Host responses to Renibacterium salmoninarum and specific components of the pathogen reveal the mechanisms of immune suppression and activation

Abstract

During infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish. Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen. It was found that the expression of msa encoding the p57 antigen of R. salmoninarum, was constitutive, while the expression of hly and rsh, encoding haemolysins, and lysB and grp was reduced after infection. Macrophages showed a rapid inflammatory response in which the expression of interleukin-1beta (IL-1beta), major histocompatibility complex class II (MHC II), inducible cyclo-oxygenase (Cox-2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor-alpha (TNF-alpha) expression was greatly reduced initially and then increased. After 5 days, except for TNF-alpha and MHC II, expression returned to levels approaching those of uninfected macrophages. We propose that R. salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF-alpha-dependent killing pathways. The effects of specific R. salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa, hly, rsh, lysB, or grp. We found that msa reduced the expression of IL-1beta, Cox-2, and MHC II but stimulated TNF-alpha while hly, rsh and grp stimulated MHC II but down-regulated TNF-alpha. Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF-alpha. The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R. salmoninarum infections rely on MHC II- and TNF-alpha-dependent pathways. Moreover, prolonged stimulation of TNF-alpha may contribute to the chronic inflammatory pathology of bacterial kidney disease.

Figure 1

Effect of increasing MOI on the expression of (a) Renibacterium salmoninarum gene hly and (b)GAPDH of trout macrophages. Trout macrophages were infected with R. salmoninarum and incubated for 2 hr, 1 day and 5 days at 15°. Total RNA was extracted, reverse transcribed to cDNA and amplified with specific primers by PCR. Data are representative of one experiment which was repeated three times. C, control samples taken immediately prior to infection of either (a) R. salmoninarum or (b) trout macrophage cDNA; +, reactions containing reverse transcriptase; −, reactions without reverse transcriptase; w, reactions with water substituted for RNA template.

Figure 2

Expression of Renibacterium salmoninarum genes msa, hly, rsh, lysB and grp following infection of trout macrophages with R. salmoninarum using MOI=5. The same result was obtained from three separate experiments. C, control samples of R. salmoninarum cDNA taken immediately prior to infection; Mø, cDNA from uninfected macrophages; +, reactions containing reverse transcriptase; −, reactions without reverse transcriptase; w, reactions with water substituted for RNA template.

Figure 3

Expression of cytokine and cytokine-related genes following infection of trout macrophages with Renibacterium salmoninarum. Mø, cDNA from uninfected trout macrophages. +, reactions containing reverse transcriptase; −, reactions without reverse transcriptase; w, reactions with water substituted for RNA template. The experiments were repeated three times with the same result.

Figure 4

Expression of DNA vaccine constructs in the tissues of rainbow trout injected with 20 µg plasmid DNA. After 7 days the muscle was fixed and observed using immunofluorescence. (a) Unstained muscle tissue from unimmunized trout; (b) a dendritic-like cell from the muscle of a fish injected with pLysB13; (c) muscle from a fish injected with pGrp11; (d) the same tissue as in (c) but with bright field illumination and fluorescence to show the structure of the muscle tissues and the localization of the staining within an individual myotome; (e) muscle from a fish injected with pHly1 showing the diffuse staining and vacuolation (*) of the surrounding tissue which was similar to the muscle from fish injected with pMsa2. All figures are × 400 by original magnification.

Figure 5

Expression of cytokine and cytokine-related genes in the pronephros of rainbow trout at time-points following the intramuscular injection of DNA vaccine constructs expressing Renibacterium salmoninarum genes hly, grp, lysB, or rsh or with the plasmid vector, pcDNA3.1, alone. Data were gathered from a single experiment and only those genes demonstrating a consistent pattern of variation between constructs and/or over time are shown. The reactions were repeated to confirm reproducibility. GAPDH expression shows the consistency of the RT-PCR for each sampling occasion. UC, samples taken from unimmunized control fish corresponding to weeks 1, 3, 5 and 7 from left to right. +, reactions containing reverse transcriptase; −, reactions without reverse transcriptase; w, reactions with water substituted for RNA template.

Figure 6

(a) Expression of cytokine and cytokine-related genes in the pronephros of rainbow trout following the intramuscular injection of pMsa2. For comparative purposes the expression of IL-1β, Cox-2, MHC II, TNF-α, TGF-β, CXC-R4 and CC-R7 in fish injected with pcDNA3.1 is presented in (b). +, reactions containing reverse transcriptase; −, reactions without reverse transcriptase; w, reactions with water substituted for RNA template.