Abstract
Many studies have indicated that the expression of interleukin-21 (IL-21) is associated with the pathogenesis of certain liver diseases. However, in alternative animal models of liver diseases, it remains unknown whether the tree shrew could be utilized to analyze the relationship between IL-21 and liver diseases. Here, the phylogenetic tree, sequence alignment and protein structure model of tree shrew and human IL-21 were analyzed using bioinformatics software. A pEGFP-N3/tsIL-21 eukaryotic expression vector of tree shrew IL-21 (tsIL-21) was constructed, and IL-21 expression by the vector-transfected Huh7 cells was evaluated using the newly established quantitative real-time PCR and immunologic protocols for assessing human IL-21. The cytokine profiles were also evaluated in tree shrew spleen lymphocytes induced by recombinant human IL-21 or concanavalin A. It was found that the coding sequence (CDS) of tsIL-21 amplified from spleen lymphocytes belonged to the predicted sequence. The tsIL-21 was closely clustered with primate IL-21 rather than rodent IL-21, and it had an alignment of 83.33% with the human IL-21 nucleotide sequence and 69.93% with the amino acid sequence. The profiles of secondary structure, hydrophobicity and surface charge of tsIL-21 were also similar with those of human IL-21. The tsIL-21 expressed by the vector-transfected Huh7 cells could be identified by their different sources of antibodies against human IL-21, which were all dose-dependent. Recombinant human IL-21 could induce the change of the cytokine profiles of tree shrew spleen lymphocytes, which showed a higher expression of IL-10 and IFN-γ rather than IL-2, IL-4, IL-17, TNF-a and IL-21 during the five-day stimulation. These results indicate that tsIL-21 has a high degree of homology, structural similarity and immunological cross-reactivity with human IL-21 and also confirm the accuracy of this predicted tsIL-21CDS. The protocols utilized in this study will lead to the experimental feasibility of further IL-21-related studies in vivo.
Highlights
Interleukin-21 (IL-21) is a type I cytokine that shares the common cytokine receptor γ chain with IL-2, IL-4, IL-7, IL-9, and IL-15[1]
In the field of hepatitis B virus (HBV)-related diseases, we found that HBV e antigen (HBeAg) seroconversion occurred in the antiviral therapy of chronic hepatitis B patients was closely related to IL-21[5], and the circulating CXCR5+CD4+ T cells benefit HBeAg seroconversion through IL-21 [6]
The phylogenetic tree showed that the tree shrew IL-21 (tsIL-21) transcripts was first clustered with human, monkey, sheep and cattle and secondly with mouse and rat (Fig 1A), which demonstrated that human IL-21 had a closer affinity to tsIL-21 than the rodents’ IL-21
Summary
Interleukin-21 (IL-21) is a type I cytokine that shares the common cytokine receptor γ chain with IL-2, IL-4, IL-7, IL-9, and IL-15[1]. It is produced by multiple CD4+ T cell subsets, including T helper 17 cells, follicular helper T cells, and natural killer T cells [1, 2]. In a recent macaque model study in vivo, recombinant IL-21 combined with antiretroviral therapy could reduce levels of simian immunodeficiency virus RNA and viral reservoirs [13]. The in vivo study of IL-21-related liver diseases has been conducted in a transgenic mouse model, but the occurrence and development of human liver diseases, hepatitis virus infection, could not be fully mimicked
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