Unknown,Transcriptomics,Genomics,Proteomics

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Zebrafish il-4 il-10 regulated immunity in gills

ABSTRACT: The evolutionary origin of vertebrate type 2 immunity is a topic of great interest. Several studies have focused on the immune cell components of evolutionary older vertebrates such as fish. However, how fish cytokines function and whether they have similar roles as in mammals is still a matter of speculation. Here, we have used the zebrafish (Danio rerio) to gain insights into il4/13a and il4/13b genes and characterized their role under both homeostatic and inflammatory conditions. We established knockouts for both il4/13a and il4/13b genes and showed that they are needed to suppress inflammation in larvae and in the gill mucosa. As a counterpoint, we examined the gills of il10 defective zebrafish and revealed the importance of il10 in maintaining homeostasis in this mucosal tissue. As in mammals, zebrafish il10 appears to have an anti-inflammatory function.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Danio rerio

SUBMITTER: Leo Zeef

PROVIDER: E-MTAB-8958 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Maternal IL10 deficiency elevates susceptibility to fetal loss induced by the model Toll-like receptor agonist lipopolysaccharide, but the mechanisms are not well elucidated. Here we show that Il10 null mutant (Il10-/-) mice exhibit altered local T cell responses in pregnancy, exhibiting pronounced hyperplasia in para-aortic lymph nodes draining the uterus with >6-fold increased CD4+ and CD8+ T cells compared with wild-type controls. Amongst these CD4+ cells, Foxp3+ Treg cells were substantially enriched, with 11-fold higher numbers at day 9.5 post coitum (pc). Lymph node hypertrophy in Il10-/- mice was associated with more activated phenotypes in dendritic cells and macrophages, with elevated expression of MHCII, scavenger receptor and CD80. Affymetrix microarray revealed an altered transcriptional profile in Treg cells from pregnant Il10-/- mice, with elevated expression of Ctse (cathepsin E), Il1r1, Il12rb2 and Ifng. In vitro, Il10-/- Treg cells showed reduced steady state Foxp3 expression, and polyclonal stimulation caused greater loss of Foxp3 and reduced capacity to suppress IL17 in CD4+Foxp3- T cells. We conclude that despite a substantially expanded Treg cell pool, diminished stability of Treg cells, increased numbers of effector T cells, and altered phenotypes in dendritic cells and macrophages in pregnancy all potentially confer vulnerability to inflammation-induced fetal loss in Il10-/- mice. These findings suggest a pivotal role for IL10 in facilitating robust immune protection of the fetus from inflammatory challenge and suggest IL10 deficiency could contribute to human gestational disorders where altered T cell responses are implicated. RNA was extracted from CD4+CD25+ lymphocytes isolated from para-aortic LNs of day 9.5 pc pregnant Il10+/+ or Il10-/- mice using miRNeasy Mini Kits (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. The concentration and purity of each RNA sample was determined using the Nanodrop Spectrophotometer (ND-1000, Nanodrop Technologies Inc., Wilmington, DE). RNA quality was determined using the RNA 6000 Pico Total RNA Kit (Agilent Technologies, Santa Clara CA) prior to use in microarray experiments. RNA with a RIN>7 was used in this study. For microarray analysis, RNA was pooled (from 2-4 mice per pool) resulting in four biological replicates of CD4+CD25+ cells from both Il10-/- and Il10+/+ mice. Microarray analysis was performed using Affymetrix Mouse Gene 2.0 ST Arrays at the Adelaide Microarray Centre. Total RNA was amplified using the Ovation PicoSL WTA System V2 (Nugen Inc., San Carlos, CA, USA) and MinElute Reaction Cleanup Kit (Qiagen Inc., Valencia, CA, USA), according to the manufacturer’s instructions, to provide 5 ?g of cRNA for each microarray. The microarray data was normalized and analyzed using Partek Genomics Suite (Partek Inc, MO). Raw data from the Affymetrix platform (.cel files) were imported and normalized using RMA background correction, Partek’s own GC content correction, and mean probe summarization.

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Zebrafish il-4 il-10 regulated immunity in gills

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