Involvement of pathogen and host-associated lipids in the macrophage immune responses in Johne’s disease

Abstract

Mycobacterium avium subsp. paratuberculosis (Mptb) infection in domestic animals is a global issue of economic and potential public health importance. A key role for macrophages in Mptb-infection has been well established. Macrophages infected with Mptb play a dual role as they are not just the site of host cellular defence mechanisms but are also the principal site of bacterial replication and persistence. An intriguing aspect of the pathogenesis of Mptb relates to the initial interaction between Mptb and the host macrophages and the ensuing mycobacterial manipulation of host immune mechanisms. This research focussed on identifying and characterising the role of the pathogen as well as host components which contribute to disease persistence. In Chapter 3 the nitric oxide response in RAW 264.7 macrophages to Mptb- specific antigen Para-LP-01 and 316v were assessed. In addition the nitric oxide response to live and heat-killed Mptb was also studied. It was found that live Mptb and its antigen Para-LP-01 and 316v induced a poor nitric oxide response. However, heat-killed Mptb induced a greater nitric oxide response. Based on this it was postulated that live Mptb might possess mechanisms to inhibit nitric oxide generation in macrophages and that cell wall-associated antigens could play a crucial role in this inhibitory effect. Since IFN-γ is an important pro-inflammatory cytokine in paratuberculosis, in Chapter 4, the effect of IFN-γ on macrophage responses was studied in RAW 264.7 macrophages after incubation with Mptb and antigens. These responses were compared to those induced to non-pathogenic mycobacterial strains. This study showed that while IFN-γ did help in overcoming the immunosuppressive effect of Mptb and enhanced responses to Mptb 316v antigen in relation to nitric oxide and cytokine production, it did not alter the TLR2 or TLR4 expression significantly and actually MHC-II expression was significantly decreased in response to live Mptb following IFN-γ pre-treatment. These features indicate that IFN-γ might have varied roles during Mptb-infection. In Chapter 5, the TLR2, TLR4 and TLR9 expression pattern in the PBMC of Mptb-exposed and unexposed sheep and cattle were assessed. TLR2 expression was significantly upregulated at 4 months post-inoculation and significantly downregulated at 8 months post-inoculation in the Mptb-exposed multibacillary sheep in comparison to the unexposed controls. This finding was similar to the results obtained in the TLR2 expression pattern in the RAW 264.7 macrophages where live Mptb caused an increase in TLR2 expression IX compared to the non-pathogenic M. smegmatis. Therefore, since it has been shown by previous studies that the TLR2 signalling can be immunosuppressive it was reasoned that increased TLR2 expression during the initial stages of Mptb-infection could be an indicator of disease susceptibility. In Chapter 6, other host responses that could favour the persistence of Mptb in cattle were assessed. Data collected from microarray studies conducted using four Mptb-exposed cattle with high IFN-γ responses and four unexposed control cattle were mined using a novel integrated approach to identify differentially regulated genes involved in pathways leading to disease progression. A total of six genes belonging to host lipid homeostasis and antibacterial defence mechanisms were identified and validated on a larger cohort of 10 unexposed and 20 Mptb-exposed cattle. GIMAP6 a gene which has been associated with disease resistance, was found to be significantly downregulated at 5 and 17 weeks post inoculation in the Mptb-exposed cattle in comparison to the unexposed control animals. Genes belonging to host cholesterol homeostasis (24DHCR, LDLR and SCD-1) were also found to be downregulated significantly at 17 weeks post inoculation. Based on this it is argued that downregulation of the host’s cholesterol homeostasis mechanism in an IFN-γ mediated manner could contribute to disease progression. This study identified novel genes which could be used as potential biomarkers for identifying disease resistance as well as those that contribute to disease persistence. In Chapter 7, the polarisation of macrophages during Mptb-infection in 10 Mptb-exposed cattle in comparison to five unexposed control cattle was studied by assessing the expression of markers specific for M1 and M2 macrophage subtypes, namely CD80, CD86 and CD163 respectively. In addition other indicators of pro- and anti-inflammatory responses like nitric oxide production, secretion of IL-10 and TNF-α were also assessed. It was found that CD80 was significantly upregulated in the monocytes isolated from the Mptb-exposed cattle as were the production of nitric oxide, IL-10 and TNF-α. Therefore it was concluded that Mptb-infection induces an atypical activation pattern in macrophages with characteristics of both classical and alternate activation patterns. Also, the phenomenon of immune tolerance and cross-tolerance may play an important role in macrophage responses to subsequent stimulation with mycobacteria and antigens and this could have important implications affecting vaccine efficacy. Therefore, the findings of this study define important biological roles for specific pathogen as well as host-associated components in modulating the macrophage immune responses to Mptb-infection which in turn direct the course of disease progression.