IL-10 suppression of IFN-γ responses in tuberculin-stimulated whole blood from Mycobacterium bovis infected cattle.

The measurement of bovine interferon-gamma (IFN-γ) forms the basis of a diagnostic test for bovine tuberculosis where Mycobacterium bovis sensitised effector T cells produce IFN-γ following in vitro stimulation with tuberculin antigens. In cattle infected with M. bovis it is also known that the anti-inflammatory IL-10 cytokine can inhibit in vitro production of IFN-γ leading to a reduced response in the IFN-γ diagnostic test. In order to investigate this in greater detail, whole blood samples from tuberculin skin test positive and negative cattle were stimulated with bovine and avian tuberculin antigens and in parallel with a neutralising anti-IL-10 monoclonal antibody. The results showed that IFN-γ protein levels increased when IL-10 activity was suppressed by Anti - IL-10. By using a standard diagnostic interpretation, the elevated levels of IFN-γ were shown to change the level of agreement between the performance of the single intradermal comparative tuberculin test (SICTT) and IFN-γ assay, depending on the tuberculin treatment. A transcriptomic analysis using RT-qPCR investigated the influence of IL-10 activity on expression of a suite of cytokine genes (IFNG, IL12B, IL10 and CXCL10) associated with antigen-stimulated production of IFN-γ. The IFNG and IL12B genes both experienced significant increases in expression in the presence of Anti-IL-10, while the expression of IL10 and CXCL10 remained unaffected.

Antigen-Specific IP-10 Release Is a Sensitive Biomarker of Mycobacterium bovis Infection in Cattle.

The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%-100%) and a specificity of 97% (95% CI, 85%-100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.

Innate gene repression associated with Mycobacterium bovis infection in cattle: toward a gene signature of disease.

BACKGROUND: Bovine tuberculosis is an enduring disease of cattle that has significant repercussions for human health. The advent of high-throughput functional genomics technologies has facilitated large-scale analyses of the immune response to this disease that may ultimately lead to novel diagnostics and therapeutic targets. Analysis of mRNA abundance in peripheral blood mononuclear cells (PBMC) from six Mycobacterium bovis infected cattle and six non-infected controls was performed. A targeted immunospecific bovine cDNA microarray with duplicated spot features representing 1,391 genes was used to test the hypothesis that a distinct gene expression profile may exist in M. bovis infected animals in vivo. RESULTS: In total, 378 gene features were differentially expressed at the P < or = 0.05 level in bovine tuberculosis (BTB)-infected and control animals, of which 244 were expressed at lower levels (65%) in the infected group. Lower relative expression of key innate immune genes, including the Toll-like receptor 2 (TLR2) and TLR4 genes, lack of differential expression of indicator adaptive immune gene transcripts (IFNG, IL2, IL4), and lower BOLA major histocompatibility complex - class I (BOLA) and class II (BOLA-DRA) gene expression was consistent with innate immune gene repression in the BTB-infected animals. Supervised hierarchical cluster analysis and class prediction validation identified a panel of 15 genes predictive of disease status and selected gene transcripts were validated (n = 8 per group) by real time quantitative reverse transcription PCR. CONCLUSION: These results suggest that large-scale expression profiling can identify gene signatures of disease in peripheral blood that can be used to classify animals on the basis of in vivo infection, in the absence of exogenous antigenic stimulation.