Comparative Study On Immune Function Of Head Kidney And Trunk Kidney During IHNV Infection

Kidney is an important immune organ of teleost,which performs excretory function in early life,and then differentiates into two parts in the development:the head kidney and the trunk kidney.The head kidney loses excretory function and becomes an important endocrine and hematopoietic organ,while trunk kidney mainly performs osmotic pressure regulation and excretory function.It has been shown that rainbow trout head kidney can generate strong immune response against pathogenic infections(Ichthyophthirius multifiliis and F.columnare),with immunoglobulins and B cells playing a major role in the process of infection resistance.But up to now,there is no report on the comparison of immune function between fish head kidney and trunk kidney in antiviral infection.IHNV is one of the most serious viral infectious pathogens in trout culture.Therefore,this study intends to establish IHNV infection model of rainbow trout,and systematically elucidates the immune response rule and mechanism of head kidney and trunk kidney immunoglobulin and different B cell subsets in the process of anti-IHNV infection from tissue,cell and protein levels by using q RT-PCR,immunofluorescence,western blot and flow cytometry.It reveals the similarities and differences between head kidney and trunk kidney in antiviral infection and enrich the immune theory of fish kidney,also provide a new idea and challenge for the study of trunk kidney function of teleost in the future.1.After IHNV infection,rainbow trout showed typical clinical symptoms such as protrusion of eyeballs,pale gill filaments and tail fin bleeding after infection.By viral immunofluorescence,we found that IHNV positive cells were detected in both head kidney and trunk kidney at 7 dpi,indicating the presence of enriched virus particles in head kidney and trunk kidney.q RT-PCR was used to analyze the viral load in the head kidney and trunk kidney,and it was found that the viral load in the head kidney and trunk kidney reached a peak at 7 dpi,and then gradually decreased at 28 dpi.2.Through paraffin and H&E staining,we conducted histopathology experiments on the head kidney and trunk kidney after IHNV infection.The results indicated that there were pathological changes in both head kidney and trunk kidney after infection.The head kidney presents with lymphatic cord disorders,vascular effusion,and sinus bleeding.The lesions begin at 4 dpi and are most severe at 7 dpi.The trunk kidney was characterized by tubular epithelial cell shedding,glomerular enlargement,and interstitial cell necrosis and bleeding.The lesions started from the 4 dpi and were most severe at 7 dpi,and then gradually recovered.3.We investigated the expression of immune-related genes in the head kidney and trunk kidney after IHNV infection at the molecular level.We selected 17 typical immune-related genes,including antiviral genes,inflammatory cytokines and immunoglobulin heavy chain genes.By analyzing the data,we found that most immune-related genes showed similar expression trends in the head kidney and trunk kidney after IHNV infection,with significantly increased expression levels at 7 dpi.It is worth nothing that the tnfαgenes expression level was higher in the trunk kidney than in the head kidney.4.Transcriptome sequencing was performed to further explore the dynamic changes of immune responses in the head kidney and trunk kidney.Similar to the results of q RT-PCR,the expression levels of antiviral genes and inflammatory factors were significantly up-regulated in both head kidney and trunk kidney at 7 dpi.The expression levels of some interferon-stimulating genes,such as dhx58,mx2 and vig1,were significantly higher in the trunk kidney than in the head kidney,which seems to indicate that the trunk kidney induces a stronger interferon response than the head kidney.Through KEGG analysis,we found that signaling pathways enriched in the head kidney and trunk kidney at 7 dpi were mainly related to immune,such as PRRs signaling pathway(Toll-like receptor signaling pathway、NOD-like receptor signaling pathway)and IL-17 signaling pathway.At 28 dpi,the main signal pathways enriched in the head kidney and trunk kidney were different.The signal pathways involved in the head kidney were mostly related to cell and signal transduction,while the trunk kidney was related to metabolism.5.By westerning blot,immunofluorescence,and flow cytometry,we compared the adaptive immune responses of the head kidney and trunk kidney at the cellular and protein levels.Our results showed that the expression levels of Ig T and Ig T~+B cells,Ig M and Ig M~+B cells were significantly up-regulated in both the head kidney and trunk kidney after IHNV infection compared to the control group.In addition,the changes of Ig T and Ig T~+B cells in trunk kidney were more significant than those in head kidney,while the changes of Ig M and Ig M~+B cells in head kidney were more obvious.Through EDU immunofluorescence,we found that Ig T~+and Ig M~+B cells were produced by the proliferation of head and trunk kidney.Taken together,our results suggest that an adaptive immune response mediated by B cells occurs in both the head kidney and trunk kidney.