Historically, infectious disease has played a major role in shaping the genetic make-up of populations due to natural selection . Susceptibility and resistance to infectious disease shows marked variation in genetically diverse populations with some individuals expressing extremes of these phenotypes . A goal of livestock breeding programs is to enhance the resistance of animals to infectious disease by selecting for resistant traits and excluding susceptible animals from these programs. The current study focused on Johne’s disease as a model to investigate the underlying biology of this resistance and susceptibility spectrum by targeting red deer which had a genetic bias towards either of these two poles.
In vitro infection of monocyte-derived macrophages with MAP resulted in quantitatively different gene expression profiles in animals with a resistant or susceptible genotype. Macrophages from genetically susceptible animals increased the expression of candidate inflammatory markers (iNOS, IL-1α, and IL-23p19, Figures 3 and 4), to a greater extent than the macrophages from genetically resistant animals. Other researchers have observed increased pro-inflammatory cytokine expression, particularly IFN-γ, IL-1α, TNF-α and IL-17 in the tissues of clinically diseased animals [22, 37, 38]. This suggests that the excessive transcription of inflammatory molecules by genetically susceptible animals observed here may lead to a dysfunctional or dysregulated innate immune response that is incompatible with the development of a protective adaptive immune response. In contrast, resistant animals may produce a more finely regulated and controlled increase in the expression of inflammatory markers in response to MAP which may be a precursor for the initiation of an appropriately controlled, protective adaptive immune response.
Given that immune responses to MAP in macrophages occur early and mycobacterial pathogens are known to express different antigens at different times in the infection process [39–41], the differences observed in gene expression between resistant and susceptible animals could also be a consequence of the sampling endpoint for the assays. At 24 h after infection, the macrophages from resistant animals may have dampened down the response which, initially, may have been considerable. By contrast, the macrophages from susceptible animals may have a persistently high inflammatory profile which could contribute to the activated state of these cells at 24 hpi. A dysregulated inflammatory response may fail to control the primary infection and trigger the immunopathology associated with chronic MAP infection. Monitoring immune markers at earlier and later time points than 24 h will be required to investigate this speculative hypothesis.
Macrophages from three animals of the purebred group displayed a very similar gene expression response to MAP to each other and to the macrophages of the resistant group. It is striking that these animals cluster so closely together and share the same sire which possessed a JBV close to neutral (0.04). On this basis, these animals were removed from the susceptible group and placed in a separate “intermediate” category (Figure 3). These animals were classified as susceptible due to the JBVs of their dams (> 0.3) but their macrophage gene expression responses suggest a strong genetic influence from their sire. While it would be interesting to explore the phenotype of these three “intermediate” animals, a limitation of the experiments presented here is that the study animals could not be classified as phenotypically susceptible or resistant. The functional phenotype could only be disclosed by experimental challenge or monitoring animals exposed to MAP infection naturally under field conditions, which was outside the scope of these experiments.
The expression of IL-10 remained relatively unchanged in response to MAP infection after 24 h and did not differ between the resistant and susceptible animals. IL-10 has been shown to be expressed at a higher level in clinically diseased animals compared to those that are sub-clinical or uninfected  as well as in bovine MDM isolated from infected cattle, in response to MAP challenge in vitro . This cytokine is also reported to be expressed transiently in MDM from cattle and sheep early in the infection process [43, 44]. The data reported here is limited to a single time point of 24 hpi so changes in IL-10 mRNA levels occurring earlier after infection would not have been detected. An alternative source of IL-10 following MAP infection in vivo may also be regulatory T cells as has previously been reported . Another cytokine that may have benefited from earlier or later time points of analysis was IL-12p35 as the expression of this cytokine, while increasing in response to MAP, did not differentiate between resistant and susceptible animals. While IL-12p35 is a key cytokine promoting the Th1 pathway thought to be protective in mycobacterial diseases , the Th1 pathway has also been implicated in the pathology of Johne’s disease [22, 46, 47]. It is difficult to pinpoint the role of this Th1-associated cytokine in protection and disease with no separation in terms of gene expression of macrophages from resistant and susceptible animals.
Two groups of animals were used in this study to assess gene expression of macrophages from genetically resistant and susceptible red deer. The purebred animals showed statistically significant differences in gene expression between the resistant and susceptible genotypes. However, while the crossbred animals displayed similar trends to the purebred group, statistically significant differences were lacking between the resistant and susceptible genotypes. The greater scatter seen in the data obtained from the crossbred animals is compatible with the concept that disease resistance or susceptibility is due to small effects involving multiple genes rather than a single contributing genetic factor. Further, the lack of separation between the resistant and susceptible crossbred animals may be a result of the increased genetic heterogeneity of this group, the smaller groups size (n = 13 compared to n = 20 in the purebred group) or could be influenced by the different method used to categorise the animals as resistant or susceptible.
The use of an exotic model species such as red deer is challenging as protein detection reagents are either not available or have limited cross-reactivity in this species. To investigate functional differences between animals of a resistant or susceptible genotype, fluorescent staining and microscopy techniques were used to assay MAP phagocytosis rates as well as cell death rates. The rate of MAP infection at an MOI of 10:1 was approximately 50% at 24 and 48 hpi, for both types of animals (Figure 6A). Other researchers have noted similar infection rates of approximately 40-70% macrophage infection [48–50]. The majority of infected macrophages from resistant and susceptible animals had ingested less than 10 bacilli (Figure 6B). However, while the numbers of MAP within infected macrophages from resistant animals did not change over the 48 h time point, MAP numbers within macrophages from susceptible animals increased between 24 and 48 h. This could represent the replication of MAP within macrophages from susceptible animals while macrophages from resistant animals are able to prevent MAP multiplication. Further investigation with larger sample numbers is warranted to determine if this trend is significant.
Following MAP infection of macrophages in vitro there are three possible outcomes for the host cell: necrosis, a form of death characterized by plasma membrane disruption, apoptosis, a form of death in which plasma membrane integrity is preserved, or survival of MAP-infected macrophages . The experiments described here were designed to distinguish these three outcomes at 24 and 48 hpi. In general, while the addition of MAP induced greater levels of macrophage apoptosis compared to uninfected controls, the proportion of apoptotic cells was low relative to the positive control (Figure 7A). MAP has been shown to induce apoptosis in bovine monocytes [52, 53] but the levels are generally low as has been noted by Berger et al. in ovine MDM . Several researchers have found that avirulent mycobacterial strains induce higher levels of apoptosis than virulent strains [54–56]. Consequently, apoptosis has been proposed to be an innate defence mechanism which can be subverted by virulent mycobacteria [56, 57]. However, macrophage apoptosis can be increased when a higher MOI of virulent mycobacteria is used in the infection protocol although it has been noted that the apoptotic process quickly converts to a necrotic cell death modality [58, 59]. A greater proportion of macrophages from resistant animals were apoptotic following MAP infection for 24 h compared to the equivalent treatment in the macrophages from susceptible animals (Figure 7A) but this effect was not evident at 48 hpi. It is possible that the macrophages from a resistant animal use apoptosis as an innate defence mechanism in the early stages of infection by depriving MAP of its preferred growth niche, by broadening the activation of other immune cells such as dendritic cells through efferocytosis and by direct anti-mycobacterial activities found in apoptotic macrophages.
Control of mycobacterial infection requires a properly balanced and regulated cytokine environment within infected tissues. The data obtained in this study infers that a dysregulated immune response characterised by excessive inflammatory gene expression occurs in macrophages from susceptible animals. This may pre-empt the development of protective immunity. In the absence of an appropriate adaptive immune response, MAP infection could invoke the chronic immunopathology observed in clinical Johne’s disease. In contrast, macrophages from resistant animals, while expressing the same inflammatory genes as those cells from susceptible animals, do so at a significantly lower level as well as exhibiting higher rates of apoptotic cell death. Together, this implies that controlled regulation of inflammation may be pivotal to protection against chronic inflammatory bowel disease caused by MAP infection.