Graphical abstract

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   Keywords: Host immune response, RNAseq, Transcriptomics of pathogenic
   fungi, Opportunistic infections, C. albicans, A. fumigatus, R. oryzae,
   Cytokines, Antifungal core host response, Pattern recognition
   receptors, Immunometabolism

Abstract

   Candidiasis, aspergillosis, and mucormycosis cause the majority of
   nosocomial fungal infections in immunocompromised patients. Using an
   unbiased transcriptional profiling in PBMCs exposed to the fungal
   species causing these infections, we found a core host response in
   healthy individuals that may govern effective fungal clearance: it
   consists of 156 transcripts, involving canonical and non-canonical
   immune pathways.

   Systematic investigation of key steps in antifungal host defense
   revealed fungal-specific signatures. As previously demonstrated,
   Candida albicans induced type I and Type II interferon-related
   pathways. In contrast, central pattern recognition receptor, reactive
   oxygen species production, and host glycolytic pathways were
   down-regulated in response to Rhizopus oryzae, which was associated
   with an ER-stress response. TLR5 was identified to be uniquely
   regulated by Aspergillus fumigatus and to control cytokine release in
   response to this fungus.

   In conclusion, our data reveals the transcriptional profiles induced by
   C. albicans, A. fumigatus, and R. oryzae, and describes both the common
   and specific antifungal host responses that could be exploited for
   novel therapeutic strategies.

1. Introduction

   Opportunistic fungal infections are worldwide causes of significant
   morbidity and mortality that equals either bacterial, viral, or
   parasitic infections [52][19], [53][108]. Among fungal diseases,
   aspergillosis, candidiasis, and mucormycosis account for the majority
   of opportunistic infections in immunocompromised patients, with the
   notable exception of HIV infected patients who primarily suffer from
   cryptococcosis in addition to oral candidiasis. Infections caused by
   the three major species Aspergillus fumigatus, Candida albicans, and
   Rhizopus oryzae, significantly increased in incidence over the past
   years from approximately 610.000 to 960.000 life-threatening invasive
   infections annually, with mortality rates ranging between 30 and 95%
   collectively [54][14], [55][19]. The number of immunocompromised
   patients is steadily increasing [56][19], [57][35] due to evolving
   medical care with more invasive procedures, increased use of
   immunosuppressive drugs [58][26], [59][75], and growing numbers of ICU
   admissions. These factors mutually contribute to the increasing
   incidence of aspergillosis, candidiasis, and mucormycosis.

   Although C. albicans is a widespread commensal in the human gut
   [60][46], [61][52], it can cause invasive candidiasis when the host
   immune system is impaired [62][55], [63][78], [64][106], [65][111] and
   the microbiome is disrupted, for example, by the use of broad-spectrum
   antibiotics [66][11], [67][117]. In contrast to C. albicans,
   Aspergillus species are saprotrophic fungi, which usually do not
   colonize the human host. However, all humans are exposed to the conidia
   of these environmental fungi on a daily basis, which can result in
   severe infections in immunocompromised hosts [68][57], [69][86],
   [70][107]. Compared to C. albicans and A. fumigatus, fungi of the genus
   Mucorales cause invasive disease in a more heterogeneous group of
   immunocompromised individuals, including diabetes and trauma patients
   [71][86]. Mucormycosis is considered one of the most aggressive forms
   of opportunistic fungal infection [72][19], [73][72] and Rhizopus
   oryzae is the most commonly isolated species [74][85].

   The outcome of opportunistic invasive fungal infections remains poor,
   and therapeutic approaches targeting the immune system have been
   recently recognized as a potentially lifesaving strategy for patients
   with invasive fungal infections [75][6], [76][103]. To improve
   antifungal immunotherapy, a global insight into the protective
   antifungal host response is needed. A common denominator of these three
   phylogenetically distinct fungal species is that they can all cause
   infections in immunocompromised patients with similar predisposing
   factors, including neutropenia, myeloablative and immunosuppressive
   therapy [77][15], [78][38], [79][51], [80][60], [81][64].

   We hypothesized that there is a core host response that governs
   resistance to these three opportunistic pathogens, which is debilitated
   in immunocompromised patients. Nevertheless, there are also intrinsic
   differences in the pathogenesis of candidiasis, aspergillosis, and
   mucormycosis among these patients. Species-specific host defense
   pathways in terms of pathogen recognition, cytokine release, and
   metabolism may be activated depending on the particular fungal species
   encountered by the immune system. The knowledge of these mechanisms
   will provide valuable insights for the selection of immunotherapies
   adapted against each species. To characterize the core host response
   and determine species-specific host responses against A. fumigatus, C.
   albicans, and R. oryzae, an unbiased transcriptional profiling approach
   was used to establish differentially expressed RNA transcripts in human
   peripheral blood mononuclear cells (PBMCs) in response to these 3
   fungal species. Subsequently, the most relevant pathways core and
   species-specific pathways have been functionally validated.

2. Results

2.1. Transcriptional profiling of host response to A. fumigatus, C. albicans,
and R. oryzae

   The genome-wide transcriptional response of human PBMCs in response to
   A. fumigatus, C. albicans, and R. oryzae was assessed using RNA
   sequencing (RNA-Seq). Unstimulated PBMCs served as reference, and the
   transcriptional profile was assessed after 4 h (4 h) and 24 h (24 h) of
   stimulation with inactivated yeast and conidia of the three fungal
   species. Transcripts that exhibited a Log[2]fold change >1 or <−1
   compared to unstimulated PBMCs and an adjusted p-value of < 0.05 were
   considered differentially regulated. A. fumigatus differentially
   regulated 472 transcripts (290 up and 182 down) at 4 h, and 506
   transcripts (467 up and 39 down) at 24 h ([82]Fig. 1A, B). C. albicans
   differentially regulated 1055 transcripts (821 up and 234 down) at 4 h,
   and 2448 transcripts (1733 up and 715 down) at 24 h ([83]Fig. 1A, B).
   R. oryzae differentially regulated a staggering amount of 10,104
   transcripts (6043 up and 4061 down) at 4 h, and 9646 transcripts (4974
   up and 4672 down) at 24 h ([84]Fig. 1A, B). Unsupervised hierarchical
   clustering revealed that the transcriptional response of PBMCs from
   various donors show stimulus dependent clustering at both the 4 h and
   24 h time points ([85]Fig. 1C, D). Of note, the response induced by R.
   oryzae of a single donor at 24 h was observed to form a separate
   cluster ([86]Fig. 1D). Following the observation that different
   expression profiles were induced by the three fungi in the unsupervised
   hierarchical cluster analysis, the inter-fungal heterogeneity in the
   response of PBMCs was investigated. Principle component analysis (PCA)
   revealed that the majority of the variance in transcription was
   stimulus-dependent, as demonstrated by the three distinct clusters
   corresponding to the three different fungal species. Of note, the
   response to A. fumigatus stimulation clustered closely to the
   unstimulated cells and the response to R. oryzae showed high
   inter-individual variation ([87]Fig. 1E).

Fig. 1.

   [88]Fig. 1
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   Transcriptional response to A. fumigatus, C. albicans, and R. oryzae
   (A-B) Volcano plots of differentially regulated genes assessed by
   RNA-Seq in human PBMCs from healthy volunteers in response stimulation
   with A. fumigatus (n = 8 donors), C. albicans (n = 8 donors), or R.
   oryzae (n = 6 and 5 donors for 4 and 24 h respectively) for 4 h (A) or
   24 h (B). The plots represent Log[2] fold change and -Log[10] of the
   corrected p-value compared to the unstimulated control. Transcripts
   with a corrected p-value < 0.05 and a mean Log[2]FC > 1 of < −1 are
   plotted in red and were considered as differentially regulated. (D-E)
   Heatmap showing unsupervised clustering analysis of the transcriptional
   responses of PBMCs to the three fungal species. Distances were defined
   as 1 - Pearson correlation and average-linkage clustering was used.
   Numbers 1–8 on the branches of the dendrogram represent numbers of the
   respective PBMC donors after 4 h (C) and 24 h (D) of stimulation. After
   QC 2 donors stimulated for 4 h and three donors stimulated for 24 h
   with R. oryzae needed to be excluded. (E) Principal component analysis
   of the transcriptional response signatures. Data from each individual
   sample is plotted along the two main principal components (PC1, PC2).
   The two principal components account for 24–34% of the total variance
   in gene expression (PC1 34%; PC2 24%). Samples from the various
   stimulations are color coded as follows: C. albicans 4 h (light beige)
   and 24 h (golden), A. fumigatus 4 h (teal blue) and 24 h (blue grey),
   R. oryzae 4 h (dark beige) and 24 h (brown), and unstimulated 4 h
   (Purple) and 24 h (light red). (For interpretation of the references to