Phylogeny And Expression Modulation Of Interleukin 1 Receptors In Grass Carp(Ctenopharyngodon Idella)

The IL-1 family plays a critical role in regulating many immune processes including inflammatory responses and immune homeostasis.In humans,it consists of 11 members,which are divided into three subfamilies,IL-1,IL-18 and IL-36,based on conserved signal sequences and binding receptors.The IL-1 family-activated receptor belongs to the Toll/IL-1 receptor(TIR)subfamily and is typically characterized by 1-3 conserved Ig domains in the extracellular region and intracellular TIR domains.The Ig domain is critical for ligand binding,and the intracellular TIR domain work for initiating cellular responses without controversy.Ten IL-1R members have been found in humans,and recently,a unified nomenclature is proposed to name them as IL-1R1-10.IL-1RAcP like(IL-1RAcPL)is also a class of receptors that are highly homologous to IL-1RAcP in the IL-1 family of receptors.Ligands of such receptors have not yet been discovered,therefore,they are referred to as orphan receptors.IL-1R8/SIGIRR contains a single Ig domain in the extracellular region and a unique anti-inflammatory receptor with a TIR domain in the cytoplasmic region.The ligand for IL-1R8/SIGIRR has not been identified.Akin IL-1R9/IL-1RAcPL1 and IL-1R10/IL-1RAcPL2 are also an orphan receptor.There are many IL-1 family receptors of fish.Due to different living environments,differences in evolution levels,and Whole genome duplication(WGD),there are different types and quantities of IL-1 family receptors among different fish species.In this study,5 IL-1 receptors including IL-1R3/ IL-1RAcP,IL-1R8/SIGIRR,IL-1R9a/IL-1RAcPL1 a,IL-1R9b/IL-1RAcPL1 b and IL-1R10/IL-1RAcPL2 were identified in grass carp(Ctenopharyngodon idella).These five receptors had a similarly sized(147 or 159 aa)conserved intracellular TIR domain and containing three Ig domains(84-110 aa)in the extracellular region(IL-1R8/SIGIRR just has a single Ig Domain).CiIL-1R9/IL-1RAcPL1a/b and CiIL-1R10/IL-1RAcPL2 were present in three different scaffolds where the chromosomal location is unclear.Similarly,IL-1R9a/IL-1RAcPL1 a,IL-1R9b/IL-1RAcPL1 b and IL-1R10/IL-1RAcPL2 are also located in three different chromosomes of zebrafish.Phylogenetic tree analysis reveals that the IL-1Rs identified in the present work grouped with their respective orthologues from other vertebrates.Another phylogenetic tree comprising Ig domains of IL-1Rs from different fish species was showed that the single domain of IL-1R8/SIGIRR grouped with Ig3 domains of other IL-1Rs.The results indicated that IL-1R9/IL-1RAcPL1 and IL-1R10/IL-1RAcPL2 were closely related sister groups.The orthologous relationships are also supported by the gene synteny where genes neighbouring the receptor genes are well conserved.Synteny of fish IL-1R9a/IL-1RAcPL1 a is conserved with mammalian IL-1R9/IL-1RAcPL1,indicating they are orthologues.However,gene synteny of grass carp and zebrafish IL-1R9b/IL-1RAcPL1 b was different from that of mammals and spotted gar which comprise a single copy of IL-1R9/IL-1RAcPL1,implying that IL-1R9/IL-1RAcPL1 could have been duplicated during the teleost specific whole genome duplication event.In the quantitative analysis section,the results showed that CiIL-1R3/IL-1RAcP was relatively highly expressed in all tissues of healthy fish compared to other receptors examined and was up-regulated by both LPS and poly(I:C)and F.cloumnare infection,suggesting that IL-1R3/IL-1RAcP was involved in inflammatory response.Consistent with the expression data in zebrafish,highly constitutive expression of CiIL-1R8/SIGIRR was detected in most tissues of healthy fish,particularly liver,head kidney and intestine,suggesting CiIL-1R8/SIGIRR could play a role in immune hemostasis.Consistent with the findings in mammals,both CiIL-1R9a/IL-1RAcPL1 a and CiIL-1R9b/IL-1RAcPL1 b were highly expressed in brain of healthy fish,suggesting they were important in maintaining normal functions of brain.This is in agreement with the indicating that IL-1R9/IL-1RAcPL1 was initially identified to be predominantly expressed in brain and was associated with mental retardation.The present study also showed that the CiIL-1R9a/IL-1RAcPL1 a expression was significantly increased in primary splenocytes after stimulation with LPS.This was supported by the in vivo study that injection of poly(I:C)or infection of F.cloumnare also resulted in increase of CiIL-1R9/IL-1RAcPL1 a expression in spleen.Interestingly,CiIL-1R10/IL-1RAcPL2 displayed some degree of similarities with CiIL-1R8/SIGIRR in expression patterns where both LPS and infection of F.cloumnare significantly inhibited expression of both receptors,suggesting that IL-1R10/IL-1RAcPL2 could function as an anti-inflammatory receptor.After the preparation of the CiIL-1R8/SIGIRR antibody,the trunk kidney and intestine of the grass carp after the infection of F.cloumnare in vivo were detected by immunohistochemistry.It was found that the positive cells detected after infection were significantly reduced,especially,it was most obvious near the renal tubules of the trunk kidney.By comparison,the results of immunohistochemistry and qPCR results confirmed each other,indicating the negative regulation of CiIL-1R8/SIGIRR.In conclusion,IL-1R9a/IL-1RAcPL1 a,IL-1R9b/IL-1RAcPL1 b were closely related to IL-1R10/IL-1RAcPL2,demonstrated that they could have evolved from an ancient receptor.IL-1R8/SIGIRR and IL-1R10/IL-1RAcPL2 exhibit similar expression patterns,suggesting that they both play a negative regulatory role in the inflammatory response.