High gene expression of inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic salmon vaccinated with oil-adjuvanted vaccines

Abstract

Background: Two decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices.Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response.

Results: Granulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-beta (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of T(H)1 T cell lineage (IFN-gamma, CD4) or T(H)2 (GATA-3) responses were differentially expressed.

Conclusions: Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon have the profile of strong expression of genes related to innate immune responses. The expression of TGF-beta, IL-17A and its receptor suggests an involvement of T(H)17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating also a T(H)2-profile. To what extent the expression of IL-17A and its receptor reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon when vaccinated with oil-based vaccines.

Figure 1

Granulomas at injection-site of Atlantic salmon at 12 weeks following injection. Well-circumscribed granulomas (G) induced by oil-adjuvanted vaccine containing standard amount of Moritella viscosa antigens. Note negative impression of oil droplets (O). Bar = 50 μm.

Figure 2

Mixed cell infiltrate at injection-site of Atlantic salmon at 12 weeks following injection. Cellular composition of inflammation consisting of macrophages (M), eosinophilic granular cells or mast cells (EGC), lymphocytes (L) and a few neutrophils (N). Fibroblasts are also distinct (F). Bar = 10 μm.

Figure 3

Severe inflammatory reaction at injection-site of Atlantic salmon at 12 weeks following injection. A severe granulomatous reaction with diffuse granulomas (G) induced by vaccine containing 6× standard M. viscosa antigens (FO-8). O- negative impression of oil droplets. Bar = 100 μm.

Figure 4

Cellular composition of severe vaccine granuloma of Atlantic salmon at 12 weeks following injection. Granulomas constitute of large macrophages (M), several lymphocytes (L), EGCs, and multinucleate cells (MGC). Bar = 15 μm.

Figure 5

Regulated genes in different groups of Atlantic salmon as determined by microarrays. FO-2 = A. salmonicida (6 × standard concentration - sc); FO-7 = M. viscosa (sc); FO-8 = M. viscosa (6 × sc).

Figure 6

Markers of inflammation expressed in the head kidney of Atlantic salmon injected with different oil-based vaccines. Quantitative real-time RT-PCR (qPCR) expression of genes identified as upregulated by microarray. FO-1 = A. salmonicida (sc); FO-2 = A. salmonicida (6 × sc); FO-7 = M. viscosa (sc); FO-8 = M. viscosa (6 × sc). Gene expression ratios normalized to Elongation factor 1α (EF1α). Error bars = standard error (SE), n= 7. *p < 0.05.

Figure 7

Antigen processing and presentation genes expressed in the head kidney of Atlantic salmon injected with different oil-based vaccines. qPCR expression studies of C-type lectin receptor (CLR), antigen recognition and processing by phagocytes (cathepsin D) and antigen presentation (CD209) genes identified as upregulated by microarray. FO-1 = A. salmonicida (sc); FO-2 = A. salmonicida (6 × sc); FO-7 = M. viscosa (sc); FO-8 = M. viscosa (6 × sc). Gene expression ratios normalized to EF1α. Error bars = SE, n= 7. *p < 0.05.

Figure 8

Cellular signaling and chemotaxin markers expressed in the head kidney of Atlantic salmon injected with different oil-based vaccines. qPCR expression studies of chemokine (CC) and leukocyte cell derived chemotaxin 2 (LECT 2) genes identified as upregulated by microarray. FO-1 = A. salmonicida (sc); FO-2 = A. salmonicida (6 × sc); FO-7 = M. viscosa (sc); FO-8 = M. viscosa (6 × sc). Gene expression ratios normalized to EF1α. Error bars = SE, n= 7. *p < 0.05.

Figure 9

Immunoglobulin genes expressed in the head kidney of Atlantic salmon injected with different oil-based vaccines. qPCR expression studies of IgM and immunoglobulin heavy chain constant region (IGHC) genes identified as upregulated by microarray. FO-1 = A. salmonicida (sc); FO-2 = A. salmonicida (6 × sc); FO-7 = M. viscosa (sc); FO-8 = M. viscosa (6 × sc). Gene expression ratios normalized to EF1α. Error bars = SE, n= 7. *p < 0.05.

Figure 10

Arginase and IL17AR genes expressed in the head kidney of Atlantic salmon injected with different oil-based vaccines. qPCR expression studies of arginase and IL17A receptor (IL17AR) identified as upregulated by microarray. FO-1 = A. salmonicida (sc); FO-2 = A. salmonicida (6 × sc); FO-7 = M. viscosa (sc); FO-8 = M. viscosa (6 × sc). Gene expression ratios normalized to EF1α. Error bars = SE, n= 7. *p < 0.05.

Figure 11

Cytokine genes expressed in the head kidney of Atlantic salmon injected with different oil-based vaccines. qPCR expression studies of IL17A and transforming growth factor β (TGF-β) genes identified as upregulated by microarray. FO-1 = A. salmonicida (sc); FO-2 = A. salmonicida (6 × sc); FO-7 = M. viscosa (sc); FO-8 = M. viscosa (6 × sc). Gene expression ratios normalized to EF1α. Error bars = SE, n= 7. *p < 0.05.

Figure 12

Microarray study design. Total RNA from 12 fish in each group was subdivided into pools of 4 fish each and subsequently used for hybridization. Vaccine A represents the reference group (FO-1 A. salmonicida standard concentration).