Several genes including complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and IgM were identified in the head kidney as associated with severe injection-site granulomatous reactions in this study. The expression of these genes corresponds with gene profiles of an active inflammation [44
] and corroborate previous reports that vaccine-based granulomas are associated with a chronic, active inflammation [2
]. Furthermore, the finding that IL-17A and its receptor (IL-17AR) representing TH
17 cells were up-regulated in fish with severe reactions while none of the genes directly reflective of TH
1 T cell lineage (IFN-γ, CD4) or TH
2 (GATA-3) differentiation were differentially expressed is interesting in light of the involvement of the TH
17 cells in autoimmune responses [13
Chronic, active inflammation is consistent with the presence of neutrophils and macrophages at the injection site of FO-8. In this group, genes encoding LECT-2 and CC chemokine were verifiably up-regulated in head kidney indicating an active inflammatory process in the "draining lymph node." Further to this, the up-regulation of mRNA transcripts of complement factors C1q and C6, mannose binding protein, lysozyme C, innate cell-associated proteins of the C-type lectin receptors, CD209, Cathepsin D and CD63, anti-oxidative genes such as metallothionein and oxidative stress-related genes are additional indications of active inflammatory processes [48
] in fish with severe granulomatous reactions.
Another intriguing result was the finding that M. viscosa
(FO-7&8) is more pro-inflammatory than A. salmonicida
(FO-1&2). In natural infection, lethality of these pathogens is in the reverse [49
]. The cause of the difference in this study is not clear but it is not unlikely that the structure or quantity of pathogen-associated molecular patterns (PAMPs) [50
], or even the orientation/presentation of antigens on the surface of oil droplets may be responsible. The importance of the different factors can only be solved in future studies.
Macrophages are the main cell type responsible for the uptake, breakdown, removal or sequestration of antigens. For an effective response, appropriate activation is necessary. The observed expression of arginase in this study is consistent with macrophages activated through the alternative pathway and is indicative of a TH
2 response [51
]. In humans, it has been documented that TH
2 cytokines bias responses towards inadequate activation of macrophages resulting in less efficient control of Mycobacterium leprae
infections and high bacterial counts inside granulomas leading to diffuse spreading of the infection [52
]. To what extent macrophage activation plays a role in clearing of the vaccine antigens in salmonids is not known in detail. It is not possible to deduce from the results of this study whether the alternative macrophage activation pathway is an appropriate response but this should be a subject of further investigation.
The massive up-regulation of IgM mRNA transcripts in the head kidneys of fish with severe injection-site reactions (Additional file 1
) is suggestive of a strong humoral response, another indication of a TH
2 response consistent with reports of others [53
]. It must be noted however that no tests to examine the specificity of antibodies were conducted in this study. It is thus unknown whether the bulk of the IgM mRNA transcripts detected in this study are indications of responses against vaccine-related antigens or if they were directed at self (autoimmune) antigens. Autoimmunity has previously been suggested as one of the etiologies of vaccine associated side effects in Atlantic salmon [36
In fish with mild injection-site reactions, microscopic examinations revealed a process of wound healing. Mast cells and macrophages were observed in abundance at the injection site of fish in these groups (FO-1, FO-2 and FO-7). These cells have previously been associated with granulomatous reactions [2
] and wound healing [42
], although they have also been associated with the initiation of inflammation in Atlantic salmon [56
]. In higher vertebrates TH
2 responses have been shown to play an important role in wound healing [57
] with mast cells producing or influencing the production of IL-4 and IL-10 tipping the response towards TH
]. In the present study, no significant differences in the expression of GATA-3 or IL-10 were observed between different groups. Similarly, no differences were observed for IL-12, IFN-γ, CD4, CD8 and Granzyme A. As similar antigen preparations per pair of vaccines were used differing only in concentrations, the anticipation is that similar TH
responses drove the inflammatory responses in all groups; the difference in severity being motivated by antigen concentration and determined mainly by the innate response.
Interestingly, the expression of IL-17A and its receptor showed similar trends as the severity of inflammatory profiles (Figures and , and Table ), being significantly up-regulated (p = 0.007 and 0.009, respectively) only in the group with severe granulomas. IL-17A is a pro-inflammatory cytokine produced predominantly by activated T cells. It acts through its receptor IL-17AR which, although ubiquitously expressed, its main responses in higher vertebrates are found in epithelial cells, endothelial cells, fibroblasts, macrophages and dendritic cells (reviewed in ref. [60
]). It is an essential component of the IL-17A signaling whose induction is required for effective responses [60
]. It has also been demonstrated to be up-regulated together with IL-17A in autoimmune diseases [65
]. IL-17A expression induces the production of an array of cytokines and metalloproteases by fibroblasts, endothelial cells, macrophages and epithelia cells resulting in the induction of inflammation and recruitment of neutrophils [68
]. These findings suggest that IL-17A/IL-17AR and therefore the TH
17 response contributes to the induction of severe side effects in line with the higher number of infiltrating neutrophils and macrophages in this group. To what extent autoimmunity as previously reported [36
] is involved in these responses remains elusive as the presence of autoantibodies was not assessed in this study.
The involvement of TGF-β in severe granulomatous reaction in this study is compelling given the expression patterns exhibited in different groups (Figure ) relative to the degree of side effect lesions (Table ). In mammals, the expression of TGF-β has previously been associated with the recruitment of polymorphonuclear cells to inflammatory sites [69
]. Together with IL-6, IL-21 or IL-23, it has also been demonstrated to be essential in the commitment of naïve T cells into TH
17 cells [14
]. TGF-β is produced by cells of the innate immune system [70
] as well as regulatory T cells (T-regs) [71
]. Although only the receptor for IL-6 and not the cytokine itself was examined in the present study and found not to be induced, it is not unlikely that a different expression pattern would have been observed with the latter. On the other hand, this finding may suggest that other cytokines such as IL-21 or IL-23 act in concert with TGF-β to regulate TH
17 cells in the induction of severe granulomatous reactions in Atlantic salmon.
The screening of differentially expressed genes for granulomatous reactions using microarrays in this study was done by examining head kidney tissues rather than the injection site. It is conceivable that more inflammatory genes would be up- or down-regulated at the latter compared to the former, as most inflammatory genes are known to act locally. In i.p. vaccinated fish, the injection site is the abdominal cavity and vaccine components localize primarily at the pyloric caeca/pancreas area [2
]. Because of the anatomical co-localization of the pylorus with the pancreas, collection of homogeneous tissue for gene expression studies is problematic. An alternative approach would be to vaccinate the fish intramuscularly and examine gene expression at the injection site as previously reported [73
]. However, for vaccines routinely administered i.p., this approach would have to be weighed against the prospect of modifying the immune response since different routes of vaccination are known to influence the resulting TH
In conclusion, the profile of the immune responses as assessed by gene expression to oil-adjuvanted vaccines in Atlantic salmon is strongly influenced by antigen content and also the type of antigens included in the vaccine preparation. There is a variation in the response along an axis of a chronic, active inflammation on one end to mild inflammation and wound healing on the other. Gene expression patterns indicative of neutrophil persistence and macrophage activation are biased towards a mixed reaction between TH2 and TH17 profiles in the head kidney.