Preliminary Characterisation of Tumor Necrosis Factor Alpha and Interleukin-10 Responses to Chlamydia pecorum Infection in the Koala (Phascolarctos cinereus)

Abstract

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

Peer Review #1 of "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 (v0.1)"

“…Although the short incubation times employed in the current study would be an advantage when stimulating samples from this wildlife species in the field, it may be that the study of IL-6 and ILwill require more extended incubation and, possibly, specific mitogens. Up-regulation of IL-10 in the present study was less than that reported by Mathew et al (Mathew et al 2013a) following antigen stimulation (by UV inactivated C. pecorum particles). In addition, although some individuals showed evidence of IL-6 up-regulation, there was no significant increase in IL-6 expression among treatment groups.…”

“…All sequences possessed key conserved residues, confirming their identity (Fig 1 and 2, Supplementary Material A). Sequences for IL-10, and IFNγ were consistent with those of Mathew et al (Mathew et al 2013a;Mathew et al 2013b). Sequences have been lodged in GenBank under the accession numbers: PhciCD4, KF731654;…”

“…We consider it most likely that further, cytokine-specific, optimization of stimulation protocols will increase up-regulation of these cytokines. The greater up-regulation of IL-10 in Mathew et al (Mathew et al 2013a) was observed after 12 hours of stimulation and production of IL-6 and IL-10 has been shown to continue to increase up to 12 hours and beyond in other studies (as opposed to the 5 hours of stimulation employed in the current study) (Baran et al 2001; Fagiolo et al 1993;Liu et al 2009). In addition to increasing the period of incubation, specific mitogens may be ideal; while PMA-ionomycin was the most potent stimulant of IFNγ and IL-4 responses, PHA-PMA showed the most promise for up-regulation of IL-6, consistent with Fagiolo et al (Fagiolo et al 1993).…”

“…Only two studies have quantified expression of koala cytokines by qRT-PCR, detecting upregulation of IFNγ, IL-10 and tumour necrosis factor-α (TNFα) in response to stimulation with chlamydial antigen (Mathew et al 2013a;Mathew et al 2013b); the range of immune genes that can be studied currently is limited, and the response of koala PBMCs to stimuli used commonly for in vitro assessment of cellular immune response in humans and other animals is largely uncharacterised. Further, no reference genes have been validated for use in lymphocyte stimulation assays in koalas.…”

Koala immunology and the koala retrovirus (KoRV)

“…Due to constant pathogen-driven selection, immune molecules are among the least conserved between species. Our ability to examine koala immune profiles has, therefore, been limited to detection of antibodies, lymphocyte proliferation assays, immunophenotyping by flow cytometry with a limited number of cross-reactive antibodies to conserved (mostly intra-cytoplasmic) domains (T cell, anti-human CD3; B cell, anti-human CD79b; MHCII, anti-human HLA-DP, DQ, DR; IFNg) (Higgins et al, 2004;Lau et al, 2012) and, very recently, qPCR for IL10, IFNg and TNFa (Mathew et al, 2013a;2013b). Our recent immunophenotypic studies on captive, KoRV-positive koalas (Lau et al, 2012) revealed some interesting features: elevated numbers of B cells relative to other species (1.0-4.9×10 6 cells/ml vs 0.17-0.56×10 6 cells/ml, respectively), and absence of MHCII expression on stimulated and nonstimulated T cells.…”