(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Single-cell transcriptomics unveils skin cell specific antifungal immune responses and IL-1Ra- IL-1R immune evasion strategies of emerging fungal pathogen Candida auris [1]

['Abishek Balakumar', 'Department Of Comparative Pathobiology', 'College Of Veterinary Medicine', 'Purdue University', 'West Lafayette', 'Indiana', 'United States Of America', 'Diprasom Das', 'Abhishek Datta', 'Abtar Mishra']

Date: 2024-12

Candida auris is an emerging multidrug-resistant fungal pathogen that preferentially colonizes and persists in skin tissue, yet the host immune factors that regulate the skin colonization of C. auris in vivo are unknown. In this study, we employed unbiased single-cell transcriptomics of murine skin infected with C. auris to understand the cell type-specific immune response to C. auris. C. auris skin infection results in the accumulation of immune cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells, T cells, and NK cells at the site of infection. We identified fibroblasts as a major non-immune cell accumulated in the C. auris infected skin tissue. The comprehensive single-cell profiling revealed the transcriptomic signatures in cytokines, chemokines, host receptors (TLRs, C-type lectin receptors, NOD receptors), antimicrobial peptides, and immune signaling pathways in individual immune and non-immune cells during C. auris skin infection. Our analysis revealed that C. auris infection upregulates the expression of the IL-1RN gene (encoding IL-1R antagonist protein) in different cell types. We found IL-1Ra produced by macrophages during C. auris skin infection decreases the killing activity of neutrophils. Furthermore, C. auris uses a unique cell wall mannan outer layer to evade IL-1R-signaling mediated host defense. Collectively, our single-cell RNA seq profiling identified the transcriptomic signatures in immune and non-immune cells during C. auris skin infection. Our results demonstrate the IL-1Ra and IL-1R-mediated immune evasion mechanisms employed by C. auris to persist in the skin. These results enhance our understanding of host defense and immune evasion mechanisms during C. auris skin infection and identify potential targets for novel antifungal therapeutics.

C. auris, an emerging fungal pathogen, preferentially colonizes human skin and causes outbreaks of systemic infections. However, the factors that regulate the pathogenesis of C. auris are still unclear. Using unbiased scRNA-seq profiling of murine skin infected with C. auris, we identified the host immune factors that are potentially involved in the regulation of C. auris in skin. Furthermore, our in vivo scRNA-seq reveals that C. auris infection upregulates IL-1Ra in the myeloid cells and limits the neutrophil antifungal activity. In addition, our results suggest that C. auris mannan layer can evade IL-1R signaling mediated skin defense. Collectively, our findings identified the immune and non-immune cells involved in the regulation of C. auris in the skin that could open the door to gain insights into the pathogenesis of this emerging fungal pathogen.

Data Availability: After our request authors have provided the following update: "The Seurat object file with annotated single-cell RNA-seq data after cell type assignments is made available through figshare: https://figshare.com/articles/dataset/Seurat_sct_slot_with_annotation_RDS/27320781?file=50047740 . DOI: 10.6084/m9.figshare.27320781 . The differentially expressed genes from pseudo-bulk analysis are provided as S10 Table .xlsx. All other data is provided in the manuscript and Supporting Information files.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Collectively, this study, for the first time, identified the previously unknown immune and non-immune cell type-specific skin responses and molecular events of skin-C. auris interactions in vivo at single-cell resolution. Furthermore, we demonstrated the IL-1Ra and IL-1R-mediated immune evasion mechanisms employed by C. auris to persist in the skin. This knowledge will be instrumental in understanding the host-pathogen interactions of C. auris. and will form a strong platform for developing novel host and pathogen-directed antifungal therapeutic approaches that potentially target IL-1Ra and fungal mannan, respectively.

To comprehensively define the transcriptome profiling of mouse skin infected with C. auris in vivo at single-cell resolution, we employed unbiased single-cell RNA sequencing (scRNA-Seq) profiling in skin tissues collected from uninfected and C. auris-infected mice. Our scRNA-Seq analysis identified immune cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells, T cells, NK cells, and non-immune cells such as fibroblasts accumulated at the site of infection. The scRNA-Seq revealed how skin reprograms genes and signaling pathways in immune and non-immune cell types following C. auris infection. The comprehensive transcriptomic profiling identified the transcriptional changes in genes that encode cytokines, chemokines, host receptors (TLRs, C-type lectin receptors, NOD receptors), antimicrobial peptides, and signaling pathways upregulated in individual myeloid cells, T cells, NK cells, fibroblast, and other non-immune cells. We identified the upregulation of the IL-1RN gene (encoding IL-1R antagonist protein) in different cell types during C. auris skin infection. Subsequently, using mouse models of C. auris skin infection and immune cell depletion studies, we elucidated the role of IL-1Ra in C. auris skin infection. We observed that the IL-1Ra level was significantly increased in the C. auris-infected skin tissue compared with C. albicans. C. auris infection induces IL-1Ra in macrophages and decreases the killing activity of neutrophils. Furthermore, C. auris evades IL-1R-mediated host defense through a unique outer mannan layer to persist the skin tissue.

The fungal cell wall components represent the predominant pathogen-associated molecular patterns (PAMPs) directly interacting with the host to orchestrate the antifungal immune response [ 12 ]. Recent evidence indicates that the cell wall of C. auris is structurally and biologically unique compared to other Candida species, including C. albicans [ 13 ]. The outer cell wall mannan layer in C. auris is highly enriched in β-1,2-linkages and contains two unique Mα1-phosphate side chains not found in other Candida species [ 13 ]. C. auris differentially stimulates cytokine production in peripheral blood mononuclear cells and has a more potent binding to IgG than C. albicans [ 14 , 15 ]. Given that C. auris possesses a unique outer cell wall layer and preferentially colonizes and persists in skin tissue long-term, understanding the skin immune responses during the dynamic pathogen infection in vivo is very important but has not been explored so far. Furthermore, classical population-based gene expression studies using in-vitro differentiated immune cells and mouse models of systemic infection do not completely represent the host defense mechanisms against skin infection in vivo. In addition, understanding the skin immune responses against C. auris is critical, as the murine skin model is widely used to study disease pathogenesis and C. auris-host interactions [ 4 , 16 – 20 ]. A closer look into cell type-specific host responses requires single-cell resolution to encompass all cell types, including immune and non-immune cells involved in host defense against C. auris skin infection in vivo.

Given that the majority of C. auris isolates exhibit resistance to several FDA-approved antifungal drugs, a deeper understanding of C. auris-host interactions is critical to understanding the pathogenesis and developing potential new host-directed therapeutic approaches to prevent and treat this newly emerging skin tropic fungal pathogen. Recent evidence from our laboratory indicates that C. auris skin infection leads to fungal dissemination, suggesting skin infection is a source of invasive fungal infection [ 9 ]. Because skin infection is a prerequisite for C. auris transmission and subsequent invasive disease, understanding the immune factors involved in skin defense against C. auris is important to understand the pathogenesis of this skin tropic fungal pathogen. Though antifungal host defense mechanisms against oral, gut, vaginal, and systemic infections of C. albicans are well known [ 10 , 11 ], to date, almost nothing is known regarding the skin immune responses against C. auris.

C. auris was recently categorized as an urgent threat by the US Centers for Disease Control and Prevention (CDC) and classified in the critical priority fungal pathogens group by the World Health Organization (WHO) [ 1 – 3 ]. Unlike other Candida species, such as Candida albicans, which colonizes the gastrointestinal tract, C. auris preferentially colonizes the human skin, leading to nosocomial transmission and outbreaks of systemic fungal infections [ 4 – 6 ]. Furthermore, unlike skin-tropic fungal pathogens such as Malassezia [ 7 ], C. auris not only colonizes the epidermis of the skin but also enters the deeper dermis, a phenomenon that was not observed previously [ 4 ]. C. auris can persist in skin tissues for several months and evade routine clinical surveillance [ 4 , 8 ].

Results

Unbiased scRNA seq profiling identified phagocytic cells, dendritic cells, T cells, NK cells, and fibroblast accumulated at the site of C. auris skin infection in vivo To identify the immune and non-immune cells involved in host defense against C. auris skin infection, we performed unbiased scRNA seq profiling from skin tissues collected from uninfected and C. auris-infected mice. A group of mice was infected intradermally with 1–2 × 106 CFU of C. auris, and another group injected with 100 μl PBS was used as an uninfected control group (Fig 1A). To capture the transcriptome profile of both innate and adaptive immune responses during C. auris skin infection in vivo, we have chosen 12 days after infection for our analysis. After 12 days post-infection (DPI), skin tissues from uninfected and infected groups (3 mice per group) were collected, minced, and digested to make single-cell suspensions as described. The single-cell suspension from infected and uninfected groups was subjected to single-cell partitioning, and RNA was sequenced using the droplet-based 10X Genomics Chromium platform (Fig 1A). After quality filtering by removing noise and batch effects, our data comprised 70,350 cells (S1 Table). The ambient RNA from the data was eliminated, and the expression level of individual genes was quantified based on the number of UMIs (unique molecular indices) detected in each cell (S2 Table). The alignment of the sequencing reads to the mouse reference genome resulted in the overall coverage of 32,285 genes. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Unbiased scRNA seq profiling identified phagocytic cells, dendritic cells, T cells, NK cells, and fibroblast accumulated at the site of C. auris skin infection in vivo. A) Schematic representation of the study groups, infection course, sample processing, and single-cell preparation for scRNA sequencing. This illustration was created using Biorender. B) Uniform Manifold Approximation and Projection (UMAP) of identified cell types in the murine skin after clustering. Each cluster was assigned as an individual cell type. After identification, 35 cell types were assigned as individual clusters. C) The dot plot represents the canonical gene marker signatures to classify cell types based on specific identities. A range of 2–7 marker gene expressions were assigned to identify a cell type in the cluster. D) The UMAP of uninfected and C. auris infected murine skin tissue with resident and recruited cell population depicting the cell type heterogenicity between the two groups. E) The composition of the individual cell types in the uninfected and C. auris infected murine skin. Cell type proportions were normalized from the total cells detected in each sample. https://doi.org/10.1371/journal.ppat.1012699.g001 After unsupervised graph-based clustering and reference-based annotation from the dataset GSE181720, 30 clusters were identified in our scRNA seq dataset, and cell-type identity was assigned to all the cell populations in the clusters (Fig 1B). To identify each cell lineage from 35 distinct cell types in our scRNA seq dataset, cell-type specific identity was assigned based on the canonical marker expression described elsewhere [21–23] (Fig 1C). After cell-type annotation, we identified 16 cell types as immune cells and the rest 19 as non-immune cells. The UMAP of C. auris infected samples indicated the recruitment of major immune cell types and resident cell populations (Fig 1D). Our analysis identified the percentage of immune and non-immune cells accumulated at the site of infection; (neutrophils– 99.9%, basophils– 80.5%, inflammatory monocytes– 93%, macrophages– 99.6%, dendritic cell 1–83.3%, dendritic cell 2–90.9%, helper T cell– 96.4%, cytotoxic T cells—81%, Tregs—84.9%, NK cells– 90.9%, B-cells—64.7%, proliferating T cells– 66.5%, resident macrophages– 58.9%, dermal dendritic cells– 51.8%, and γδ T cells—47.8%). Surprisingly, we identified fibroblast 3 cell types (87.9%) as major non-immune cells that showed increased accumulation in the skin tissue of infected groups (Fig 1D and 1E) (S1 Table). The recruitment of neutrophils, inflammatory monocytes, macrophages, dendritic cells, and T cell subsets following C. auris skin infection was validated by flow cytometry (S1 Fig). The proportion of cell types between the C. auris infected and uninfected groups varies drastically (Fig 1E) and displays cellular heterogeneity of the resident and recruited cell population. Collectively, our scRNA seq identified the accumulation of various innate and adaptive immune cells at the site of infection. In addition to phagocytic cells, DCs, and T cells, which are known to play a critical role in antifungal defense, our analysis identified an accumulation of NK cells and fibroblast during C. auris skin infection.

scRNA seq revealed the host immune transcriptomic signatures in individual myeloid cells during C. auris skin infection To identify the host immune genes regulated in individual myeloid cells identified during C. auris skin infection in vivo, we performed pseudo-bulk analysis by normalizing UMI counts for the target genes and identifying differentially expressed genes (DEGs) between the infected and uninfected groups. For the downstream analysis, we filtered DEGs with FDR < 5%, and the log2 fold change ≥ 2 was considered upregulated, and ≤ -2 was considered downregulated. In neutrophils, 1107 genes (1105 upregulated and 2 downregulated), inflammatory monocytes, 299 genes (217 upregulated and 82 downregulated), macrophages, 1340 genes (1322 upregulated and 18 downregulated), resident macrophages, 357 genes (143 upregulated and 214 downregulated), dendritic cell 1, 454 genes (260 upregulated and 194 downregulated), dendritic cell 2, 265 genes (244 upregulated and 21 downregulated) and dermal dendritic cells 262 (91 upregulated and 171 downregulates) were differentially expressed in the C. auris infected groups. The top 10 upregulated and downregulated genes in the myeloid subsets were highlighted in the volcano plot (Fig 2A). Among the top 10 DEGs in myeloid cells, chemokine genes such as Ccl4 and Cxcl3 in neutrophils, Cxcl9 and Ccl5 in inflammatory monocytes, and Cxcr2 in dermal dendritic cells were significantly upregulated in the C. auris infected group (Fig 2A). In macrophages, resident macrophages, and dermal dendritic cells, Nos2 and Arg1 involved in nitric oxide metabolism were upregulated genes after infection. Arg1 was also significantly upregulated in dermal dendritic cell 1 (Fig 2A). In addition, Inhba, slpi, and aw112020 genes known to shift the nitric oxide metabolism were upregulated in macrophages (Fig 2A). Furthermore, the gene encoding for serum amyloid A3 protein (Saa3) was significantly upregulated in all the phagocytic cells (S2A Fig) (S10 Table). We examined the genes that were upregulated in all phagocytic cells. Spp1, Egln3, Slpi, Upp1, Inhba, F10, Acod1, AA467197, Ppp1r3b, Nos2, Slc2a1, Tarm1, Cd300lf, and Ly6a were significantly upregulated in all the phagocytic cells during C. auris infection (S2A Fig) (S10 Table). We identified AA467197, Plac8, Arg1, Ccl17, Slpi, and AW112010 were significantly upregulated in dendritic cell 1, dendritic cell 2, and dermal dendritic cells (S2B Fig) (S10 Table). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. scRNA seq revealed the host immune transcriptomic signatures in individual myeloid cells during C. auris skin infection. A) Volcano plots indicating significant differentially expressed genes (DEGs) in the infected and uninfected neutrophils, monocytes, macrophages, resident macrophages, dendritic cell 1, dendritic cell 2 and dermal dendritic cells highlighting the top 10 upregulated and downregulated genes; The Dot plot represents the expression of selected B) chemokines and its receptors, C) cytokines and its receptors, D) fungal recognizing receptors and antimicrobial peptides (AMPs) in the myeloid subsets (Y-axis). The dot size represents the percentage of cells with expressions, and the color indicates the scaled average expression calculated from the 3 uninfected and 3 infected samples. E) The bubble plot represents the KEGG pathway of the enriched upregulated genes of the myeloid subsets (X-axis). DEGs from pseudo-bulk analysis with threshold Log 2-fold change ± 2 and FDR > 5% were considered as significant DEGs, and Log 2-fold change ≥ 2 and FDR > 5% as upregulated genes. https://doi.org/10.1371/journal.ppat.1012699.g002 Next, we compared the expression of chemokines, cytokines, pattern-recognizing receptors (PRRs), and antimicrobial peptides (AMPs) among different myeloid subsets [11, 24]. The gene list from the mouse genome database was used to explore the expression of the host immune genes [25]. Among the chemokines and chemokine receptors, Ccr1, Ccrl2, Cxcl2, Cxcl3, and Cxcr2 were highly expressed in neutrophils. Ccr1, Ccr2, Ccr5, and Cxcr4 were highly expressed in inflammatory monocytes, whereas Ccr1, Ccrl2, Ccr5, Ccl5, Ccl9, Cmklr1, Cxcl16 and Cxcr4 were highly expressed in the inflammatory macrophage and Ccl2, Ccl6, Ccl7, Ccl8, Ccl9, Ccl12, and Ccl24 were highly expressed in the resident macrophages. Dendritic cell 1 showed increased expression of Ccr5, Ccr9, and Ccl16, whereas Ccl5, ccl17, Ccl22, Ccr7, and Cxcl16 were highly expressed in dendritic cell 2. Ccl6, Ccl9, Ccr2, and Ccr5 were highly expressed in the dermal dendritic cells (Fig 2B). Among the cytokines and cytokine receptors, Il1a, Il1b, Il1r2, and Il1rn were highly expressed in neutrophils. Il1a, Il1b, and Il1rn were highly expressed in macrophages, whereas Il1b and Il1rn were highly expressed in monocytes and dendritic cells 1. We identified increased expression of Il10 in the resident macrophages, Tnf and Csf1 were increased in neutrophils, and Tgfbi expression was upregulated in monocytes and macrophages (Fig 2C). Next, we compared the expression of TLRs, C-type lectin receptors, NOD-like receptors (NLRs), and complement receptor 3 among myeloid subsets. Neutrophils showed an increased Tlr2, Tlr4, Tlr6, and Tlr13 expressions. Tlr2, Tlr4, Tlr7, Tlr13, and the adaptor molecules such as MyD88 and Traf6 were highly expressed in the inflammatory monocytes. Tlr2 was highly expressed in macrophages, whereas Tlr2 and Tlr7 were highly expressed in resident macrophages. Tlr7 and Tlr2 were highly expressed in dendritic cell 1 and dermal dendritic cells, respectively. Among the C-type lectin receptors, Clec4d (Dectin-2), Clec4n (Dectin-3), and Clec4e (Mincle) were highly expressed in neutrophils, monocytes, and macrophages. The Syk, an adopter protein involved in Dectin-1 signaling, is highly expressed in all myeloid cells. Card9, the other downstream signaling protein of Dectin-1, is selectively expressed in all the myeloid subsets except neutrophils. The Clec4a1, Clec4a2, and Clec4a3 were highly expressed in monocytes, macrophages, resident macrophages, and dermal dendritic cells, whereas the Clec4b1 was only expressed in the dermal dendritic cells. The mannose-binding receptor Mrc1 (Mannose receptor) was highly expressed in resident macrophages and dermal dendritic cells. The cd209a (DC-SIGN1), cd209d (SIGN-R3), cd209f (SIGN-R8), and cd209g encoding DC-SIGN were highly expressed in resident macrophages. The Lgals3 (Galectin-3) was highly expressed in monocytes and macrophages, followed by dermal dendritic cells and dendritic cell 1. We identified increased expression of intracellular PRR, Nod1, and Nod2 in the neutrophils and monocytes. The Itgam (complement receptor 3) was highly expressed in neutrophils, monocytes, macrophages, resident macrophages, and dermal dendritic cells. We identified increased expression of Nlrp3 in neutrophils. Among the AMPs, the Lcn2, S100a8, and S100a9 were highly expressed in neutrophils, and the Ang was selectively expressed in the resident macrophages (Fig 2D). To explore the enrichments of DEGs in the myeloid cells, KEGG pathway analysis was performed for significantly upregulated genes (5% FDR, Log2Fc ≥ 2) in neutrophil, monocytes, macrophages, resident macrophages, dendritic cell 1, dendritic cell 2 and dermal dendritic cells 2 to identify the upregulated pathways (Fig 2E). Among myeloid subsets, we identified several pathways involved in pathogen recognition and immune signaling that were highly enriched in neutrophils. The major innate pathways enriched in the neutrophils during C. auris skin infection: 1) phagocytosis (phagosome, endocytosis, efferocytosis, ubiquitin-mediated proteolysis, Fc gamma R-mediated phagocytosis, and neutrophil extracellular trap formation), 2) PRRs (Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and NOD-like receptor signaling pathway), 3) inflammasome activation (HIF-1 signaling pathway, TNF signaling pathway, and NF-kappa B signaling pathway), 4) cytokine and chemokine signaling (chemokine signaling, cytokine-cytokine receptor pathway, and JAK-STAT signaling pathway), 5) T-helper cell (Th) differentiation (Th1 and Th2 cell differentiation, Th17 cell differentiation and IL17 signaling pathway). In addition, we identified the enriched upregulated genes in these KEGG pathways (S3 Table). Collectively, our single-cell transcriptomics identified the AMPs, cytokines, chemokines, and fungal recognition receptors upregulated in myeloid subsets during C. auris skin infection. We identified the genes involved in nitric oxide metabolism and acute phase proteins, which were highly upregulated in different myeloid cells. Furthermore, our analysis revealed the enrichment of several pathways involved in fungal recognition, phagocytosis, cytokine and chemokine signaling, and T-cell differentiation in individual myeloid cells during C. auris skin infection in vivo.

C. auris skin infection induces IL-17 and IFNγ signaling pathways in lymphoid cells To identify the host immune genes and transcription factors (TFs) regulated in T cell subsets and NK cells during C. auris skin infection, we sub-clustered the lymphoid cells at 0.2 resolution to identify the subsets of CD4+ Th cells, CD8+, γδ T cells, Tregs, and NK cells (Fig 3A). Sub-clustering the lymphoid population identified 203 genes in CD4+ Th cells (118 upregulated and 85 downregulated), 41 genes in Tregs (34 upregulated and 7 downregulated), 18 genes in CD8+ cells (14 upregulated and 4 downregulated), 92 genes in γδ T cells (73 upregulated and 19 downregulated) and 16 genes in NK cells (9 upregulated and 7 downregulated) were differentially expressed, and the heatmap shows the significantly upregulated genes (log2 fold change ≥ + 2, FDR < 5% or 1%) in CD4+ Th cells, γδ T cells, Tregs, CD8+ cells, and NK cells (Figs 3D, S3B and S3C). We examined the genes upregulated in lymphoid subsets (S3A Fig). The Nfkbia was upregulated in CD4+ Th cells and CD8+ cells. Epsti1 and Enox2 were significantly upregulated in CD4+ Th cells and NK cells (S3A Fig) (S10 Table). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Single-cell profiling of lymphoid subsets. A) Uniform Manifold Approximation and Projection (UMAP) represents the sub-clustering analysis of lymphoid subsets at 0.2 resolution. The CD4+ Th cells, Tregs, CD8+ cells, γδ T cells, and NK cells were identified in the subpopulation. The Dot plot represents the expression of selected B) chemokines and their receptors, transcription factors (TFs), and cytolytic enzymes, and C) cytokines and their receptors in the lymphoid subsets (Y-axis). The dot size represents the percentage of cells with expressions, and the color indicates the scaled average expression calculated from the 3 uninfected and 3 infected samples. D) The heatmap represents the expression of the significantly upregulated genes in CD4+ Th cells, γδ T cells, and Tregs in uninfected and infected groups. The normalized gene counts were plotted in the heatmap, and the scale indicates red for high, blue for low, and white for moderate expression in the samples. Each column represents a different sample. E) The bubble plot represents the KEGG pathway enrichment analysis of significantly upregulated genes in the lymphoid subsets (X-axis). F) Violin plots depict the scaled count of Il17a, Il17f, and Ifng in CD4+ Th cells, Tregs, CD8+ cells, γδ T cells, and NK cells in both uninfected and infected samples. The scaled count were normalized using SCTransform method. DEGs from pseudo-bulk analysis with threshold Log 2-fold change ≥ 1 and FDR > 5% were considered as significant upregulated genes for KEGG pathways. Upregulated genes with Log 2-fold change ≥ 2 and FDR > 1% for CD4+ Th cells and FDR > 5% for Tregs, and γδ T cells were represented for the heatmap. https://doi.org/10.1371/journal.ppat.1012699.g003 Next, we compared the expression of chemokines, cytokines, and TFs among lymphoid subsets. Cxcr6 was highly expressed in CD4+ Th cells and γδ T cells. We identified increased expression of Xcl1, Ccl3, Ccl4, Ccl5, Ccr5, and Cxcr4 in NK cells, and Ccr2, Ccr3, and Ccr4 in the Tregs. TFs such as Stat4, Stat5a, Stat5b, Stat6, Rora, Rorc, and Ahr were highly expressed in the CD4+ Th cells. Tregs showed increased expression of Stat1, Gata3, Stat5a, Stat5b, Batf, Foxp3, Ikzf2, Irf4 and Ahr. We identified the increased Tbx21, Stat4, Stat6, and Stat3 expression in NK cells. Gzma, Gzmb, and prf1 in NK cells and Gzmb were highly expressed in the Tregs (Fig 3B). Il16, Csf1, Il1b, Il17a, Tnf, and Csf2 were highly expressed in the CD4+ Th cells. Tregs showed a higher expression of Il10. Il17a and Il17f were selectively expressed in γδ T cells. We identified increased expression of tgfb1 and ifng in the NK cells (Fig 3C). The upregulated pathways enriched in CD4+ Th cells, CD8+ cells, γδ T cells, Tregs, and NK cells were identified from KEGG pathway analysis of the upregulated genes with Log2Fc ≥ 1 and 5% FDR threshold (Fig 3E). We identified pathways involved in Th17 cell differentiation, cytokine-cytokine receptor interaction, Th1, and Th2 cell differentiation, and HIF-1 signaling pathway were highly enriched in CD4+ Th cells during C. auris skin infection (Fig 3E). Other pathways involved in TNF, JAK-STAT, NF-kappa B and HIF-1 signaling were enriched in CD4+ Th cells, Tregs and γδ T cells (Fig 3E). In addition, we examined the upregulated DEGs enriched in these KEGG pathways (S4 Table). Next, we used violin plots to identify the cell type level expression of Il17a, Il17f, and Ifng in CD4+ Th cells, CD8+ cells, γδ T cells, Tregs, and NK cells (Fig 3F). Our analysis revealed that Il17a and Il17f were mainly expressed in CD4+ Th cells and γδ T cells during C. auris skin infection. Our analysis identified that NK cells showed an increased expression of Ifng, followed by CD4+ Th cells, CD8+ cells, and γδ T cells (Fig 3F). IL-17 signaling pathway, which is known to play a critical role in antifungal defense, is upregulated in CD4+ Th cells and γδ T cells. Taken together, our scRNA analysis identified the expression of cytokines, chemokines, and TFs upregulated in T cells and NK cells.

scRNA seq revealed the transcriptomic signatures in fibroblast and other non-immune cells during C. auris skin infection Our unbiased scRNA profiling identified fibroblast as a major non-immune cell accumulated at the site of skin infection. To understand the transcriptomic signatures of fibroblast during C. auris skin infection, the gene expression profiling of fibroblast sub-clusters, fibroblast 1, fibroblast 2, fibroblast 3, and fibroblast 4, were explored. The DEGs from the pseudo-bulk analysis with a threshold of FDR < 5% and the log2 fold change ≥ 2 were used for further analysis. Log2 fold change of ≤ -2 were considered downregulated for the volcano plots. Among the DEGs in fibroblast subsets, 370 genes in fibroblast 1 (150 upregulated and 220 downregulated), 68 genes in fibroblast 2 (12 upregulated and 56 downregulated), 389 genes in fibroblast 3 (294 upregulated and 95 downregulated) and 9 genes in fibroblast 4 (1 upregulated and 8 downregulated) were regulated upon C. auris infection. The top 10 upregulated and downregulated genes in fibroblast subsets were denoted in the volcano plots (Fig 4A). The antimicrobial peptide S100a9 is the top upregulated genes in fibroblast 1, fibroblast 2 and fibroblast 4. The S100a8, which forms a complex with S100a9 as calprotectin, is among the top 10 upregulated genes in fibroblast 2. The serum amyloid protein Saa3 is the highest upregulated gene in fibroblast 3, and the Saa1 and Saa3 were highly upregulated in fibroblast 1 (Fig 4A). Further, the antimicrobial peptides NOS2 and Lcn2 are highly upregulated in fibroblast 1 and fibroblast 3 (Fig 4A) (S5A Fig). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. scRNA seq revealed the transcriptomic signatures in fibroblast and other non-immune cells during C. auris skin infection. A) The Volcano plots indicate significant differentially expressed genes (DEGs) in the infected and uninfected fibroblast subsets, highlighting the top 10 upregulated and downregulated genes. The Dot plot represents the expression of selected B) fibroblast lineage markers, C) cytokines and their receptors, and D) chemokines and their receptors, fungal recognizing receptors, and antimicrobial peptides (AMPs) in the fibroblast subsets (Y-axis). The dot size represents the percentage of cells with expressions, and the color indicates the scaled average expression calculated from the 3 uninfected and 3 infected samples. E) The bubble plot represents the KEGG pathway enrichment of the upregulated genes of fibroblast subsets (X-axis).; Violin plots depict the scaled count of F) IL17ra and G) Cxcl1, Cxcl2, Cxcl5, Cxcl12, Lcn2, and IL33 in fibroblast subsets and other non-immune cells in both uninfected and infected samples. The scaled count were normalized using SCTransform method; DEGs from pseudo-bulk analysis with threshold Log 2-fold change ± 2 and FDR > 5% were considered as significant DEGs and Log 2-fold change ≥ 2 and FDR > 5% as upregulated genes. https://doi.org/10.1371/journal.ppat.1012699.g004 We classified the fibroblast subsets based on their lineage-specific marker expression. Fibroblasts 1 and 3 highly expressed most adipocyte lineage markers, and fibroblast 4 substantially expressed papillary lineage markers (Fig 4B). Next, we compared the expression of chemokines, cytokines, fungal-recognizing receptors, and AMP among four fibroblast subsets (Fig 4C and 4D). Il33, Csf1, and Tgfb2 were highly expressed in fibroblast 3 and fibroblast 1, whereas Il17d was selectively expressed in fibroblast 2 and fibroblast 4. Among the cytokine receptors, Il1r1, Il1rl2, Il6st, Il11ra1, Il17ra, Tgfbr2, and Tgfbr3 were highly expressed in fibroblast 3 and fibroblast 1, whereas Il11ra1 and Tgfbr3 were highly expressed in fibroblast 2 (Fig 4C). Among the chemokines, Ccl2, Ccl7, Ccl11, Cxcl1, and Cxcl12 were highly expressed in fibroblast 1 and fibroblast 3. Cxcl5, Cxcl9, Cxcl10, and Cxcl14 were highly expressed in fibroblast 3. Among the fungal recognizing receptors, Clec3b, Mrc2, Lgals1, Lgals9, Lman1, and Clec11a were highly in fibroblast 3 and fibroblast 1 (Fig 4D). We identified increased expressions of Clec11a, Mrc2, Lgals1, and Lman1 in fibroblast 2 and selective expression of Nod2 in fibroblast 3. Among the AMPs, fibroblast 3 showed increased expression of Lcn2, Nos2, and Amd, whereas Amd and Ang were highly expressed in fibroblast 1 (Fig 4D). To identify the upregulated pathways in fibroblast subsets during C. auris skin infection, KEGG pathway enrichment was performed for upregulated genes with Log2Fc ≥ 2 and 5% FDR in fibroblast 1, fibroblast 2, fibroblast 3, and fibroblast 4 (Fig 4E). We identified pathways involved in HIF-1 signaling, PI3K-Akt signaling, cytokine-chemokine receptor interaction, and complement coagulation cascade pathways that were highly enriched in fibroblast 3 and fibroblast 1. ECM-receptor interaction pathway was enriched in fibroblast 3. The IL-17 signaling pathway was enriched in all three fibroblast subtypes except fibroblast 4 (Fig 4E). Next, we analyzed other non-immune cells, such as BEC, outer bulge cells, and pericytes, that showed DEGs during C. auris skin infection (S4 Fig). We examined the upregulated DEGs enriched in these KEGG pathways (S5 Table). KEGG pathways, such as Th17 cell differentiation and cytokine-cytokine receptor interactions, were highly enriched in BEC, pericytes, and outer bulge cells (S5B–S5D Fig). To understand if fibroblast and other non-immune skin cells play a role in the recruitment of immune cells during C. auris infection, we analyzed the expression of genes such as IL-17ra and chemokines involved in recruiting immune cells such as neutrophils. Among non-immune cells, fibroblast subsets and LEC showed increased expression of the IL-17ra gene (Fig 4E). The chemoattractants such as Cxcl1, Cxcl2, Cxcl5, Cxcl12, Lcn2, and Il33 involved in the recruitment of neutrophils were highly expressed in different non-immune cells, but fibroblast subsets showed relatively higher expression (Fig 4G). Taken together, our unbiased scRNA seq revealed the increased expression of cytokines, chemokines, PRRs, and AMPs in fibroblasts and other non-immune cells that could either directly (or) indirectly contribute to the host defense against C. auris skin infection.

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012699

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.