- Email: [email protected]
Behaviour, individual variation and immunity
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
Economic Animals, College of Life Sciences, Sun Yat-Sen University, Guangzhou, PR China Abstract Innate immune response, the first-line of defense against pathogenic microorganisms, requires macrophages to sense and respond to pathogenassociated molecule patterns (PAMPs) by a broad repertoire of pattern recognition receptors (PRR), especially Toll-like receptor (TLR). Interferon-g (IFNg) is one of the most potent macrophage-activating factors. IFNg-priming macrophages are more sensitive to intracellular pathogens and functions to amplify the TLR-induced cellular responses. However, the involvement of IFNg in teleost TLR recognition system is still unknown. In contrast to single copy of IFNg in mammalian genomes, we have cloned two IFNg isoforms in Tetraodon nigroviridis by analysing its genome database. Herein, we made a comparison of the two IFNg in the regulation of TLR recognition system. For functional analysis, Tetraodon recombinant IFNg1 (rIFNg1) and IFNg2 (rIFNg 2) was produced and purified. Proliferation assays revealed the distinct influence of two rIFNgs. The analysis of downstream gene expressions showed that rIFNg1 regulated ISG15 and MX expression, while rIFNg2 only augmented ISG15 and no effects on MX. To estimate the influence of rIFNg1 or rIFNg2 on TLR system at transcriptional level, six TLR family members and two TLR signaling molecules were selected: TLR1-TLR3, TLR7-TLR9, MyD88 and TRAF6. Realtime PCR showed that mRNA levels of TLRs and signaling molecules were affected by rIFNgs in varying degrees. The rIFNg1 slowly increased eight TLR gene expression, functioned at least 1 h after stimulation. Whereas, rIFNg2 had rapid effects within 0.5 h resulting in sharp increases of mRNA transcripts of TLR1, TLR2, TLR3, TRAF6 or decreases of TLR7, TLR8, TLR9 and MyD88. Distinct regulatory ability of two rIFNgs suggested that they probably have different roles in the regulation of TLR system at transcriptional level. To further confirm the involvement of rIFNg in TLR-mediated signal transduction, western blot and EMSA analysis were employed to characterize the changes of MyD88, TFAF6 and NF-kB and the activation of NF-kB upon exposure to two rIFNgs. The rIFNg1-treatment gradually stimulated MyD88-, TFAF6- and NF-kB-protein synthesis. But exposure to rIFNg2 led to the quick changes of MyD88, TFAF6 and NF-kB within 1 h. EMSA also indicated the tardy influence of rIFNg1 and rapid effects of rIFNg2 on the activation of NF-kB. In summary, we have demonstrated that the two Tetraodon IFNgs regulated the TLR pathogen-recognition system at multilevels and varied paces.infection. * Corresponding author. E-mail address: [email protected] (B. Novoa)
O-137. MAPK-activated CA-TSP, hemocyanin and lectin-like proteins form a protein complex in the hemocyte of oyster Crassostrea ariakensis Gould M. Luo 1, S. Ye 1,2, T. Xu 1, X. Wu 1, *. 1
Laboratory of Marine Life Science and Technology, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China; 2 College of Life Science and Biotechnology, Dalian Ocean University, Dalian, Liaoning, China Abstract Tetraspanin proteins regulate cell morphology, motility, invasion, fusion and signaling as organizers of multi molecular membrane complexes. A novel tetraspanin from the oyster, Crassostrea ariakensis Gould that named Ca-TSP was identified previously in our laboratory. In order to further study the functional role of Ca-TSP, the Co-immunoprecipitation (Co-IP) assays, Immunofluorescence assay using confocal laser scanning system, and haemagglutination (HA) and adsorption assays were carried out. CaTSP-specific antibody was made. The results showed that Ca-TSP and hemocyanin were colocalized close to the nucleus of the hemocytes. Although not all hemocytes were strongly positive, hemocytes positive for anti-Ca-TSP were also positive for anti-Hemocyanin staining, indicating the presence of hemocyanin and Ca-TSP in one single cell. In addition, CaTSP and hemocyanin colocalized in positive granules. The Co-IP assays with Ca-TSP-specific antibody showed that two proteins, hemocyanin, and
1663
an unknown protein, were specifically bound with Ca-TSP protein. The Western blot indicated that the protein band corresponding to hemocyanin was observed in the Co-IP eluate using hemocyanin-specific antibody, showing the existence of Ca-TSP-hemocyanin complex. To confirm the binding of hemocyanin with Ca-TSP, the specific antibody against hemocyanin was used in Co-IP. The Western blot results showed hemocyanin not only binded with Ca-TSP also with their multimer. In an attempt to address isolated unknown protein, except for above Co-IP assays, the haemagglutination (HA) of whole haemolymph and isolated unknown protein against the rabbit erythrocytes were determined. The results showed that isolated unknown protein is about 40 KD. Both oyster haemolymph and the isolated protein agglutinated rabbit erythrocytes. The highest titre of the isolated unknown protein was about 4.5. In addition, here Ca-TSP was found to be up-regulated by LPS, but was inhibited by MEK inhibitors, for example, pre-incubation with MEK inhibitor U0126 and PD98059 resulted in a reduction of p-MAPK after 1h of treatment, but the level of p-MAPK increased a little after 12h of treatment. And Ca-TSP level was down-regulated in hemocytes after 12h of MEK inhibitor treatments in vitro. This is in agreement with our previous study, where challenge of Crassostrea ariakensis hemocytes with the outer membrane protein (ompR) of rickettsia-like organism was carried. The ompR induced a rapid and persistent increase in the level of phosphorylated P38 and a large decrease in the level of phosphorylated JNK. The ompR mediated increase in NF-kB DNA binding activity was significantly inhibited by the addition of SB203580, an inhibitor of the P38 pathway (Zhu and Wu, 2008). According to above analyses, it was suggested that the MAP kinase pathway may be involved in the regulation of Ca-TSP. * Corresponding author. E-mail address: [email protected] (X. Wu)
KS-3. Behaviour, individual variation and immunity S. MacKenzie. Institute of Aquaculture, University of Stirling, UK
Abstract Understanding individual susceptibility to disease is a major challenge in biological research with significant implications for animal and human health. To understand the complex interactions between healthy and disease states it is of utmost importance to address biological variation within the population under relevant environmental conditions. Recent studies incorporating elements of behavioural ecology into fish immunology have shed light upon the underlying molecular mechanisms of the immune response from two distinct viewpoints. Firstly, fish are ectothermic, as are the vast majority of animal species, and can only manipulate their body temperature by choice of an appropriate environmental temperature. Under infectious conditions, fish express behavioural fever where thermo-coupling of the immune response, at a gene-environment level, acts to increase the efficiency of the response to infection. This leads to increased survival. Secondly, consistent individual variability in behaviour and physiological response to challenge over time and context referred to as individual coping style has been reported within animals of the same species, sex and population including fish. Two distinct coping styles, proactive and reactive, have been described and individuals display significant differences in stress reactivity and neuroendocrine regulation. Recently coping styles have also been correlated to immunity and welfare. Thus coping styles provide a framework toward understanding biological variation in a population by helping to resolve observed variance in measured variables such as gene expression. Our aim is to explore the interface between environmental complexity and the individual's capacity to adapt and meet these demands in the context of the immune response and survival. Future studies aim to shed light upon the underpinning molecular mechanisms that determine susceptibility and resistance to disease within and between animal populations.
E-mail address: [email protected] (S. MacKenzie)
Economic Animals, College of Life Sciences, Sun Yat-Sen University, Guangzhou, PR China Abstract Innate immune response, the first-line of defense against pathogenic microorganisms, requires macrophages to sense and respond to pathogenassociated molecule patterns (PAMPs) by a broad repertoire of pattern recognition receptors (PRR), especially Toll-like receptor (TLR). Interferon-g (IFNg) is one of the most potent macrophage-activating factors. IFNg-priming macrophages are more sensitive to intracellular pathogens and functions to amplify the TLR-induced cellular responses. However, the involvement of IFNg in teleost TLR recognition system is still unknown. In contrast to single copy of IFNg in mammalian genomes, we have cloned two IFNg isoforms in Tetraodon nigroviridis by analysing its genome database. Herein, we made a comparison of the two IFNg in the regulation of TLR recognition system. For functional analysis, Tetraodon recombinant IFNg1 (rIFNg1) and IFNg2 (rIFNg 2) was produced and purified. Proliferation assays revealed the distinct influence of two rIFNgs. The analysis of downstream gene expressions showed that rIFNg1 regulated ISG15 and MX expression, while rIFNg2 only augmented ISG15 and no effects on MX. To estimate the influence of rIFNg1 or rIFNg2 on TLR system at transcriptional level, six TLR family members and two TLR signaling molecules were selected: TLR1-TLR3, TLR7-TLR9, MyD88 and TRAF6. Realtime PCR showed that mRNA levels of TLRs and signaling molecules were affected by rIFNgs in varying degrees. The rIFNg1 slowly increased eight TLR gene expression, functioned at least 1 h after stimulation. Whereas, rIFNg2 had rapid effects within 0.5 h resulting in sharp increases of mRNA transcripts of TLR1, TLR2, TLR3, TRAF6 or decreases of TLR7, TLR8, TLR9 and MyD88. Distinct regulatory ability of two rIFNgs suggested that they probably have different roles in the regulation of TLR system at transcriptional level. To further confirm the involvement of rIFNg in TLR-mediated signal transduction, western blot and EMSA analysis were employed to characterize the changes of MyD88, TFAF6 and NF-kB and the activation of NF-kB upon exposure to two rIFNgs. The rIFNg1-treatment gradually stimulated MyD88-, TFAF6- and NF-kB-protein synthesis. But exposure to rIFNg2 led to the quick changes of MyD88, TFAF6 and NF-kB within 1 h. EMSA also indicated the tardy influence of rIFNg1 and rapid effects of rIFNg2 on the activation of NF-kB. In summary, we have demonstrated that the two Tetraodon IFNgs regulated the TLR pathogen-recognition system at multilevels and varied paces.infection. * Corresponding author. E-mail address: [email protected] (B. Novoa)
O-137. MAPK-activated CA-TSP, hemocyanin and lectin-like proteins form a protein complex in the hemocyte of oyster Crassostrea ariakensis Gould M. Luo 1, S. Ye 1,2, T. Xu 1, X. Wu 1, *. 1
Laboratory of Marine Life Science and Technology, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China; 2 College of Life Science and Biotechnology, Dalian Ocean University, Dalian, Liaoning, China Abstract Tetraspanin proteins regulate cell morphology, motility, invasion, fusion and signaling as organizers of multi molecular membrane complexes. A novel tetraspanin from the oyster, Crassostrea ariakensis Gould that named Ca-TSP was identified previously in our laboratory. In order to further study the functional role of Ca-TSP, the Co-immunoprecipitation (Co-IP) assays, Immunofluorescence assay using confocal laser scanning system, and haemagglutination (HA) and adsorption assays were carried out. CaTSP-specific antibody was made. The results showed that Ca-TSP and hemocyanin were colocalized close to the nucleus of the hemocytes. Although not all hemocytes were strongly positive, hemocytes positive for anti-Ca-TSP were also positive for anti-Hemocyanin staining, indicating the presence of hemocyanin and Ca-TSP in one single cell. In addition, CaTSP and hemocyanin colocalized in positive granules. The Co-IP assays with Ca-TSP-specific antibody showed that two proteins, hemocyanin, and
1663
an unknown protein, were specifically bound with Ca-TSP protein. The Western blot indicated that the protein band corresponding to hemocyanin was observed in the Co-IP eluate using hemocyanin-specific antibody, showing the existence of Ca-TSP-hemocyanin complex. To confirm the binding of hemocyanin with Ca-TSP, the specific antibody against hemocyanin was used in Co-IP. The Western blot results showed hemocyanin not only binded with Ca-TSP also with their multimer. In an attempt to address isolated unknown protein, except for above Co-IP assays, the haemagglutination (HA) of whole haemolymph and isolated unknown protein against the rabbit erythrocytes were determined. The results showed that isolated unknown protein is about 40 KD. Both oyster haemolymph and the isolated protein agglutinated rabbit erythrocytes. The highest titre of the isolated unknown protein was about 4.5. In addition, here Ca-TSP was found to be up-regulated by LPS, but was inhibited by MEK inhibitors, for example, pre-incubation with MEK inhibitor U0126 and PD98059 resulted in a reduction of p-MAPK after 1h of treatment, but the level of p-MAPK increased a little after 12h of treatment. And Ca-TSP level was down-regulated in hemocytes after 12h of MEK inhibitor treatments in vitro. This is in agreement with our previous study, where challenge of Crassostrea ariakensis hemocytes with the outer membrane protein (ompR) of rickettsia-like organism was carried. The ompR induced a rapid and persistent increase in the level of phosphorylated P38 and a large decrease in the level of phosphorylated JNK. The ompR mediated increase in NF-kB DNA binding activity was significantly inhibited by the addition of SB203580, an inhibitor of the P38 pathway (Zhu and Wu, 2008). According to above analyses, it was suggested that the MAP kinase pathway may be involved in the regulation of Ca-TSP. * Corresponding author. E-mail address: [email protected] (X. Wu)
KS-3. Behaviour, individual variation and immunity S. MacKenzie. Institute of Aquaculture, University of Stirling, UK
Abstract Understanding individual susceptibility to disease is a major challenge in biological research with significant implications for animal and human health. To understand the complex interactions between healthy and disease states it is of utmost importance to address biological variation within the population under relevant environmental conditions. Recent studies incorporating elements of behavioural ecology into fish immunology have shed light upon the underlying molecular mechanisms of the immune response from two distinct viewpoints. Firstly, fish are ectothermic, as are the vast majority of animal species, and can only manipulate their body temperature by choice of an appropriate environmental temperature. Under infectious conditions, fish express behavioural fever where thermo-coupling of the immune response, at a gene-environment level, acts to increase the efficiency of the response to infection. This leads to increased survival. Secondly, consistent individual variability in behaviour and physiological response to challenge over time and context referred to as individual coping style has been reported within animals of the same species, sex and population including fish. Two distinct coping styles, proactive and reactive, have been described and individuals display significant differences in stress reactivity and neuroendocrine regulation. Recently coping styles have also been correlated to immunity and welfare. Thus coping styles provide a framework toward understanding biological variation in a population by helping to resolve observed variance in measured variables such as gene expression. Our aim is to explore the interface between environmental complexity and the individual's capacity to adapt and meet these demands in the context of the immune response and survival. Future studies aim to shed light upon the underpinning molecular mechanisms that determine susceptibility and resistance to disease within and between animal populations.
E-mail address: [email protected] (S. MacKenzie)