Transcriptional Profiling of Disease-Induced Host Responses in Bovine Tuberculosis and the Identification of Potential Diagnostic Biomarkers

Abstract

Bovine tuberculosis (bTb) remains a major and economically important disease of livestock. Improved ante-mortem diagnostic tools would help to underpin novel control strategies. The definition of biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bTb. We have used a murine bTb model to identify promising candidates in the host transcriptome post-infection. RNA from in vitro-stimulated splenocytes and lung cells from BALB/c mice infected aerogenically with Mycobacterium bovis were probed with high-density microarrays to identify possible biomarkers of disease. In antigen-stimulated splenocytes we found statistically significant differential regulation of 1109 genes early (3 days) after infection and 1134 at a later time-point post-infection (14 days). 618 of these genes were modulated at both time points. In lung cells, 282 genes were significantly modulated post-infection. Amongst the most strongly up-regulated genes were: granzyme A, granzyme B, cxcl9, interleukin-22, and ccr6. The expression of 14 out of the most up-regulated genes identified in the murine studies was evaluated using in vitro with antigen-stimulated PBMC from uninfected and naturally infected cattle. We show that the expression of cxcl9, cxcl10, granzyme A and interleukin-22 was significantly increased in PBMC from infected cattle compared to naïve animals following PPD stimulation in vitro. Thus, murine transcriptome analysis can be used to predict immunological responses in cattle allowing the prioritisation of CXCLl9, CXCL10, Granzyme A and IL-22 as potential additional readout systems for the ante-mortem diagnosis of bovine tuberculosis.

Analysis of Cytokine Gene Expression using a Novel Chromogenic In-situ Hybridization Method in Pulmonary Granulomas of Cattle Infected Experimentally by Aerosolized Mycobacterium bovis

“…CXCL10, also known as IFN-g-induced protein-10 (IP-10), is a chemokine detected within human and bovine tuberculoid lymph node granulomas (Ferrero et al, 2003;Aranday-Cortes et al, 2012a). Produced by monocytes, it attracts activated T cells and monocytes to granulomas and promotes a Th1 response.…”

“…More targeted investigations have examined tissues or organs containing tuberculoid granulomas, focusing on regions within or near lesions (Widdison et al, 2006(Widdison et al, , 2009Shu et al, 2014). Others have focused on the granuloma itself, through the use of immunohistochemistry (IHC) or laser capture microdissection (LCM) combined with real time quantitative polymerase chain reaction (qPCR) (Palmer et al, 1999;Wangoo et al, 2005;Johnson et al, 2006Johnson et al, , 2007Aranday-Cortes et al, 2012a). Relatively few studies have focused on granulomas of different stages of development (Palmer et al, 1999;Johnson et al, 2007;ArandayCortes et al, 2012a).…”

“…Relatively few studies have focused on granulomas of different stages of development (Palmer et al, 1999;Johnson et al, 2007;ArandayCortes et al, 2012a). Moreover, previous studies have examined cytokine expression by stimulated PBMCs or cells from tuberculous lymph nodes Boddu-Jasmine et al, 2008;Witchell et al, 2010;Aranday-Cortes et al, 2012a, 2012b. Few studies have examined cytokine expression at the primary site of infection (i.e.…”

“…Other cytokines and chemokines shown to be associated with an anti-mycobacterial response in cattle include tumour necrosis factor (TNF)-a, interleukin (IL)-17A, CXCL9 and CXCL10 (Thacker et al, 2007;Aranday-Cortes et al, 2012a;Shu et al, 2014). Using lymph nodes from experimentally infected cattle, one study showed that the expression of certain cytokines and chemokines varied among different stages of granuloma development (Aranday-Cortes et al, 2012a). In most cases, changes in expression were not linear in that there was not a trend of increasing or decreasing expression from stages I through IV.…”

Interleukin-17A as a Biomarker for Bovine Tuberculosis

“…PPD-specific IL-17 mRNA responses at 60 and 90 days after experimental M. bovis infection correlated with the presence of gross tuberculous lesions at necropsy 4 months after challenge (7). Using laser capture microdissection followed by qPCR, Aranday-Cortes et al demonstrated increased IL-17A and IL-22 expression within tuberculous granulomas versus nonaffected tissues from experimentally infected cattle, particularly in more advanced lesions (50). In the present study, M. bovis infection also elicited IL-17A protein responses that correlated with infection, similar to IFN-␥ responses (Table 5).…”