Data from: Analyses Xenopus laevis mast cells, neutrophils, and mast cell-enriched, chytrid infected skin
Data files
Jul 30, 2024 version files 2.82 MB
Abstract
Global amphibian declines are compounded by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). Much has been learned about the roles of amphibian skin-produced antimicrobial components and microbiomes in controlling Bd, yet almost nothing is known about the roles of skin-resident immune cells in anti-Bd defenses. Mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Accordingly, we investigated the roles of Xenopus laevis frog mast cells during Bd infections. Our findings indicate that enrichment of X. laevis skin mast cells confers anti-Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. This includes a significant reduction in infiltration of Bd-infected skin by neutrophils, promoting mucin content within cutaneous mucus glands, and preventing Bd-mediated changes to skin microbiomes. Mammalian mast cells are known for their production of the pleiotropic interleukin-4 (IL4) cytokine and our findings suggest that the X. laevis IL4 plays a key role in conferring the effects seen following cutaneous mast cell enrichment. Together, this work underscores the importance of amphibian skin-resident immune cells in anti-Bd defenses and illuminates a novel avenue for investigating amphibian host-chytrid pathogen interactions.
Frog bone marrow-derived mast cells and neutrophils were generated and transcriptionally compared. Bone marrow isolation, culture conditions, and establishment of neutrophil cultures have been previously described68. Briefly, adult X. laevis (approximately one year old) were euthanized in 5% tricaine mesylate followed by cervical dislocation. Femurs were removed and washed in ice-cold Amphibian-PBS (A-PBS) in sterile conditions. Each femur was flushed with 5 mL of A-PBS. Red blood cells were removed from culture via a differential gradient generated with 51% Percoll. Bone marrow cell counts were generated using trypan blue exclusion and cells were seeded at a density of 104cells/well for gene expression experiments, 5x104 cells/well for histology analyses, and 105 cells/well for electron microscopy analyses.
Mast cell cultures were generated according to protocols adapted from Koubourli et al. (2018) and Meurer et al. (2016)69,70. Isolated bone marrow cells were treated with 250 ng/µl of rSCF on Day 0, Day 4, Day 7, and collected for further analysis on Day 9. Cell cultures were maintained at 27°C with 5% CO2 in amphibian medium supplemented with 10% fetal bovine serum and 0.25% X. laevis serum. Neutrophil-like granulocytes were generated as above but with 250 ng/µl of rCSF3 on Day 0, Day 3, and collected for further analysis on Day 5. Cell cultures were maintained at 27°C with 5% CO2 in amphibian serum-free medium supplemented with 10% fetal bovine serum, 0.25% X. laevis serum, 10 μg/mL gentamicin (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Thermo Fisher Scientific).
X. laevis were subcutaneously injected between the skin and muscle layers with 5 µg/animal of rSCF or r-ctrl in 5 µL of saline using finely pulled glass needles. For *in vivo *infection studies, zoospores were harvested by flooding confluent tryptone agar plates with 2 mL sterile A-PBS for 10 minutes. Twelve hours post rSCF, or r-ctrl injection, animals were infected with 10E7zoospores or mock-infected in 100 mL of water (105 zoospores/mL). After 3 hrs, 400 mL of water was added to each tank. Skins were collected forgene expression analyses after 21 dpi.
For transcriptomic profiling, bone marrow-derived neutrophil and mast cell cultures were generated as described above and FACS-sorted according to preestablished size and internal complexity parameters to isolate the respective subsets for further analyses. Sorted cells were immediately processed to extract and purify RNA. Flash frozen samples were sent to Azenta Life Sciences for all library preparation, RNA sequencing, and analyses. In short, polyadenylated RNA was isolated using Oligo dT beads. Enriched mRNAs were then fragmented for first and second strand cDNA synthesis. cDNA fragments were end repaired, 5’ phosphorylated, and dA-tailed. Fragments were then ligated to universal adaptors and PCR-amplified. 150-bp paired-end sequencing was performed on an Illumina HiSeq platform.
FastQC was used to evaluate raw data quality. Adaptors sequences and poor-quality nucleotides were removed from reads using Trimmomatic v.0.36. The STAR aligner v.2.55.2b was used to map these reads to the Xenopus_laevis_9_2 reference genome from ENSEMBL. To determine differential gene expression, featureCount (Subread package v.1.5.2) was first used to count unique gene hits, which were then used with DESeq2 to calculate absolute log2-fold change.
Comparison of Xenopus laevis control (V) and mast cell-enriched (S) skin: frogs were injected subcutaneously with a recombinant (vector) control (V) or recombinant stem cell factor, infected with Batrachochytrium dendrobatidis and skin transcriptomics examined 21 days post infection.
In vitro derived mast cells (ML) vs neutrophils (GL): Xenopus laevis bone marrow-derived mast cells (ML) and neutrophils (GL) were compared.
cells with 'n/a' represent 'not available', based on data generated by Azenta.
Description of the data and file structure:
Comparison of Xenopus laevis control (V) and mast cell-enriched (S) skin:
Column A: names of differentially expressed genes
Column B: abbreviations of gene names
Column C: full gene names
Column D: baseMean
Column E: log2FoldChange of gene expression
Column F: Standard error
Column G: statistical output
Column H: p-value
Column I: adjusted p-value
cells with 'n/a' represent 'not available', based on data generated by Azenta.
In vitro derived mast cells (ML) vs neutrophils (GL):
Column A: abbreviated gene name
Column B: log2FoldChange
Column C: pvalue
Column D: adjusted pvalue
Column E-H:reads of given gene in bone marrow-derived neutrophils (GL), each column representing reads derived from cells generated from an individual Xenopus laevis frog
Column I-L: reads of given gene in bone marrow-derived mast cell (SL), each column representing reads derived from cells generated from an individual Xenopus laevis frog
cells with 'n/a' represent 'not available', based on data generated by Azenta.
For transcriptomic profiling, bone marrow-derived neutrophil and mast cell cultures were generated as described above and FACS-sorted according to preestablished size and internal complexity parameters to isolate the respective subsets for further analyses. Sorted cells were immediately processed to extract and purify RNA. Flash frozen samples were sent to Azenta Life Sciences for all library preparation, RNA sequencing, and analyses. In short, polyadenylated RNA was isolated using Oligo dT beads. Enriched mRNAs were then fragmented for first and second strand cDNA synthesis. cDNA fragments were end repaired, 5’ phosphorylated, and dA-tailed. Fragments were then ligated to universal adaptors and PCR-amplified. 150-bp paired-end sequencing was performed on an Illumina HiSeq platform.
FastQC was used to evaluate raw data quality. Adaptors sequences and poor-quality nucleotides were removed from reads using Trimmomatic v.0.36. The STAR aligner v.2.55.2b was used to map these reads to the Xenopus_laevis_9_2 reference genome from ENSEMBL. To determine differential gene expression, featureCount (Subread package v.1.5.2) was first used to count unique gene hits, which were then used with DESeq2 to calculate absolute log2-fold change.