Short-Term Feed Deprivation Alters Immune Status of Surface Mucosa in Channel Catfish (<i>Ictalurus punctatus</i>)

<div><p>Short-term feed deprivation (or fasting) is a common occurrence in aquacultured fish species whether due to season, production strategies, or disease. In channel catfish (<i>Ictalurus punctatus</i>) fasting impacts susceptibility to several bacterial pathogens including <i>Flavobacterium columnare</i>, the causative agent of columnaris disease. As columnaris gains entry through the gills and skin of fish, we examined here changes in transcriptional regulation induced in these surface mucosal tissues due to short-term (7 day) fasting. RNA-seq expression analysis revealed a total of 1,545 genes perturbed by fasting. Fasting significantly altered expression of critical innate immune factors in a manner consistent with lower immune fitness as well as dysregulating key genes involved in energy metabolism and cell cycling/proliferation. Downregulation of innate immune actors such as iNOS2b, Lysozyme C, and peptidoglycan recognition protein 6 is predicted to impact the delicate recognition/tolerance balance for commensal and pathogenic bacteria on the skin and gill. The highlighted expression profiles reveal potential mechanistic similarities between gut and surface mucosa and underscore the complex interrelationships between nutrition, mucosal integrity, and immunity in teleost fish.</p></div

Summary of gene identification and annotation of assembled catfish contigs based on BLAST homology searches against various protein databases (Zebrafish, UniProt, nr).

<p>Putative gene matches were at E-value ≤ 1e-5. Hypothetical gene matches denote those BLAST hits with uninformative annotation. Quality unigene hits denote more stringent parameters, including score ≥100, E-value ≤ 1e-20.</p

Key differentially expressed genes in the gill and skin between fasted and fed channel catfish in different functional classifications.

<p>Positive/negative values indicate upregulation and downregulation, respectively, in fasted fish relative to the fed control. When reads number equaled to 0 in fed or fasted group, the fold change is presented by average normalized read number in fed/average normalized read number in fasted. All fold changes were significant at p-value <0.05.</p

Statistics of differently expressed genes between fasted and fed channel catfish with fed catfish serving as the control.

<p>Values indicate contigs/genes passing cutoff values of fold change ≥1.5 (p<0.05). Average contig size refers to reads/contig.</p

Comparison of relative fold changes between RNA-seq and QPCR results in channel catfish.

<p>Gene abbreviations are: Lysozyme g-like 1, LYGL1; Taxilin beta b, TXLNBB; Twinfilin-2, TWF2; Endothelial lipase precursor, LIPG; Stress-associated endoplasmic reticulum protein 2, SERP1; Anterior gradient protein 2 homolog precursor, AGR2; Phosphomannomutase 2, PMM2; Complement c4, C4; Nitric oxide synthase 2 b, inducible, iNOS2b; Vitelline membrane outer layer protein 1 homolog, VMO1.</p

Summary of <i>de novo</i> assembly results of Illumina RNA-seq data from channel catfish gill and skin using Trans-ABySS and Trinity assemblers.

<p>Summary of <i>de novo</i> assembly results of Illumina RNA-seq data from channel catfish gill and skin using Trans-ABySS and Trinity assemblers.</p

Flow cytometric evaluation of the γδ T cell receptor (TCR) repertoire in control vs. early and late stage tumor-bearing mice (distribution on left panels, x ± SD, right panels, * = p<0.05).

<p>There were no significant changes in the Vγ1, Vγ4, or Vγ7 subsets from controls either post-tumor injection day 11±1 or at end stage. Additionally, there were no changes in the proportions of Vδ1, Vδ4, and Vδ6.3 at day 10. A significant decrease of the %Vδ6.3 population at end stage was offset by a parallel increase in %Vδ4 cells. TCR subsets Vγ3 and Vδ3 were also measured, however, relative proportions were negligible <i>(data not shown)</i>.</p

Cytotoxicity and growth inhibition assessment of activated γδ T cells against GL261 tumors.

<p><i>(a)</i> Activated γδ T cells were incubated with GL261 cells either in suspension (closed circles) or attached to plastic (closed squares) show robust <i>in vitro</i> cytotoxicity against the non-adherent GL261 cells in a 4h standard assay (x ± SD, * = p<0.05). The insert is a high-power confocal micrograph of adherent GL261 cells cytoplasm stained with wheat germ agglutinin (green) showing microfilopodia spreading from the attached cells as well as tumor from an athymic nude mouse was harvested 10 days following GL261 cells, chopped and disaggregated in media, fixed/post-fixed in in glutaraldehyde and osmium tetroxide, dehydrated in ethanol, and embedded in epoxy resin. Filopodia (clear arrows) and microspikes (filled arrows) are seen on the cell surface in these transmission electron micrographs from tumor sections. <i>(b)</i> Injection of 5 x 10⁵ GL261 cells followed 15m later by 1.5 x 10⁶ activated γδ T cells (dashed line) did not improve median survival when compared to saline-injected mice (solid line). <i>(c)</i> Evidence of local tumor inhibition was noted in histologic examination. Representative histologic specimens show a much smaller tumor at the injection site (arrow) in the γδ T cells injected mouse (B) than from a control mouse (A) at 25 days.</p

Circulating γδ T cell enumeration and function in tumor-bearing mice.

<p><i>(a)</i> The circulating T cell count (distribution on left panels, x ± SD, right panels, * = p<0.05) was increased in tumor-bearing mice at 10 days post-GL261 injection independent of the total T cell count. Terminal γδ T cell counts in tumor-bearing mice also fell significantly lower than controls and day 10 glioma-bearing mice. <i>(b)</i> Approximately 12% of γδ T cells constitutively produced IFN-γ with no additional increase following PMA/Ionomycin stimulation. <i>(c)</i> Annexin V expression was upregulated on γδ T cells from glioma-bearing mice, also independently from the total T cell population indicating simultaneous γδ T cell proliferation and apoptosis likely due to activation-induced cell death (AICD). <i>(d)</i> The γδ T cell count and Annexin V expression were measured in unmanipulated (e.g. no intracranial injection) mice and 10 days following IC injection of the methylcellulose vehicle in which GL261 cells were suspended to determine if IC injection-related injury produced the same increase in γδ T cell count and Annexin V expression as tumor injection. There was no difference in either parameter between sham-injected mice and mice that received intracranial methylcellulose vehicle alone.</p