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Rainbow trout (Oncorhynchus mykiss) represents the third most produced species of diadromous fish, with the total production of 0,732 million tonnes in 2009. More than one third of this production comes from Europe, where it is dominated by Norway, Italy and France. Germany is the fifth biggest producer in Europe, producing 21 thousand tonnes of rainbow trout in the value of 6,1 million Euro. However, the conditions in the intensive aquaculture often increase the disease susceptibility to many pathogens. One of the highest economic threats for a salmonids aquaculture is the causative agent of furunculosis, Aeromonas salmonicida subsp. salmonicida. Several strategies have been developed to protect the fish, but the traditional methods are either laborious or represent a potential risk for the environment. The selective breeding established more than 35 years ago in the brackish waters of Baltic Sea represent a attractive alternative, delivering a novel strain of rainbow trout better adapted to the brackish environment and exhibiting reduced mortality in the infection with A.salmonicida. Nevertheless, no information was available about the fundaments of this phenomenon. Thus, the aim of presented study was the identification of immune adaptations, which occurred during the 30 years of selection and favoured increased survival of “born” trout to the bacterial diseas es. In the presented work, the peritoneal cavity of rainbow trout has been used as a model for the investigation of disease resistance in fish. In the first chapter, the peritoneal cavity has been described as a unique niche of teleost immune system and the kinetic of peritoneal leukocytes induced by the stimulation has been analysed. Furthermore, a unique set of monoclonal antibodies has been used to evaluate the contribution of distinct cell populations on the inflammation and its resolution. In the second part of the study, the transcriptional changes of peritoneal leukocytes have been evaluated using the GRASP microarray. The following analysis provided unique insights into the local immune response in rainbow trout. The unprecedented combination of both data sets offers an unparalleled description of the local immune response in teleost fish and can be summarized into following facts. In general, the obtained results revealed, that the unstimulated peritoneal cavity is populated predominantly by lymphocytes with IgM+ Bcells being the major cells type. The rapid changes in the composition induced by the stimulation were underlined by the upregulation of major proinflammatory molecules such as IL1β, IL8 and TNFα within 12hpi. Although the initial phase of the reaction was dominated by myeloid cells, the cavity underwent within 72 hours two complete changes in the composition corresponding with the massive changes in the transcriptome. Eventually, the resolution of inflammation was marked by an increasing number of lymphocytes and correlated with the downregulation of pro-inflammatory genes to the initial level and upregulation of anti-inflammatory cytokines IL10 and TGFβ. Besides the general observations common to all treatments and both strains, our experiments revealed also remarkable differences between the antigenic stimulation and reaction towards pathogen. From these differences following conclusions can be drawn; the infection induces comparable reaction pattern as the stimulation, although the intensity of the reaction and number of cells is higher. These observations correlated with the higher expression of inflammatory molecules after the infection. Viable bacteria also prolong the myeloid phase of the reaction and delay the resolution of inflammation. Finally, model of peritoneal inflammation caused by A. salmonicida has been applied also to the second strain of rainbow trout, known for its higher resistance to infection. The comparison of obtained data suggested that resistant trout reacted to the antigenic stimulation and infection with a lower number of cells despite minor differences in the expression level of major pro-inflammatory molecules during early stages of the infection. Eventually, the resolution of inflammation and onset of adaptive immune response occurred in resistant trout almost 24 hours earlier and was correlating with an increased expression of anti-inflammatory cytokines IL10 and TGFβ. Notably, the increased survival of resistant strain correlates with the increased expression of antibacterial proteins such as NRAMP and hepcidin. Taken together, obtained data provided unprecedented insights into the local immune response in teleost fish and identified features conserved during the selection breeding in the brackish water of Baltic Sea. Additionally, combination of cellular and molecular data elucidates the peritoneal inflammation in fish and suggested high conservation of the immune response in the evolution.
Challenge of immunized mice with H. pylori induces protective gastric inflammation that is histologically indistinguishable from chronic H. pylori-associated gastritis in non-immune mice. To identify mechanisms of protective immunity gene expression in the gastric tissue from infected mice and mice vaccinated prior to challenge was compared by DNA array analysis. Message RNA was used to screen over 10,000 murine genes. Major Histocompatibility Complex antigens and IFN-γ dependent GTP binding proteins were strongly upregulated in both infected and immunized/challenged mice compared to naive controls. Differences in gene expression were also observed in novel T cell genes, which were exclusively upregulated in immunized/challenged mice. Both IFN I and II associated genes like the IFN-a/ßreceptor or IFN dependent transcription factors mIRF-1 and ISGF3 were also predominantly expressed in this group. These results were confirmed for several candidate genes by semi-quantitative RT-PCR. Additionally, H. pylori-stimulation of CD4+ T cells from immune mice induced significantly more IFN-γ production than stimulation of cells from infected mice. The present study provides evidence that the inflammatory infiltrate that arises in the gastric mucosa when immunized mice are challenged with H. pylori is associated with specific T cells sets and protein families that are distinct from those present in Helicobacter-associated chronic inflammation. Gene array profiles and in vitro assays indicate that immunized mice are more readily poised than infected mice to promote IFN-γ production and IFN related events and thus promote a strong proinflammatory THi response. This study supports recent findings that an immune response dominated by THi cytokines is essential for protection from H. pylori infection. This insight could facilitate the choice of the appropriate adjuvants for the development of vaccines against H. pylori, which are efficient and safe for use in humans. The mechanisms by which THi cells induce protective immunity or reduce Helicobacter colonization remain poorly understood and will be subject of future research.