Altered Immune Cytokine Expression Associated with KoRV B Infection and Season in Captive Koalas

Koala (Phascolarctos cinereus) populations are increasingly vulnerable and one of the main threats is chlamydial infection. Koala retrovirus (KoRV) has been proposed as an underlying cause of the koala’s susceptibility to infection with Chlamydia and high rates of lymphoid neoplasia; however, the regionally ubiquitous, endogenous nature of this virus suggests that KoRV A infection is not sufficient for immune suppression to occur. A recently discovered exogenous variant of KoRV, KoRV B, has several structural elements that cause increased pathogenicity in related retroviruses and was associated with lymphoid neoplasia in one study. The present study assesses whether KoRV B infection is associated with alterations in immune function. Cytokine gene expression by mitogen stimulated lymphocytes of KoRV B positive (n = 5–6) and negative (n = 6–7) captive koalas was evaluated by qPCR four times (April 2014-February 2015) to control for seasonal variation. Key immune genes in the Th1 pathway (IFNγ, TNFα), Th2 pathway (IL 10, IL4, IL6) and Th17 pathway (IL17A), along with CD4:CD8 ratio, were assessed. KoRV B positive koalas showed significantly increased up-regulation of IL17A and IL10 in three out of four sampling periods and IFNγ, IL6, IL4 and TNFα in two out of four. IL17A is an immune marker for chlamydial pathogenesis in the koala; increased expression of IL17A in KoRV B positive koalas, and concurrent immune dysregulation, may explain the differences in susceptibility to chlamydial infection and severity of disease seen between individuals and populations. There was also marked seasonal variation in up-regulation for most of the cytokines and the CD4:CD8 ratio. The up-regulation in both Th1 and Th2 cytokines mirrors changes associated with immune dysregulation in humans and felids as a result of retroviral infections. This is the first report of altered immune expression in koalas infected by an exogenous variant of KoRV and also the first report of seasonal variation in cytokine up-regulation and CD4:CD8 ratio in marsupials.

考拉（Phascolarctos cinereus）种群的生存脆弱性日益加剧，其中衣原体感染是主要威胁之一。考拉逆转录病毒（Koala retrovirus, KoRV）被认为是考拉易感染衣原体以及淋巴组织肿瘤高发的潜在诱因；然而该病毒在区域内广泛存在且为内源性病毒的特性表明，仅KoRV A感染不足以引发免疫抑制。近期发现的KoRV外源性变体KoRV-B，携带有若干可提升相关逆转录病毒致病性的结构元件，且在一项研究中被发现与淋巴组织肿瘤存在关联。本研究旨在评估KoRV-B感染是否与免疫功能异常相关。研究通过定量聚合酶链反应（quantitative real-time PCR, qPCR），在2014年4月至2015年2月间开展四次采样检测，对KoRV-B阳性（n=5~6）与阴性（n=6~7）圈养考拉的丝裂原刺激淋巴细胞的细胞因子基因表达水平进行评估，以控制季节波动带来的影响。本研究同时检测了辅助性T细胞1（Th1）通路（干扰素γIFNγ、肿瘤坏死因子αTNFα）、辅助性T细胞2（Th2）通路（白细胞介素10IL-10、白细胞介素4IL-4、白细胞介素6IL-6）以及辅助性T细胞17（Th17）通路（白细胞介素17AIL-17A）中的关键免疫基因，以及CD4:CD8比值。结果显示，KoRV-B阳性考拉在四次采样中的三次，均呈现出IL-17A与IL-10的显著上调；而在四次采样中的两次，则呈现出IFNγ、IL-6、IL-4以及TNFα的显著上调。IL-17A是考拉衣原体发病的免疫标志物；KoRV-B阳性考拉体内IL-17A表达升高且伴随免疫功能失调，这或可解释不同个体与种群之间在衣原体感染易感性以及疾病严重程度上的差异。此外，多数细胞因子的上调水平以及CD4:CD8比值均存在显著的季节波动。无论是Th1还是Th2类细胞因子的上调模式，均与逆转录病毒感染引发的人类及猫科动物免疫失调特征相符。本研究首次报道了感染KoRV外源性变体的考拉出现免疫表达异常，同时也是首个关于有袋类动物细胞因子上调及CD4:CD8比值存在季节波动的研究报道。