M.K. Chadzinska

CXCL8 Chemokines in Teleost Fish: Two Lineages with Distinct Expression Profiles during Early Phases of Inflammation

Background During the inflammatory process, chemokine CXCL8 plays a pivotal role in recruitment of human neutrophilic granulocytes. A diversity of sequences similar to CXCL8 was reported in fish, but their evolutionary relationships and functional homology with their human homolog remain unclear. Principal Findings We screened fish genomes to seek for sequences related to CXCL8. A first lineage was retrieved in all teleosts, while a second CXCL8 lineage was found in zebrafish and carp only. An early inflammatory function for both lineages was indicated by several lines of evidence. The induction of carp CXCL8s, CXCb, and CXC receptor-1 and -2 was analyzed after in vitro stimulation of leukocyte subpopulations and in two in vivo inflammation models. Recombinant proteins of carp CXCL8 proteins were produced and showed significant chemotactic activity for carp leukocytes. Conclusions While both carp CXCL8s appear to be functional homologs of mammalian CXCL8, their different induction requirements and kinetics evoke a gene-specific sub-functionalization.

Functional analysis of carp interferon-γ: evolutionary conservation of classical phagocyte activation.

In teleost fish two IFN-gamma gene sequences were found for which two phylogenetic clusters can be distinguished. Our previous analysis of expression of these in carp led us to hypothesize that a classical IFN-gamma function is associated with the IFN-gamma2 cluster. We investigated the evolutionary conservation of the IFN-gamma function, inducing classical activation of phagocytes, thus skewing towards a Th1-like profile of immune activation. Recombinant proteins for the carp IFN-gamma sequences of both clusters were made and we studied their effects on expression of proinflammatory mediators. Carp IFN-gamma2, in contrast to carp IFN-gamma1, was powerful in inducing a proinflammatory reaction in phagocytes: a classical synergistic response with lipopolysaccharide was observed for the induction of iNOS expression and NO release, for expression of CXCL9-11-like chemokines and the expression of proinflammatory cytokines IL-1beta, TNFalpha and the IL-12 subunits p35 and p40. In contrast, like in mammals, the CXCL8-like cytokines are LPS but not IFN-gamma sensitive. These results corroborate an evolutionary conserved nature of IFN-gamma function in lower vertebrates including classical activation of phagocytes.

The effect of elevated reproductive effort onhumoral immune function in collared flycatcher females

Abstract In order to test whether high reproductive investments impair immune function in naturally breeding collared flycatchers, we performed a brood manipulation experiment and simultaneously induced an immune response by challenging birds with a non-pathogenic antigen – sheep red blood cells (SRBC). Females rearing experimentally enlarged number of nestlings showed significantly lower level of specific anti-SRBC antibodies than control females attending unaltered broods, but only in one of the two study years. The haemoconcentration of leukocytes did not differ between the two groups in both study years. The significant difference in immunological responsiveness between control and enlarged group coincided with differences in survival probability to the next breeding season: females attending enlarged broods showed lower probability of survival than control females, but there was no relationship between the level of immune response and survival probability. Our results indicate that reproduction may indeed trade for resources with immune functions at least in terms of specific antibody production. However, as in the other studies on reproductive costs, these costs seem not always to be pronounced.

Delayed effects of cold stress on immune response in laboratory mice

This study was undertaken to examine the trade–off between the cost of thermoregulation and immune function in laboratory mice. Mice were maintained either at 23°C or cold exposed at 5°C for 10 days. Then, they were immunized with sheep red blood cells. Thus, the cold–exposed mice had either experienced or not experienced cold stress prior to immunization. Cold stress elicited a substantial increase in food intake, accompanied by a significant reduction in food digestibility. An increase in mass of metabolically active internal organs (small intestines, heart and kidney) was observed in cold–exposed mice. These findings reassured us that costs of increased thermoregulation caused by cold stress were substantial. The immune response of mice exposed to long–lasting cold stress was significantly lower, but immune response was not affected in short–exposed mice. Differences in immune response between experimental groups accompanied changes in mass of immunocompetent organs (thymus and spleen). Our findings indicate that studies of trade–offs should account for the fact that resource reallocation in response to an environmental challenge may not be immediate. In fact, resource reallocation may be postponed until the new environmental state becomes permanent or until an organism attains physiological adaptation to the current conditions.

Morphine modulation of peritoneal inflammation in Atlantic salmon and CB6 mice.

Peritoneal inflammation is a convenient model for comparisons of modulatory effects of morphine in phylogenetically distant vertebrates. Both in salmon and mice morphine injected intraperitoneally together with an irritant (thioglycollate) significantly inhibits inflammation as estimated by the number of peritoneal leukocytes. The low number of exudate cells in morphine‐treated animals seems to be compensated by their high activity, as evidenced by the enhanced phorbol myristate acetate‐induced respiratory burst. The morphine‐inhibited influx of leukocytes into the irritated peritoneal cavity correlates with the morphine‐lowered level of plasma chemotactic factors both in fish and mice. It implies that morphine impairs the level of plasma chemotactic factor either directly (affecting their release from the resident peritoneal cells) or indirectly (decreasing the number of inflammatory leukocytes by inhibition of their migration from hemopoietic sites). The inhibitory effects of morphine on both the cell number and chemoattractant level are completely reversed by the naltrexone pretreatment, which implicates the involvement of opioid receptors. J. Leukoc. Biol. 65: 590–596; 1999.

Expression profiles of matrix metalloproteinase 9 in teleost fish provide evidence for its active role in initiation and resolution of inflammation

Matrix metalloproteinase 9 (MMP‐9) belongs to a family of zinc‐dependent endopeptidases. As a consequence of its ability to cleave structural extracellular matrix molecules, mammalian MMP‐9 is associated with vital inflammatory processes such as leucocyte migration and tissue remodelling and regeneration. Interestingly, MMP‐9 genes have been identified in fish, but functional data are still limited and focus on the involvement of MMP‐9 in embryonic development, reproduction and post‐mortem tenderization. Here, we describe the involvement of MMP‐9 in the innate immunity of carp. In carp, MMP‐9 was most notably expressed in classical fish immune organs and in peritoneal and peripheral blood leucocytes, indicating a role of MMP‐9 in immune responses. In our well‐characterized zymosan‐induced peritonitis model for carp, we analysed expression of the MMP‐9 gene and the gelatinolytic levels of both pro‐ and activated forms of MMP‐9. The biphasic profile of MMP‐9 mRNA expression indicated involvement during the initial phase of inflammation and during the later phase of tissue remodelling. Also, in vitro stimulation of carp phagocytes with lipopolysaccharide or concanavalin A increased MMP‐9 gene expression, with a peak at 24 hr. The increase of MMP‐9 mRNA correlated with the peak of MMP‐9 gelatinolytic level in culture supernatants. These results provide evidence for an evolutionarily conserved and relevant role of MMP‐9 in the innate immune response.

The immune response differentially regulates Hsp70 and glucocorticoid receptor expression in vitro and in vivo in common carp (Cyprinus carpio L.).

Heat shock or stress proteins and glucocorticoids (cortisol) regulate a sequential pro-inflammatory and anti-inflammatory cytokine expression profile to effectively kill pathogens, whilst minimizing damage to the host. Cortisol elicits its effects through the glucocorticoid receptor (GR) for which Hsp70 and Hsp90 are required as chaperones. In common carp, (Cyprinus carpio) duplicated glucocorticoid receptor genes and splice variants with different cortisol sensitivities exist. We investigated the expression profiles of heat shock proteins Hsp70, Hsc70, Hsp90alpha and Hsp90beta and the three different variants of GR in vitro in and in vivo to define their role in immune modulation. A rapid transient induction of GR1 (a and b) and Hsp70 was seen after LPS treatment in vitro in head kidney phagocytes, whereas cortisol treatment did not affect constitutive or LPS-induced expression of Hsp70 or GR1 expression. In vivo zymosan-induced peritonitis upregulated GR and Hsp70 expression which appears to increase sensitivity for cortisol-induced immune modulation. Indeed, the increased GR and Hsp70 expression correlates with inhibition of both LPS- and zymosan-induced expression of pro-inflammatory cytokines. Infection with the blood parasite T. borreli decreases GR1a expression in thymus, but increases GR2 expression in spleen. Differentially regulated expression of Hsp70 and of glucocorticoid receptor variants with different cortisol sensitivities, underlines their physiological importance in a balanced immune response.

Morphine affects the inflammatory response in carp by impairment of leukocyte migration

Opioid peptides are evolutionary conserved and in teleost fish their specific receptor types have been identified not only on neuroendocrine cells but also on immunocytes. In the present work we have studied the effects of morphine, ligand for the mu3 opioid receptor, on innate immune responses of common carp. Both in vitro and in vivo, during zymosan-induced peritonitis, morphine reduced gene expression of pro-inflammatory cytokines/chemokines and chemokine receptors. Furthermore, in vitro morphine administration also affects nitric oxide production, chemotaxis and apoptosis of head kidney leukocytes. These results provide evidence for an anti-inflammatory function of morphine and suggest an evolutionary conserved cross-talk between chemokines and opioids.

Neuroendocrine–immune interaction in fish: Differential regulation of phagocyte activity by neuroendocrine factors

Coping with physical, chemical and biological disturbances depends on an extensive repertoire of physiological, endocrinological and immunological responses. Fish provide intriguing models to study bi-directional interaction between the neuroendocrine and the immune systems. Macrophages and granulocytes are the main actors in the first and rapid innate immune response. They are resident in different organs and are moreover rapidly recruited and activated upon infection. They act in response to recognition of pathogen-associated molecular patterns (PAMPs) via a repertoire of surface and intracellular receptors by inducing a plethora of defense reactions aiming to eradicate the pathogen. Subsequent production of inflammatory mediators stimulates other leukocytes required to develop an adaptive and specific antibody response. The type of phagocyte reaction will therefore depend on their differentiation state, specific receptor repertoire and their specific location. Apart from these pathogen induced responses, immune reactivity may be modulated by neuroendocrine factors. Over the last years we extensively studied changes in carp stress axis activity and the effect of its end-products on the immune system in an acute stress paradigm. We focus on specific neuroendocrine receptors on leukocytes and their effect on crucial phagocyte activities. We performed identification and functional analyses of different glucocorticoid, opioid and adrenergic receptors on carp phagocytes. Results show that their ligands of neuroendocrine origin may have substantial impact on specific phagocyte functions in a differential way. Inflammatory and microbicidal responses fight pathogens but may be detrimental to the host tissue. Neuroendocrine modulation may regulate inflammation to reach an optimum defense while preventing excessive host cell damage.

Trypanosomiasis-Induced Th17-Like Immune Responses in Carp

Background In mammalian vertebrates, the cytokine interleukin (IL)-12 consists of a heterodimer between p35 and p40 subunits whereas interleukin-23 is formed by a heterodimer between p19 and p40 subunits. During an immune response, the balance between IL-12 and IL-23 can depend on the nature of the pathogen associated molecular pattern (PAMP) recognized by, for example TLR2, leading to a preferential production of IL-23. IL-23 production promotes a Th17-mediated immune response characterized by the production of IL-17A/F and several chemokines, important for neutrophil recruitment and activation. For the cold blooded vertebrate common carp, only the IL-12 subunits have been described so far. Methodology/Principal Findings Common carp is the natural host of two protozoan parasites: Trypanoplasma borreli and Trypanosoma carassii. We found that these parasites negatively affect p35 and p40a gene expression in carp. Transfection studies of HEK293 and carp macrophages show that T. carassii-derived PAMPs are agonists of carp TLR2, promoting p19 and p40c gene expression. The two protozoan parasites induce different immune responses as assessed by gene expression and histological studies. During T. carassii infections, in particular, we observed a propensity to induce p19 and p40c gene expression, suggestive of the formation of IL-23. Infections with T. borreli and T. carassii lead to an increase of IFN-γ2 gene expression whereas IL-17A/F2 gene expression was only observed during T. carasssii infections. The moderate increase in the number of splenic macrophages during T. borreli infection contrasts the marked increase in the number of splenic neutrophilic granulocytes during T. carassii infection, along with an increased gene expression of metalloproteinase-9 and chemokines. Conclusion/Significance This is the first study that provides evidence for a Th17-like immune response in fish in response to infection with a protozoan parasite.