Expression profiles of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus) by qRT-PCR

Abstract

Investigation of the immune response of the koala (Phascolarctos cinereus) is needed urgently, but has been limited by scarcity of species-specific reagents and methods for this unique and divergent marsupial. Infectious disease is an important threat to wild populations of koalas; the most widespread and important of these is Chlamydial disease, caused by Chlamydia pecorum and Chlamydia pneumoniae. In addition, koala retrovirus (KoRV), which is of 100% prevalence in northern Australia, has been proposed as an important agent of immune suppression that could explain the koala’s susceptibility to disease. The correct balance of T regulatory, T helper 1 (Th1) and Th2 lymphocyte responses are important to an individual’s susceptibility or resistance to chlamydial infection. The ability to study chlamydial or KoRV pathogenesis, effects of environmental stressors on immunity, and the response of koalas to vaccines under development, by examining the koala’s adaptive response to natural infection or in-vitro stimulation, has been limited to date by a paucity of species- specific reagents. In this study we have used cytokine sequences from four marsupial genomes to identify mRNA sequences for key T regulatory, Th1 and Th2 cytokines interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFNγ) along with CD4 and CD8β. The koala sequences used for primer design showed >58% homology with grey short-tailed opossum, >71% with tammar wallaby and 78% with Tasmanian devil amino acid sequences. We report the development of real-time RT-PCR assays to measure the expression of these genes in unstimulated cells and after three common mitogen stimulation protocols (phorbol myristate acetate/ionomycin, phorbol myristate acetate/phytohemagglutinin and concanavalin A). Phorbol myristate acetate/ionomycin was found to be the most effective mitogen to up-regulate the production of IL-4, IL-10 and IFNγ. IL-6 production was not consistently up-regulated by any of the protocols. Expression of CD4 and CD8β was down-regulated by mitogen stimulation. We found that the reference genes GAPDH and 28s are valid for normalising cytokine expression by koala lymphocytes after mitogen stimulation.

Tumor‐associated macrophages secrete CC‐chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer

“…After reviewing the relevant literature, we selected seven cytokines (CXCL-1, CCL2, CCL5, interleukin [IL]-6, IL-8, IL-17, and CXCL-5) that may promote endocrine resistance in breast cancer cells. [37][38][39][40][41][42][43] Transcription of these seven cytokines in the three macrophages (MΦ, MS, and MR) was assessed by RT-PCR to identify the cytokines whose transcription levels were consistent with the trends from the CCK-8 and apoptotic flow cytometry assays. Only CCL2 was consistent with the previous results and the difference was statistically significant (Figure 2A).…”

“…As a well-known chemokine abundantly present in macrophages, CCL2 is responsible for the recruitment of monocytes. [37][38][39][40][41][42]46 The concentration of CCL2 is positively correlated with its chemotaxispromoting potential. We have already determined that the levels of CCL2 secreted by MΦ, MS, and MR are different.…”

Identification of stable reference genes for quantitative PCR in koalas

“…ACTB and GAPDH , the most commonly used reference genes for normalization of quantitative expression studies in koalas 29,37,38 were not ranked as the most stable genes with any of the algorithms or in any experimental group. The BestKeeper algorithm chose ACTB as the second most stable gene in all experimental conditions but in other algorithms and the final comprehensive analysis, its ranking position indicated lower stability.…”

Gene polymorphisms and expression levels of interleukin-6 and interleukin-10 in lumbar disc disease: a meta-analysis and immunohistochemical study

“…Subsequently, real-time PCR amplification was performed using specific primers of target genes and a SYBR Green real-time PCR kit (Quanta Biosciences, MD, USA). The primer sequences were selected according to previous study [24].…”