Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses caused by the ectoparasite Ichthyophthirius multifiliis

Real time quantitative PCR (RQ-PCR) assays were developed for the measurement of differential real-time expression of immune-related genes in skin and whole blood from Cyprinus carpio during an infection with the ectoparasite Ichthyophthirius multifiliis. The target genes included the chemokines CXCa and CXCb, the chemokine receptors CXCR1 and CXCR2, the pro-inflammatory cytokines interleukin 1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha) and the enzymes inducible nitric oxide synthase (iNOS) and arginase 2. The strongest up-regulation in skin was observed in the IL-1beta, CXCR1 and iNOS genes at 36-48h post-exposure to theronts. A significant up-regulation of the genes CXCa and TNF-alpha was also observed. An up-regulation of the expression of the genes CXCa, CXCR1, IL-1beta and iNOS was likewise found in blood, although the increase in the expression levels was more moderate and the expression peak was detected earlier in comparison with the skin. In addition, CXCR2 and the arginase 2 genes were specifically induced in blood. Our results confirm the role of CXCR1 and IL-1beta as two prominent molecules involved in the initiation of the inflammatory process in fish in relation to an ectoparasite infection. Moreover, this study confirms the role of carp skin as an important source of pro-inflammatory molecules as well as an active modulator of the local inflammation. Finally, expression and regulation of the evaluated genes in blood confirm the important role of the migrated leucocytes in the immune response against I. multifiliis.     

Cloning and expression of TNF-alpha, IL-1beta and COX-2 in an anadromous and landlocked strain of Atlantic salmon (Salmo salar L.) during the smolting period

The parr-smolt transformation involves complex modulation of immune parameters, affecting both cell populations and humoral factors. The expression of cytokines was studied in salmon cells and tissues during this period using an anadromous and a landlocked freshwater resident dwarf strain of Atlantic salmon (Salmo salar L.). The constitutive activity of three immunoregulatory genes encoding the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) and the cyclo-oxygenase (COX) isoform COX-2 was investigated in head kidney, spleen and gill tissue from healthy, unvaccinated fish by real-time PCR. The TNF-alpha gene was generally lower expressed than COX-2 and IL-1beta1, which were approximately expressed at equal levels and constitutive expression was seen for COX-2 and IL-1beta1 in all tissues examined and at all sampling dates. The expression of all three genes in head kidney and spleen tissue seemed to be highest at the sampling in May for both strains around the time of seawater transfer suggesting an influence of smolting related hormones on cytokine expression. The gill tissue experienced the highest expression of IL-1beta1 and COX-2 at all sampling dates indicating that this organ is immunologically important.     

IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence

IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1β, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1β and IL-8 did not rise in the spleen although an increase of IL-1β, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1β and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1β and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.     

Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida

Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1beta, IL-8, TNF-alpha and IFN-gamma was increased in the proximal intestine. TGF-beta was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1beta, IL-8, TNF-alpha and TGF-beta. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection.     

Evidence for pituitary adenylate cyclase-activating peptide as a direct immunoregulator in teleost head kidney

In mammals, pituitary adenylate cyclase activating polypeptide (PACAP) is a potent anti-inflammatory factor, showing that it inhibits the expression and release of proinflammatory cytokines and enhances the production of anti-inflammatory factors. However, whether fish PACAP plays similar regulatory roles as seen in mammals remains unclear. In the present study, expression of PACAP-specific receptor PAC1-R was shown in grass carp head kidney and spleen, supporting that PACAP may have a direct effect on fish immune cells. To test this hypothesis, the immunoregulatory role of grass carp PACAP (gcPACAP) was examined in head kidney leucocytes (HKLs). Results showed that gcPACAP inhibited basal and further attenuated lipopolysaccharide (LPS)-stimulated cell viability of HKLs, indicating that gcPACAP may possess similar inhibitory property at cellular level as seen in mammals. Curiously, in vitro and in vivo studies revealed that gcPACAP stimulated proinflammatory factors (IL-1β and TNF-α) but not IL-10 mRNA expression in HKLs and head kidney. Moreover, bacterial infection and LPS enhanced IL-1β, TNF-α and IL-10 mRNA expression in grass carp head kidney and HKLs, respectively, and these stimulatory effects were not influenced by gcPACAP. These findings suggest that PACAP plays distinct roles, at least does not function as an anti-inflammatory factor, in fish compared with that in mammals.     

Inducible nitric oxide synthase mediates cytokine release: the time course in conscious and septic rats

Nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-beta (IL-1beta) are postulated to play a key pathophysiologic role during sepsis. In this study, we examined the time course of inducible NO synthase (iNOS) mRNA expression and the plasma TNF-alpha and IL-1beta in lipopolysaccharide (LPS)-treated conscious rats. The hemodynamic pattern in septic shock is more similar to clinical conditions without anesthesia. The data showed that a significant increase in iNOS mRNA levels was found in the spleen, lung, liver, with slight elevation in the heart and kidney at 3 h after LPS administration. However, iNOS mRNA levels were not elevated significantly in all tissues examined at 24 h. In the plasma, TNF-alpha and IL-1beta culminated within 1 h, and reduced gradually to baseline levels in a relatively short period (within 9 h). The results suggest that local NO production by activation of iNOS mRNA expression and cytokine release may contribute to LPS-induced organ dysfunction at various time points.     

The effect of IL-1beta on the expression of inflammatory cytokines and their receptors in human chondrocytes

Cytokines released at sites of inflammation and infection can alter the normal processes of cartilage turnover, resulting in pathologic destruction or formation. Interleukin (IL)-1beta plays a central role in the pathophysiology of cartilage damage and degradation in arthritis. In the present study, we examined the effect of IL-1beta on the expression of IL-1beta, IL-6, IL-8, IL-11, tumor necrosis factor-alpha (TNF-alpha), and their receptors in human chondrocytes. The cells were cultured either with or without 100 U/ml of IL-1beta for up to 28 days. The level of expression of the cytokines and their receptors was estimated by determining mRNA levels using real-time PCR or by determining protein levels using ELISA. The expression of IL-1beta, IL-8, and TNF-alpha markedly increased in the presence of IL-1beta after day 14 of culture. The expression of IL-6 and IL-11 increased greatly in the presence of IL-1beta on day 1 and after day 14 of culture. The expression of IL-1beta, IL-8, IL-11, and TNF-alpha receptors significantly decreased in the presence of IL-1beta after day 14 of culture, whereas the expression of IL-6 receptor significantly increased. The expression of these cytokines, except for IL-6, decreased with the addition of human IL-1 receptor antagonist. These results suggest that IL-1beta promotes the resolution system of cartilage matrix turnover through an increase in inflammatory cytokine production by chondrocytes and that it also may promote the autocrine action of IL-6 through an increase in IL-6 receptor expression in the cells.     

Temperature-dependent expression of immune-relevant genes in rainbow trout following Yersinia ruckeri vaccination

The gene expression of immune-relevant genes in rainbow trout Oncorhynchus mykiss following vaccination with a bacterin of Yersinia ruckeri, a bacterial pathogen causing enteric red mouth disease (ERM), was investigated at 5, 15, and 25 degrees C. Rainbow trout were immunized by i.p. injection of a water-based Y. ruckeri (serotype O1) bacterin, and gene expression profiles were compared to control groups injected with phosphate buffered saline (PBS). Blood and tissue samples (spleen and head kidney) were taken for subsequent analysis using solid phase enzyme-linked immunosorbent assay (ELISA) and real-time PCR, respectively. The up-regulation of cytokine genes was generally faster and higher at high water temperature, with major expression at 25 degrees C. The proinflammatory cytokine interleukin (IL)-1beta and interferon (IFN)-gamma were significantly up-regulated in all immunized groups, whereas the cytokine IL-10 was only up-regulated in fish kept at 15 and 25 degrees C. The gene encoding the C5a (anaphylatoxin) receptor was expressed at a significantly increased level in both head kidney and spleen of immunized fish. The secreted immunoglobulin M (IgM)-encoding gene was significantly up-regulated in the head kidney of immunized trout reared at 25 degrees C, and a positive correlation (r = 0.663) was found between gene expression of secreted IgM in the head kidney and Y. ruckeri-specific antibodies in plasma measured by ELISA. However, no regulation of the teleost specific immunoglobulin T (IgT), which was generally expressed at a much lower level than IgM, could be detected. The study indicated that expression of both innate and specific adaptive immune-response genes are highly temperature-dependent in rainbow trout.     

Outer membrane vesicles from Neisseria meningitidis: effects on cytokine production in human whole blood

The Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine consists of outer membrane proteins (OMPs) as main antigens with significant amounts of lipopolysaccharide (LPS; 5-9% relative to protein). We have studied the ability of this OMV vaccine preparation to induce secretion of pro-inflammatory cytokines, tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), interleukin 6 (IL-6), interleukin 8 (IL-8) and anti-inflammatory cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 13 (IL-13) in a human whole blood model. Plasma levels of TNF-alpha, IL-1beta, IL-6 and IL-8 were massively increased; mean peak levels of TNF-alpha 44 696+/-7764, IL-1beta 38 043+/-5411, IL-6 10 057+/-1619 and IL-8 30 449+/-5397 pg/ml were obtained with an OMV-LPS concentration of 1 microg/ml; corresponding levels in control plasmas were below the detection limit of the assay. Mean maximal level of IL-10 (2540+/-144 pg/ml) was obtained at OMV-LPS concentration of 10 microg/ml, after 24 h; while the level in control plasma was below detection limit. OMV-LPS did not induce release of IL-4 and IL-13 in doses from 0.001-10 microg/ml. The present results show that OMVs from meningococci have potent pro-inflammatory properties and are likely to contribute to the observed local and systemic inflammatory effects.     

The effects of infection of thermal injury by Pseudomonas aeruginosa PAO1 on the murine cytokine response.

Pseudomonas aeruginosa infection, one of the major complications of burn wounds, may lead to sepsis and death. Using the Multi-Probe Template/RNase protection assay, we have compared the expression of different cytokine genes within the skin and livers of thermally injured mice infected with P. aeruginosa PAO1. Thermal injury alone enhanced or up-regulated certain cytokines, including macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1)RI, IL-1 beta, macrophage inflammatory protein (MIP)-1 beta and MIP-2; while PAO1 challenge alone up-regulated tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) expression. The combination of thermal injury plus PAO1 infection enhanced the expression of several pro-inflammatory and haematopoietic cytokines [stem cell factor (SCF), leukocyte inhibitory factor (LIF), IL-6 and TNF-alpha]; induced the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF by 5 h and the expression of additional cytokines, including TGF-beta, TNF-beta, lymphotoxin beta (LT-beta), interferon gamma (IFN-gamma), and IFN-beta by 40 h post-burn/infection. While the most intense cytokine expression occurred in the skin, the majority of cytokines tested were also expressed in the liver by 40 h post-burn/infection. These results suggest that in P. aeruginosa infection of burn wounds: (1) up-regulation of the expression of different cytokines, locally and within the livers of burned mice, is an indication of P. aeruginosa -induced sepsis; and (2) IL-6 and G-CSF play an important role in the host response mechanism.     

IL-10 and IL-4 regulate type-I and type-II IL-1 receptors expression on IL-1β-activated mouse primary astrocytes

When activated by its ligand, the interleukin receptor type I (IL-1RI) transduces signals in cooperation with the IL-1 receptor accessory protein (IL-1RacP). In contrast, IL-1RII functions as a decoy receptor without participating in IL-1 signalling. Brain astrocytes are cellular targets of IL-1 and play a pivotal role in brain responses to inflammation. The regulation of IL-1 receptors on astrocytes by anti-inflammatory cytokines such as IL-4 and IL-10 has not been studied, despite its importance for understanding the way these cells respond to IL-1. Using RT-PCR, we first showed that the expression of IL-1RI and IL-1RII, but not IL-1RacP, mRNAs are up-regulated by IL-1 beta in a time-dependent manner. Using a radioligand binding technique, we then showed that astrocytes display an equivalent number of IL-1RI and IL-1RII. IL-1 beta decreases the number of IL-1RI binding sites, whereas it increases those of IL-1RII. IL-4 and IL-10 both up-regulate IL-1RII IL-1 beta-induced, but only IL-4 does so for IL-1RI. At the protein level, IL-4 and IL-10 dramatically reverse the ability of IL-1 beta to inhibit expression of IL-1RI but neither affects the ability of IL-1 beta to enhance the number of IL-1RII. Collectively, these results establish the existence of receptor cross-talk between pro- and anti-inflammatory cytokines on a critical type of cell that regulates inflammatory events in the brain.