DOI: 10.5061/dryad.bcc2fqznc
title: Data from: Analyses Xenopus laevis mast cells, neutrophils, and mast cell-enriched, chytrid infected skin

description: 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.
description: 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 log<sub>2-</sub>fold
 change.
description: # Data from: Analyses Xenopus laevis mast cells, neutrophils, and mast
 cell-enriched, chytrid infected skin [Access this dataset on
 Dryad](https://doi.org/10.5061/dryad.bcc2fqznc) 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.