culture conditions

The study was performed on two *Scedosporium* strains: *S. apiospermum* IHEM 14462, originally isolated from a sputum sample from a CF patient and previously used for whole-genome sequencing [25] and *S. dehoogii* UA 110354521 isolated from a soil sample from Meknes (Morocco).

The strains were maintained at 37°C by weekly passages on potato dextrose agar (PDA) (Condalab, containing in g/L: dextrose, 20; potato-infusion extract, 4; and bacteriological agar, 15), supplemented with 0.5 g/L chloramphenicol. Conidia used in each experiment were collected from 8-day-old cultures. Conidia were harvested from agar plates using sterile water; then the obtained suspensions were filtered through sterilized Miracloth® (pore size
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22-25 µm) to remove hyphae, and conidia in the filtrates were enumerated by haematocytometer counts.

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## 2.2 M1-like macrophage generation and co-culture assays

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Peripheral blood mononuclear cells (PBMC) were obtained from healthy human volunteers (Blood Collection Center, Angers, France; agreement CPDL-PLER-2021 038 approved by the Ethics Committee of the University Hospital of Angers) by standard density gradient centrifugation on Lymphocyte Separation medium (Eurobio). To generate macrophages, monocytes were purified from PBMC by positive selection using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotec). Monocytes were cultured for 5 days in a complete medium consisting of RPMI 1640 (Lonza) supplemented with 10% fetal calf serum (Eurobio), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES, 100 U/mL penicillin and 100 µg/mL streptomycin (all from Lonza) in the presence of GM-CSF (50 ng/mL, R&D Systems) to generate M1-like macrophages. For interaction studies, M1-like macrophages (5 × 10⁵/well in 24-well plates) were incubated with S. apiospermum or S. dehoogii conidia at a 1:1 ratio at 37°C under 5% CO₂. Higher macrophage-conidia ratios (e.g. 1:10) were not considered in this study since we observed important cell death in M1-like macrophages 24 h after co-incubation.

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## 2.3 Analysis of immune-relevant gene expression by qPCR

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Total RNA was isolated from M1-like macrophages after 3 h and 24 h of co-culture with *S. apiospermum* and *S. dehoogii* conidia using the PureLink RNA Mini Kit (Thermo Scientific). The Qubit® 2.0 Fluorometer (Invitrogen) was used for RNA quantification. Total RNA was reverse transcribed using the High-capacity cDNA Reverse Transcription Kit (Applied Biosystems). After a 5-fold dilution, cDNA samples were used as templates for real-time quantitative PCR (qPCR) using the StepOnePlus® thermocycler (Applied Biosystems). Each qPCR reaction was performed in a final volume of 20 µL containing PowerUp SYBR Green Master Mix (Applied Biosystems), 250 nM or 500 nM of each primer (primer sequences are listed in Table 1), 6 µL of water, and 2 µL of diluted cDNA. PCR cycling parameters were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 3 s, and cooled to 60°C for 30 s (Fast cycling mode). Then, the PCR products were heated to 95°C for 15 s, cooled to 60°C for 1 min, and heated to 95°C to measure the dissociation curves. Five-point serial dilutions of cDNA were used as templates to construct a standard curve for each gene and determine the amplification efficiency (E). Thanks to its limited
variation between the different samples, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as an endogenous reference. The relative gene expression level (RQ) was calculated using the 2⁻⁰·⁰⁰Cycle.Threshold (CT) method according to the following formula.

$$ \Delta \text{CT experiment (stimulated macrophages)} = \text{CT target gene} - \text{CT GAPDH} $$

$$ \Delta \text{CT control (untreated macrophages)} = \text{CT target gene} - \text{CT GAPDH} $$

$$ \text{RQ} = 2^{-(\Delta \text{CT experiment} - \Delta \text{CT control})} $$

## 2.4 Multiplex cytokine/chemokine analysis

Supernatants were collected after 3 h and 24 h of M1-like macrophage co-culture with S. apiospermum and S. dehoogii conidia. They were centrifuged at 1,700 rpm at 4°C for 10 min, and stored at -80°C until use. Secreted cytokines and chemokines were measured in the supernatants by multiplex fluorescent bead-based technology (Luminex 200), using a commercial Luminex Screening Assay Kit from R&D Systems according to the manufacturer's recommendations. Briefly, the samples were diluted 1:2 before incubation with specific antibody-coated fluorescent beads. After washing, 50 beads were analyzed using the Luminex 200 analyzer and Bio-Plex Manager software version 6 (Bio-Rad Laboratories), and the analyte concentrations of the samples were estimated through the serial dilution of cytokine standards: IL-1β, sensitivity < 10 pg/mL; IL-6, sensitivity < 5 pg/mL; IL-8 (also called C-X-C motif Ligand 8 (CXCL8)), sensitivity < 10 pg/mL; IL-18, sensitivity < 5 pg/mL; IL-23, sensitivity < 100 pg/mL; TNFα, sensitivity < 5 pg/mL; C-C motif chemokine Ligand 2 (CCL2), sensitivity < 25 pg/mL; CCL3, sensitivity < 50 pg/mL; CCL5, sensitivity < 10 pg/mL; and CXCL2, sensitivity < 70 pg/mL.

## 2.5 Conidial killing experiments

S. apiospermum and S. dehoogii conidia were stained with 40 µg/mL of fluorescein isothiocyanate (FITC; Sigma-Aldrich) in phosphate-buffered saline (PBS, 1X) for 30 min in the dark with constant shaking (120 rpm), washed twice with PBS, and enumerated. FITC-labeled conidia (5 × 10⁵ cells) were co-cultured with adherent M1-like macrophages (cell ratio of 1:1). The cells were then incubated for 2 h at 37°C under 5% CO₂ to allow the internalization of the conidia. The medium containing the non-ingested conidia was removed, wells were washed twice with pre-warmed PBS and a new medium was added. To measure the conidiacidal capacity, M1-like macrophages were allowed to kill the ingested conidia for 3 h at 37°C under 5% CO₂. After the second incubation period, the medium was removed, and the wells were washed twice with PBS. After microscopic examination to visualize adherence
and ingestion of the conidia, cells were lifted from the wells by scraping, transferred to Eppendorf tubes, and centrifuged at 4,600 rpm for 5 min to remove PBS. The cell pellets were resuspended with 500 µL of water and incubated for 30 min to allow lysis of M1-like macrophages and release of intracellular conidia. After microscopic control of macrophage lysis, the resulting suspensions were centrifuged at 4,600 rpm for 5 min to pellet the conidia. Macrophage debris was removed by treating the suspensions with DNase, RNase, and proteinase K (all from Invitrogen). A negative control reflecting the live conidia (conidia not exposed to macrophages) and a positive control referring to heat-killed conidia (85°C, 30 min) were included in each assay. Conidia were then labelled with 25 µg/mL propidium iodide (PI; Sigma-Aldrich) for 30 min at room temperature in the dark prior to flow cytometry analysis. Both controls were used to establish cut-offs for positivity of each marker. Conidia were considered alive if they were only FITC positive and dead if they were double positive (FITC⁺PI⁺). The percentage of killing was defined as the number of conidia killed by the macrophages divided by the total number of conidia released by macrophages × 100.

## 2.6 Protein-protein interaction (PPI) networks and biological process analysis

Significantly differentially expressed genes and cytokines in response to conidial challenge were used to construct PPI networks. The Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING v11.5) was used to construct and analyze all PPI networks. PPIs were restricted to those with a confidence of more than 90% as calculated by the String system. We also performed pathway enrichment analysis for each PPI network to identify significantly enriched Gene Ontology (GO) biological processes in each set of genes.

## 2.7 Statistical analysis

All experiments were performed in triplicate at least four times independently. Statistical analysis and visualization were performed using the R 4.3.1 and GraphPad Prism9. To assess the global transcriptomic response, principal component analysis was performed on log-transformed fold-change (log₂(FC)) data obtained from qPCR. To illustrate gene expression, heatmaps were generated based on log₂(FC) and using the ComplexHeatmap R package. Hierarchical clustering was performed based on complete linkage and Euclidean distance measure. A significant difference in gene expression levels was defined as at least a 2- fold change up to or down, corresponding to log₂ (FC) ≥ 1 or log₂ (FC) ≤ -1 and a *p*-value
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< 0.05 (treated M1-like macrophages vs. untreated cells). Differences between groups were determined by a Mann-Whitney test (2 groups) or a one-way analysis of variance Kruskall-Wallis test followed by Dunn's multiple comparison test (3 groups) using the R package ggpurb. In all analyses, *p*-values < 0.05 were considered statistically significant.

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# 3 Results

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## 3.1 *Scedosporium apiospermum* and *Scedosporium dehoogii* induce distinct temporal transcriptomic profiles in M1-like macrophages

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To assess both the early (3 h) and late (24 h) macrophage response, we measured the relative transcript levels of 19 receptors, cytokines, and chemokines from macrophages challenged with conidia of both *S. apiospermum* and *S. dehoogii*. The overall expression profile was subsequently explored by performing multivariate analysis based on principal component analysis (PCA). PC1 accounted for 33.2% of the variability while PC2 explained 25.6% of variability (Fig. 1A).

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Despite the overlapping confidence ellipses, the non-close centroids discriminated the early and the late M1-like macrophage response against the two fungi (Fig. 1A, B and Supplementary Table S1).

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Furthermore, our global analysis highlighted a segregation in the early macrophage immune response against *S. apiospermum* and *S. dehoogii* (Fig. 1A).

Taken together, these results suggest a distinct temporal M1-like macrophage transcriptional profile after *Scedosporium* challenge.

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## 3.2 *Scedosporium dehoogii* is a more potent inducer of the early M1-like macrophage transcriptomic response

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We further investigated the transcriptional profiles of M1-like macrophages after co-culture with *S. apiospermum* or *S. dehoogii* by generating heatmaps of gene expression. We then used the Mann-Whitney U test to determine the statistical differences between untreated and infected macrophages, and between macrophages infected with *S. apiospermum* or *S. dehoogii* conidia.

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After 3 h of co-culture, 9 genes were upregulated in response to *S. apiospermum* conidia compared with the untreated control (Fig. 2A, B and Supplementary Table S2). These genes
encode chemokines (IL-8, CXCL1, CXCL2, CCL3, and CCL5) and pro-inflammatory
cytokines (IL-1α, IL-1β, IL-6, and TNFα). With the exception of CXCL1, a similar profile
was observed for *S. dehoogii* (Fig. 2A and B). At the gene level, *S. dehoogii* conidia induced
significantly higher expression levels of genes encoding IL-8, IL-1α, IL-1β, CCL5, and TNFα
in M1-like macrophages than *S. apiospermum* conidia after 3 h of stimulation (Fig. 3A and
Supplementary Table S4). These differences were particularly observed for IL-1α (572 fold
for *S. dehoogii* vs. 102 fold for *S. apiospermum*, *p*-value = 0.0079) and IL-8 expression (304
fold for *S. dehoogii* vs. 47 fold for *S. apiospermum*, *p*-value = 0.0079).

After 24 h of co-culture, 9 genes (encoding IL-1α, IL-1β, IL-6, IL-8, CXCL2, CCL3, CCL5, TNFα, and IL-23A) were upregulated and 5 (encoding CCL2, IL-18, Dectin-1, HLA-DRA, and TLR4) were downregulated in response to both fungal species compared to the untreated control (Fig. 2C, D and Supplementary Table S3). Among these genes, IL-8 expression was statistically more induced when M1-like macrophages were challenged with *S. dehoogii* compared to *S. apiospermum* (1,700 fold for *S. dehoogii* vs. 154 fold for *S. apiospermum*, *p*-value = 0.0285) (Fig. 3B and Supplementary Table S5). No significant changes were detected in transcription of the genes encoding CXCL9, interferon gamma (IFNγ), IL-15, and TLR2, regardless of the interactions and experimental time point.

Collectively, these data show that *S. apiospermum* and *S. dehoogii* conidia induce a common early and late transcriptome signature (stimulated vs. untreated cells). However, the intensity of the early induced response is species-specific.

## 3.3 The cytokine release profile of M1-like macrophages confirms the strong early pro-inflammatory potential of *Scedosporium dehoogii*

Since the transcriptomic analysis suggested that both *S. apiospermum* and *S. dehoogii* stimulate the inflammatory response of macrophages, we aimed to verify these observations at the protein level. In this line, we assessed the M1-like macrophage production of cytokines and chemokines, which the corresponding gene expression level changed significantly, 3 h and 24 h after challenge with conidia. The results are summarized in Figure 4. After exposure to *S. dehoogii* conidia for 3 h, M1-like macrophages produced significantly higher levels of the pro-inflammatory cytokines TNFα, IL-6, IL-23, and IL-1β and the chemokines CCL3, CCL5, and CXCL2 compared to unstimulated macrophages. Except for CCL3, CXCL2, and TNFα, *S. apiospermum* conidia did not induce higher levels of pro-inflammatory cytokines and
chemokines after 3 h of M1-like macrophages co-culture (Fig. 4A and Supplementary Table S6). Furthermore, *S. dehoogii* conidia tended to induce a strong and early pro-inflammatory response (but not statistically significant) compared to *S. apiospermum* (Fig. 4B). IL-8 and CCL2 cytokine levels were too highly expressed after macrophages challenge with both fungi to be evaluated precisely by the assay. At the 24 h time point, higher levels of IL-1β, IL-6, IL-18, IL-23, CXCL2, TNFα, CCL3 and CCL5 were produced by M1-like macrophages when compared to the control (Fig. 4C and Supplementary Table S7). Interestingly, *S. apiospermum* induced a higher production of the majority of the studied cytokines and chemokines than *S. dehoogii* after 24 h of co-culture with M1-like macrophages (Fig. 4D).

Overall, these results are consistent with the transcriptomic profile of stimulated macrophages and support that *S. dehoogii* exhibits a stronger early pro-inflammatory response compared to *S. apiospermum*.

## 3.4 *Scedosporium dehoogii* polysaccharides may orchestrate the specific early M1-like macrophage response

Given that *S. dehoogii* induced a strong early inflammatory response, we aimed to investigate potentially relevant interactions between the induced mediators of inflammation. The cytokines and chemokines, which were statistically upregulated (expression and/or production vs. control) after macrophage co-culture with *S. dehoogii* conidia for 3 h, were used to construct a protein-protein interaction (PPI) network. A high confidence threshold (90%) was applied to counteract potential background. The network in Figure 5 represents 11 cytokines rapidly stimulated by *S. dehoogii* conidia (IL-1α, IL-1β, IL-6, IL-8, IL-18, IL-23, CXCL2, CCL2, CCL3, CCL5 and TNFα).

GO analysis showed that the induced cytokines and chemokines were part of many biological processes especially cellular response to lipopolysaccharide, neutrophil chemotaxis, and myeloid leukocyte migration.

Taken together, these results suggest the involvement of *S. dehoogii* polysaccharides in the induction of a strong early M1-like macrophage inflammatory response.

## 3.5 M1-like macrophages can engulf and kill conidia of both *Scedosporium* strains

Microscopic analysis showed that *Scedosporium* conidia adhered to M1-like macrophages. The macrophages were also able to internalize both *S. apiospermum* and *S.*
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*dehoogii* conidia. Some macrophages appeared to interact with or engulf many fungal conidia while others were not able to engulf any conidia (Fig. 6A and B).

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No significant differences in killing activity were observed between the two strains, with values of 16.26% for *S. apiospermum* vs. 18.96% for *S. dehoogii* (Fig. 6C).

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Collectively, these results show that M1-like macrophages interact with *S. apiospermum/S. dehoogii* conidia and that the two strains are capable of surviving equally inside phagocytic cells.

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# 4 Discussion

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Although *Scedosporium* species are the second most common filamentous fungi colonizing the airways of CF patients, the immune mechanisms involved in host defense against these fungi are largely unknown. The prevalence of these species is highly variable, *S. apiospermum* being the most commonly recovered in the clinical setting [26,27]. To date, *S. dehoogii* is considered as an environmental species and is not commonly associated with human infections, despite the high virulence observed in murine models [26]. Myeloid cells, including macrophages, express different PRRs that are important for *Scedosporium* detection. Upon recognition of their respective ligands, these receptors induce cytokine and chemokine production, which in turn regulate leukocyte trafficking and activation [17,28]. Our data showed that *S. apiospermum* and *S. dehoogii* conidia strongly stimulated the expression of a number of pro-inflammatory cytokines and chemokines 3 h after incubation with M1-like macrophages. The most significant gene expression changes were observed in IL-1α, IL-1β, and IL-8, supporting their early involvement in the innate immune response against *Scedosporium* fungi. Indeed, IL-8 attracts neutrophils to sites of infection and activates them to kill the fungal cells [29]. While IL-1α/β play an important role in mediating immune responses, each cytokine possesses a distinct function. IL-1α acts as an alarmin, driving leukocyte recruitment and preventing fungal growth [30]. IL-1β signaling enhances phagocytosis and killing, increases reactive oxygen species/nitrogen oxide synthase (ROS/NOS) production and promotes Th1 and Th17 immunity [31]. In our study, *S. dehoogii* tended to be more potent in stimulating the early release of pro-inflammatory cytokines and chemokines compared to *S. apiospermum*. This result may be related to the different structural characteristics of the two species. Indeed, the presence of different pathogen-associated molecular patterns (PAMPs) on the cell wall of the two *Scedosporium* conidia may
be related to different cytokine and chemokine signatures. In this context, a comparative
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genomic analysis of the major *Scedosporium* species revealed several mutations in the *S.
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dehoogii* genome that may lead to a loss of gene function. Some of these genes encode cell
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wall components [10]. Given the importance of the cell wall in invasion of the conidia to the
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immune response, the absence of certain molecules may rapidly expose *S. dehoogii* to
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immune cells. Overall, it is tempting to speculate that the early abundant release of pro-
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inflammatory cytokines and chemokines in response to *S. dehoogii* is associated with a lower
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virulence. Despite the complexity of the immune mechanisms, it is currently assumed that, in
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general, in the early immune response, the fewer pro-inflammatory cytokines are released by
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an immune cell, the more virulent is the behaviour of the microorganism. *A. fumigatus*, the
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most frequent filamentous fungus colonizing the airways of pwCF, and *Lomentospora
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prolificans* have been shown to differ in their capacity to stimulate the early production of
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pro-inflammatory cytokines (*L. prolificans* induces higher levels of TNFα and IL-1β than *A.
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fumigatus*) [21]. Interestingly, a recent study showed that *A. fumigatus* induces a lower
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cytokine production in human lung adenocarcinoma cell line A549 compared to *S.
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apiospermum* during the initial invasion period [32]. In term of cytokine levels, IL-21 levels
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are extremely high with *S. apiospermum* co-culture whereas IL-1α and IL-22 levels are
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moderately elevated in the pneumocytic cells [32]. These observations may explain, at least in
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part, *A. fumigatus* ranking the first among the filamentous fungi causing human respiratory
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infections [21]. Similarly, Bruno *et al*. suggested that the early cytokine production induced
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by yeasts of the *Candida auris* clade is inversely correlated with their virulence [33]. Indeed,
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*C. auris* belonging to clade III, which is associated with invasive infections, shows a reduced
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*in vitro* induction of cytokine production compared to other clades [33,34]. This is consistent
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with the fact that the early production of pro-inflammatory cytokines and chemokines plays
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an important role in protection against infection [12,24]. However, it is important to note that
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a protective early response to fungal infection depends on several factors, including the host
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immune status, site of infection, cell wall complexity, and fungal virulence traits [35]. A
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previous study showed that *S. apiospermum* can efficiently destroy host proteins such as the
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complement factors C3 and C1q, presumably by proteolytic degradation [36]. Furthermore,
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some pathogenic *Scedosporium* species express surface glycoproteins with
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immunomodulatory effects and produce some molecules masking PAMPs, which may make
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recognition by the innate immune system difficult [37, 38].
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By evaluating the late (24 h) *Scedosporium*-induced inflammatory response, we observed substantial production of pro-inflammatory cytokines and chemokines. Considering that M1-like macrophages did not inhibit the germinating process, this late response was probably mediated by phagocytized conidia and *Scedosporium* hyphae. Indeed, *S. apiospermum* and *S. dehoogii* conidia were able to germinate and to produce long hyphae during 24 h of contact with M1-like macrophages. It should be pointed out that, in general, the morphology and density of the monolayers of all M1-like macrophages used herein were preserved at the end of the 24 h of interaction with *S. apiospermum* or *S. dehoogii*. Interestingly, our observations indicated that, contrary to the early response, the late inflammatory pattern induced by *S. apiospermum* was more intense than the one induced by *S. dehoogii*. As hyphae are the invasive morphotype of *Scedosporium*, we can assume that the marked late pro-inflammatory response by M1-like macrophages against *S. apiospermum* is aimed at enhancing the recruitment of other immune cells, most of the innate defenses against hyphae relying on the activity of other immune cells [17,39]. Although the design of our study did not allow us to investigate the mechanisms by which M1-like macrophages recognize the two fungi, our PPI network and pathway analysis suggested the involvement of *Scedosporium* polysaccharides in the inflammatory response. Nevertheless, this predictive model may be subject to selection bias due to the small number of genes investigated in this study. Further analysis based on RNA sequencing would allow for a more comprehensive prediction of *Scedosporium* structures involved in the pro-inflammatory response of M1-like macrophages.

Cytokine induction is important after pathogen recognition, but the induction of phagocytosis and killing is also critical [33]. Our data indicated that *S. apiospermum* and *S. dehoogii* conidia were able to adhere and to be internalized by M1-like macrophages after only 2 h of contact. A previous study conducted by Pinto *et al.* [40] reported that the surface PAMPs are recognized by host cell PRRs, such as TLR4, characterizing it as a *S. apiospermum* adhesin. Furthermore, different types of macrophages are capable of phagocytosing other *Scedosporium* species despite the much larger size of their conidia [17,41]. However, the intracellular outcome of the conidia is species-specific. Indeed, ingested conidia of *S. aurantiacum* and *L. prolificans* are less likely to be killed than those of *S. minutisporum* [22]. This suggests specific mechanisms allowing *S. aurantiacum* and *L. prolificans* conidia to survive inside macrophages [22]. In fact, several microbial systems including superoxide dismutases (SODs) and components of the thioredoxin system can convert ROS into less toxic products [39]. Interestingly, the expression of two thioredoxin-like proteins has been shown to
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be affected in *S. dehoogii* [10]. Hence, one might have predicted an enhanced killing of *S. dehoogii* conidia by M1-like macrophages. Surprisingly, our flow cytometry-based protocol showed that the macrophage-mediated killing rates were similar for *S. dehoogii* and *S. apiospermum* conidia. To the best of our knowledge, this study provides a first understanding of the immune response against *S. dehoogii*.

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There are several important limitations to the current work, including our exclusive focus on the M1-like macrophage response to the two *Scedosporium* species. In fact, the immune response to fungi involves many myeloid cells, such as dendritic cells and neutrophils, and lymphoid cells, such as NK cells and lymphocytes. Furthermore, our study of the immune response to *S. apiospermum* and *S. dehoogii* was limited to cells derived from healthy donors. In fact, macrophages from pwCF differ significantly from healthy cells. They display overexpression of several pro-inflammatory cytokines and have altered phagocytic capacity and microbial killing [42]. We also selected only two well-characterized *S. apiospermum* and *S. dehoogii* isolates for this study, which may have masked some intraspecific variations in terms of virulence. Furthermore, the two strains used in our study were of different origin: *S. apiospermum* was a clinical isolate, while *S. dehoogii* was an environmental one. Indeed, the source of isolation (clinical or environmental) could affect the host-pathogen interaction and, consequently, could also influence the induced immune response profile. Nevertheless, *S. dehoogii* has been reported very rarely in humans, mainly as a cause of cutaneous or subcutaneous infections due to traumatic inoculation of soil fungal elements, and only one isolate has been reported as recovered from the respiratory tract [9]. In addition, a unique study compared the susceptibility of clinical and environmental isolates of the main pathogenic *Scedosporium* species to reactive oxidative species and no differences were seen between isolates of the same species in their susceptibility to chemically induced oxidative stresses or even a lower susceptibility of the environmental isolates [43]. Obviously, replication of this work after incubation of different immune cells from pwCF with resting or germinating conidia, and hyphae from clinical strains would allow a more comprehensive perspective of the immune response against *Scedosporium* species.

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In conclusion, our study showed that M1-like macrophages can rapidly initiate a host defense response against both *S. apiospermum* and *S. dehoogii*. This response is characterized by a similar killing efficiency of internalized conidia and a tendency to a species-specific cytokine production. Our data pave the way for future research aiming at assessing the detailed
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mechanisms of the macrophage inflammatory response against these underestimated
emerging pathogens.

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Declaration of interest

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The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

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Acknowledgment

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We thank the members of the PACeM (Platforme d'Analyse Cellulaire et Moléculaire) of the
University of Angers, Nathalie Clément, Charlotte Godon and Julia Razafimandimby for the
technical assistance. This work was supported by a grant from SFR ICAT (University of
Angers, Angers, France) and by institutional grants from Inserm and the University of
Angers. The funders had no role in the design and conduct of the study, nor the decision to
prepare and submit the manuscript for publication. This work was realized in the context of
the Laboratory of excellence Immunotherapy Graft-Oncology (LabEx IGO) program
supported by the National Research Agency (ANR) via the investments for the future
program ANR-11-LABEX-0016-01 and by the University of Angers - University Hospital of
Angers joint program (3I-Impact).

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Credit authorship contribution statement

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**Dorra Elhaj Mahmoud:** Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review & editing. **Meriem Hanachi:** Formal analysis, Writing original draft. **Hajar Yaakoub:** Formal analysis, Writing-review & editing.

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**Simon Blanchard:** Formal analysis, Writing-original draft. **Pascale Pignon:** Formal analysis, Writing-review & editing. **Oussama Souiri:** Formal analysis. **Yves Delneste:** Funding acquisition, Conceptualization, Writing-review & editing. **Jean-Philippe Bouchara:** Conceptualization, Writing-original draft, Writing-review & editing. **Nicolas Papon:**

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Supervision, Writing-original draft, Writing-review & editing. **Anaïs Hérivaux:** Funding acquisition, Conceptualization, Project administration, Supervision, Validation, Writing-original draft, Writing-review & editing.

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449
450 References

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591 **Figure legends**

592 **Figure 1. Principal component analysis (PCA) of the M1-like macrophage response** 593 during *S. apiospermum* and *S. dehoogii* co-culture. The PCA was performed on scaled gene 594 expression counts (log<sub>2</sub>(FC)) obtained from qPCR. (A) Individual plot: samples are depicted 595 as dots, the largest of which represents the group mean point. The dots are color coded 596 according to each experimental time point encountered in a given species. (B) Variables plot: 597 the principal components PC1 (or Dim1) and PC2 (or Dim2) of the most significant variables 598 are represented by vectors. The ellipses reflect a 95% confidence level.

599 **CCL**: (C-C motif) Ligand; 600 **CLEC**: C-Type Lectin Domain Containing 7A; 601 **CXCL**: (C-X-C motif) Ligand; 602 **HLA-DRA**: Major Histocompatibility Complex, Class II, DR Alpha; 603 **IFNG**: Interferon Gamma; 604 **IL**: Interleukin; 605 **IL1A**: Interleukin 1 Alpha; 606 **IL 1B**: Interleukin1 Beta; 607 **TLR**: Toll-like receptor; 608 **TNFA**: Tumor Necrosis Factor Alpha.

603 **Figure 2. Analysis of chemokine, chemokine and immune receptor mRNA expression by** 604 M1-like macrophages after *Scedosporium* co-culture. The expression heatmap was 605 generated using the log<sub>2</sub> (FC) of 19 genes encoding cytokines, chemokines and immune 606 receptors and boxed based on distinct expression profiles. Genes were ordered using full 607 hierarchical clustering. The color scale used for log<sub>2</sub> (FC) is green to red, where green 608 represents 1 and red represents - 1. (A and C) Heatmaps of 19 genes expressed by M1-like 609 macrophages after *S. apiospermum* and *S. dehoogii* conidia challenge for 3 h and 24 h, 610 respectively. (B and D) Venn diagrams illustrating common and specific differentially 611 expressed genes between M1-like macrophages infected with *S. apiospermum* and M1-like 612 macrophages infected with *S. dehoogii* for 3 h and 24 h respectively. Upregulated genes: 613 log<sub>2</sub>(FC) ≥ 1 and a *p*-value < 0.05 (treated M1-like macrophages vs. untreated cells) are 614 shown in green. Downregulated genes: log<sub>2</sub>(FC) ≤ -1 and a *p*-value < 0.05 (treated M1-like 615 macrophages vs. untreated cells) are shown in red. Significance levels were calculated using 616 the Mann-Whitney U test (n=4-5 independent experiments).

617 **Figure 3. Comparative analysis of M1-like macrophage gene expression after** 618 *Scedosporium apiospermum* and *Scedosporium dehoogii* co-culture. (A and B) Boxplots 619 comparing log₂(FC) levels of the common up or downregulated genes after M1-like 620 macrophage challenge with *S. apiospermum* and *S. dehoogii* for 3 h (8 genes) and 24 h (14
genes). The boxes extend from the 25th to the 75th percentile, and the line in the middle is the median. The statistical significance of the expression differences between the two species was determined using the Mann-Whitney U test. (n=4-5 independent experiences). Levels of significance were allocated as follow: ns, not significant, * p-value < 0.05, ** p-value <0.01.

**Figure 4. Analysis of cytokine and chemokine release by M1-like macrophages after *Scedosporium* co-culture.** (A and C) Boxplots showing cytokine concentrations in untreated macrophages (CTRL), M1-like macrophages infected with *S. apiospermum* conidia and M1-like macrophages infected with *S. dehoogii* conidia for 3 h and 24 h, respectively. The boxes extend from the 25th to the 75th percentile, and the line in the middle is the median. (B and D) Plot of relative cytokine abundance. The cytokine level in M1-like macrophages infected with *S. apiospermum* for 3 h and 24 h, respectively (1) is represented by the vertical red line. Bar graphs represent the mean of cytokine levels detected after *S. dehoogii* co-culture (3 h and 24 h) relative to the mean of cytokine levels released after *S. apiospermum* co-culture (3 h and 24 h). Significance levels were assigned as follows: * p < 0.05 and ** p< 0.01 by Kruskall-Wallis test. (n=4-5 independent experiments).

**Figure 5. Protein-protein interaction (PPI) networks** of cytokines and chemokines up-regulated (in expression and or in release) by *S. dehoogii* after 3 h of co-culture. The PPI network was constrained to a 90% confidence level as calculated by the StringDB system.

**Figure 6. Internalization and killing of *Scedosporium* conidia by M1-like macrophages.**
(A and B) Interaction between *Scedosporium* conidia and M1-like macrophages after 2 h of co-culture. (C) Killing percentage was defined as the number of macrophage-killed conidia defined by the total number of macrophage-released conidia × 100; killing percentage data were collected from four independent experiments. Levels of significance were allocated using the Mann-Whitney U test.
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<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>
A

Cytokine concentration (pg/mL)

<figure><img src="image_37.png" /></figure>

3h

B

<figure><img src="image_38.png" /></figure>

C

Cytokine concentration (pg/mL)

<figure><img src="image_39.png" /></figure>

24h

D

<figure><img src="image_40.png" /></figure>

S. apiospermum
<figure><img src="image_41.png" /></figure>

PPI Legend

Known interactions

from curated databases

experimentally determined

Predicted interactions

gene neighborhood

gene fusions

gene co-occurrence

Others

textmining

co-expression

protein homology
A

*S. apiospermum*

<figure><img src="image_42.png" /></figure>

B

*S. dehoogii*

<figure><img src="image_43.png" /></figure>

C

<figure><img src="image_44.png" /></figure>
<table><caption>Table 1: Primers used in qPCR</caption><thead><tr><th>Gene</th><th>Forward primer</th><th>Reverse primer</th><th>Product size (bp)</th></tr></thead><tbody><tr><td>CCL2</td><td>CAGCCAGATGCAATCAATGCC</td><td>TGGAATCCTGAACCCACTTCT</td><td>190</td></tr><tr><td>CCL3</td><td>TTCCGTCACCTGCTCAGAATC</td><td>GTGATGCAGAGAACTGGTTGC</td><td>94</td></tr><tr><td>CCL5</td><td>CCTGCTGCTTTGCCTACATTGC</td><td>ACACACTTGGCGGTTCTTTCGG</td><td>125</td></tr><tr><td>CXCL1</td><td>CTGGCTTAGAACAAAGGGGCT</td><td>TAAAGGTAGCCCTTGTTTCCCC</td><td>112</td></tr><tr><td>CXCL2</td><td>TTCACAGTGTGTGGTCAACAT</td><td>TCTCTGCTCTAACACAGAGGGA</td><td>150</td></tr><tr><td>CXCL9</td><td>TGAGAAAGGGTCGCTGTTCC</td><td>GGGCTTGGGGCAAATTGTTT</td><td>91</td></tr><tr><td>Dectin1 (CLEC7A)</td><td>ACAATGCTGGCAACTGGGCTCT</td><td>AGAGCCATGGTACCTCAGTCTG</td><td>143</td></tr><tr><td>GAPDH</td><td>ACTTCAACAGCGACACCCACTC</td><td>TACCAGGAAATGAGCTTGACAAAG</td><td>88</td></tr><tr><td>HLA-DRA</td><td>CCGATCACCAATGTACCTCCA</td><td>CGAAGCCACGTGACATTGAC</td><td>128</td></tr><tr><td>IFNγ</td><td>ACTGTCGCCAGCAGCTAAAA</td><td>TATTGCAGGCAGGACAACCA</td><td>96</td></tr><tr><td>IL-1α</td><td>TGTATGTGACTGCCCAAGATGAAG</td><td>AGAGGAGGTTGGTCTCACTACC</td><td>96</td></tr><tr><td>IL-1β</td><td>AAATACCTGTGGCCTTGGGC</td><td>TTTGGGATCTACACTCTCCAGCT</td><td>101</td></tr><tr><td>IL-6</td><td>GTAGCCGCCCCACACAGA</td><td>CATGTCTCCTTTCTCAGGGCTG</td><td>101</td></tr><tr><td>IL-8</td><td>ATAAAGACATACTCCAAACCTTTCCAC</td><td>AAGCTTTACAATAATTTCTGTGTTGGC</td><td>102</td></tr><tr><td>IL-15</td><td>AACAGAAGCCAACTGGGTGAATG</td><td>CTCCAAGAGAAAGCACTTCATTGC</td><td>148</td></tr><tr><td>IL-18</td><td>TGCAGTCTACACAGCTTCGG</td><td>ACTGGTTCAGCAGCCATCTT</td><td>129</td></tr><tr><td>IL-23A</td><td>GAGCCTTCTCTGCTCCCTGATA</td><td>GACTGAGGCTTGGAATCTGCTG</td><td>121</td></tr><tr><td>TLR2</td><td>CTTCACTCAGGAGCAGCAAGCA</td><td>ACACCAGTGCTGTCCTGTGACA</td><td>146</td></tr><tr><td>TLR4</td><td>CCCTGAGGCATTTAGGCAGCTA</td><td>AGGTAGAGAGGTGGCTTAGGCT</td><td>126</td></tr><tr><td>TNFα</td><td>CACAGTGAAGTGCTGGCAAC</td><td>GATCAAAGCTGTAGGCCCCA</td><td>75</td></tr></tbody></table>

Abbreviations: bp: basepair; CCL: (C-C motif) Ligand; CLEC7A: C-Type Lectin Domain Containing 7A; CXCL: (C-X-C motif) Ligand;

Abbreviations: bp: basepair; CCL: (C-C motif) Ligand; CLEC: C-Type Lectin Domain Containing 7A; CXCL: (C-X-C motif) Ligand;

GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; HLA-DRA: Major Histocompatibility Complex, Class II, DR Alpha; IFNγ:

Interferon Gamma; IL: Interleukin; TLR: Toll-like receptor; TNFα: Tumor Necrosis Factor Alpha.