Temporal Expression of Peripheral Blood Leukocyte Biomarkers in a Macaca fascicularis Infection Model of Tuberculosis; Comparison with Human Datasets and Analysis with Parametric/Non-parametric Tools for Improved Diagnostic Biomarker Identification

Abstract

A temporal study of gene expression in peripheral blood leukocytes (PBLs) from a Mycobacterium tuberculosis primary, pulmonary challenge model Macaca fascicularis has been conducted. PBL samples were taken prior to challenge and at one, two, four and six weeks post-challenge and labelled, purified RNAs hybridised to Operon Human Genome AROS V4.0 slides. Data analyses revealed a large number of differentially regulated gene entities, which exhibited temporal profiles of expression across the time course study. Further data refinements identified groups of key markers showing group-specific expression patterns, with a substantial reprogramming event evident at the four to six week interval. Selected statistically-significant gene entities from this study and other immune and apoptotic markers were validated using qPCR, which confirmed many of the results obtained using microarray hybridisation. These showed evidence of a step-change in gene expression from an ‘early’ FOS-associated response, to a ‘late’ predominantly type I interferon-driven response, with coincident reduction of expression of other markers. Loss of T-cell-associate marker expression was observed in responsive animals, with concordant elevation of markers which may be associated with a myeloid suppressor cell phenotype e.g. CD163. The animals in the study were of different lineages and these Chinese and Mauritian cynomolgous macaque lines showed clear evidence of differing susceptibilities to Tuberculosis challenge. We determined a number of key differences in response profiles between the groups, particularly in expression of T-cell and apoptotic makers, amongst others. These have provided interesting insights into innate susceptibility related to different host `phenotypes. Using a combination of parametric and non-parametric artificial neural network analyses we have identified key genes and regulatory pathways which may be important in early and adaptive responses to TB. Using comparisons between data outputs of each analytical pipeline and comparisons with previously published Human TB datasets, we have delineated a subset of gene entities which may be of use for biomarker diagnostic test development.

Rhesus Macaques Are More Susceptible to Progressive Tuberculosis than Cynomolgus Macaques: a Quantitative Comparison

“…MCM originate from the island of Mauritius, and they have limited major histocompatibility complex diversity due to extreme founder effects that created a tight genetic bottleneck (36). Aerosol challenge with M. tuberculosis has been described in these animals (21,37), and they appeared to be quite susceptible to TB.…”

Quantitative and qualitative iNKT repertoire associations with disease susceptibility and outcome in macaque tuberculosis infection

“…To investigate the relationship between iNKTs and disease outcome in the macaque model of Mtb infection, we first determined peripheral blood ex vivo iNKT cell frequencies across four genetically distinct groups of mycobacteria-naive macaques comprising rhesus and cynomolgus species [15, 18, 20]. We employed a highly specific strategy of iNKT identification, by flow cytometry staining of PBMC with CD1d-αGC tetramers and anti-Vα24 after gating on live, CD3 + T cells (Figure 1a).…”

“…Importantly, animals that fall into rhesus and cynomolgus macaque species differ with regard to their genetic background and susceptibility to Mtb, a divergence also observed after challenge with other microorganisms, including human immunodeficiency virus and Shigella species [15–19]. Such differences may range from a strong ability to control infection in cynomolgus of Chinese or Indonesian genotype to reduced resistance in Mauritian cynomolgus [15, 20, 21]. Variations in susceptibility are likely due to immunological differences.…”

Concordant and discordant gene expression patterns in mouse strains identify best-fit animal model for human tuberculosis

“…T cell proliferation is known to vary in lungs of susceptible mouse strain I/St and resistant mouse strain A/Sn, however the genes underlying those differences have not been identified 35 . Recently, differences in transcriptome regulation in peripheral blood leukocytes of susceptible and resistant lineages of Macaca fascicularis were described 36 . In TB-susceptible macaques down-regulation of T-cell related genes was observed at week 6 p.i., when animals had lost 10% body weight.…”