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IL10, A Tale of an Evolutionarily Conserved Cytokine across Vertebrates
Abstract
IL10 was discovered in 1989, and since then it has been the subject of intense investigation, which has revealed its potent anti-inflammatory and regulatory activities in most immune processes during infection and disease. In 2003, the first non-mammalian IL10 sequence was identified in teleost fish, followed in 2004 by the chicken IL10 sequence. In this review, we summarize the work performed in non-mammalian vertebrates in which the IL10, IL10 receptors (IL10Rs), and their signaling components have been identified. We review the genomic organization, genes, and protein structure of IL10(Rs); we focus on studies providing a functional characterization of their biological activities. In addition, we describe the activities of viral IL10s identified in viruses infecting non-mammalian hosts. Altogether, our analysis reveals remarkable conservation of the anti-inflammatory and regulatory activities of (viral) IL10 across vertebrates, confirming the crucial role of IL10 throughout evolution. Interestingly, in some teleost fish, the presence of multiple copies of IL10(Rs) adds an additional degree of complexity. In fact, the evidence suggests that gene duplication does not necessarily imply functional redundancy, and leaves teleosts with additional possibilities to fine tune IL10 activities. Finally, we discuss the use of zebrafish (Danio rerio) as a complementary animal model for the study of IL10 activities in non-mammalian vertebrates.
... A word of caution in light of similarity of genes or cells of different species is worth giving here. As for similarity of genes between (host) species, it has to be noted that (automatic) annotation might not mean functional conservation as has been shown for the IL-10R2 (Piazzon et al., 2016). In terms of receptor similarity it is shown that for IL-10R1 functional homology exists between fish and mammals. ...
... Since the two putative IL-10R2 receptors (CRFB4 and CRFB5) are very similar in protein structure and genomic organization both are annotated in the zebrafish database 1 as IL-10R2 (ENSDARG00000078042 and ENSDARG00000068711). Studies performed in grass carp actually showed that CRFB4 is probably the functional homolog of IL-10R (Wei et al., 2014;Piazzon et al., 2016). Additionally, the lack of cell markers for specific cell types in zebrafish sometimes leads to assumptions on the presence of certain cell types only on the basis of gene expression data. ...
... Innate immunity develops from 2 dpf and adaptive immunity develops from 10 dpf onward (at this time CD4 + lymphocytes have been shown to leave the thymus). IL-22 and IL-10 seem to be conserved, although their regulation may differ (Costa et al., 2013;Siupka et al., 2014;Piazzon et al., 2016). ...
Our world is filled with microbes. Each multicellular organism has developed ways to interact with this microbial environment. Microbes do not always pose a threat; they can contribute to many processes that benefit the host. Upon colonization both host and microbes adapt resulting in dynamic ecosystems in different host niches. Regulatory processes develop within the host to prevent overt inflammation to beneficial microbes, yet keeping the possibility to respond when pathogens attempt to adhere and invade tissues. This review will focus on microbial colonization and the early (innate) host immune response, with special emphasis on the microbiota-modifying roles of IL-10 and IL-22 in the intestine. IL-10 knock out mice show an altered microbial composition, and spontaneously develop enterocolitis over time. IL-22 knock out mice, although not developing enterocolitis spontaneously, also have an altered microbial composition and increase of epithelial-adherent bacteria, mainly caused by a decrease in mucin and anti-microbial peptide production. Recently interesting links have been found between the IL-10 and IL-22 pathways. While IL-22 can function as a regulatory cytokine at the mucosal surface, it also has inflammatory roles depending on the context. For example, lack of IL-22 in the IL-10–/– mice model prevents spontaneous colitis development. Additionally, the reduced microbial diversity observed in IL-10–/– mice was also reversed in IL-10/IL-22 double mutant mice ( Gunasekera et al., 2020 ). Since in early life, host immunity develops in parallel and in interaction with colonizing microbes, there is a need for future studies that focus on the effect of the timing of colonization in relation to the developmental phase of the host. To illustrate this, examples from zebrafish research will be compared with studies performed in mammals. Since zebrafish develop from eggs and are directly exposed to the outside microbial world, timing of the development of host immunity and subsequent control of microbial composition, is different from mammals that develop in utero and only get exposed after birth. Likewise, colonization studies using adult germfree mice might yield different results from those using neonatal germfree mice. Lastly, special emphasis will be given to the need for host genotype and environmental (co-housing) control of experiments.
... The tight control of cellular processes is an essential pillar of large physiological programs, not only with regard to immunity, but also in terms of development or metabolism. An increasingly huge number of factors [2][3][4][5][6][7][8][9] dispose of the potential to interfere with inflammatory responses, thereby also modulating adaptive immune responses and establishing tolerance. ...
... Signalling pathways mediated by pattern-recognition receptors result in a variety of cellular responses, above all the rapid synthesis of pro-inflammatory cytokines directing eventually adaptive immune response. Cytokines are doubtlessly the best-studied immune factors in fish, and serve as highly informative biomarkers to assess the health status and well-being as documented by recent review articles [5,212,213]. The aforementioned reports conclude that the functions of pro-inflammatory cytokines are quite well conserved among vertebrates and majorly involve the chemotactic attraction of leucocytes, vascular dilation or the enhanced production of prostaglandins and reactive oxygen species. ...
... Interleukin-10 (IL10) was originally termed 'cytokine synthesis inhibitory factor' [230], and though the name may suggest otherwise, its functional spectrum is much broader covering inhibited monocyte differentiation, impaired phagocytosis, decreased production of toxic radicals, suppressed expression of genes coding for MH class II molecules and pro-inflammatory cytokines [5]. Orthologs have been characterised in many fish species encompassing tetraodontid [231], cyprinid [232][233][234][235], salmonid [236], moronid [237], gadid [238], and carangid representatives [239], few of which encode (at least) two il10 paralogs [240,241]. ...
... The tight control of cellular processes is an essential pillar of large physiological programs, not only with regard to immunity, but also in terms of development or metabolism. An increasingly huge number of factors [2][3][4][5][6][7][8][9] dispose of the potential to interfere with inflammatory responses, thereby also modulating adaptive immune responses and establishing tolerance. ...
... Signalling pathways mediated by pattern-recognition receptors result in a variety of cellular responses, above all the rapid synthesis of pro-inflammatory cytokines directing eventually adaptive immune response. Cytokines are doubtlessly the best-studied immune factors in fish, and serve as highly informative biomarkers to assess the health status and well-being as documented by recent review articles [5,212,213]. The aforementioned reports conclude that the functions of pro-inflammatory cytokines are quite well conserved among vertebrates and majorly involve the chemotactic attraction of leucocytes, vascular dilation or the enhanced production of prostaglandins and reactive oxygen species. ...
... Interleukin-10 (IL10) was originally termed 'cytokine synthesis inhibitory factor' [230], and though the name may suggest otherwise, its functional spectrum is much broader covering inhibited monocyte differentiation, impaired phagocytosis, decreased production of toxic radicals, suppressed expression of genes coding for MH class II molecules and pro-inflammatory cytokines [5]. Orthologs have been characterised in many fish species encompassing tetraodontid [231], cyprinid [232][233][234][235], salmonid [236], moronid [237], gadid [238], and carangid representatives [239], few of which encode (at least) two il10 paralogs [240,241]. ...
The innate immune response involves a concerted network of induced gene products, preformed immune effectors, biochemical signalling cascades and specialised cells. However, the multifaceted activation of these defensive measures can derail or overshoot and, if left unchecked, overwhelm the host. A plenty of regulatory devices therefore mediate the fragile equilibrium between pathogen defence and pathophysiological manifestations. Over the past decade in particular, an almost complete set of teleostean sequences orthologous to mammalian immunoregulatory factors has been identified in various fish species, which prove the remarkable conservation of innate immune-control concepts among vertebrates. This review will present the current knowledge on more than 50 teleostean regulatory factors (plus additional fish-specific paralogs) that are of paramount importance for controlling the clotting cascade, the complement system, pattern-recognition pathways and cytokine signalling networks. A special focus lies on those immunoregulatory features that have emerged as potential biomarker genes in transcriptome-wide research studies. Moreover, we report on the latest progress in elucidating control elements that act directly with immune-gene-encoding nucleic acids, such as transcription factors, hormone receptors and micro- and long noncoding RNAs. Investigations into the function of teleostean inhibitory factors are still mainly based on gene-expression profiling or overexpression studies. However, in support of structural and in-vitro analyses, evidence from in-vivo trials is also available and revealed many biochemical details on piscine immune regulation. The presence of multiple gene copies in fish adds a degree of complexity, as it is so far hardly understood if they might play distinct roles during inflammation. The present review addresses this and other open questions that should be tackled by fish immunologists in future.
... Another important effect of rVP1 immunization was the upregulation of il-10a in the head kidney of rainbow trout, which suggests that a well-regulated and limited immune response has been elicited. This is supported by several functional studies of IL-10 in goldfish, common carp, grass carp, tongue sole (Cynoglossus semilaevis) and spotted knifejaw (Oplegnathus punctatus), showing that fish IL-10 inhibits pro-inflammatory gene expression, and reduces respiratory burst, nitrogen radical production and phagocytic activity [50][51][52][53] as occurs in higher vertebrates [52,54]. Up-regulation of tgf-β1 gene expression was also observed in response to rVP1 immunization. ...
... Another important effect of rVP1 immunization was the upregulation of il-10a in the head kidney of rainbow trout, which suggests that a well-regulated and limited immune response has been elicited. This is supported by several functional studies of IL-10 in goldfish, common carp, grass carp, tongue sole (Cynoglossus semilaevis) and spotted knifejaw (Oplegnathus punctatus), showing that fish IL-10 inhibits pro-inflammatory gene expression, and reduces respiratory burst, nitrogen radical production and phagocytic activity [50][51][52][53] as occurs in higher vertebrates [52,54]. Up-regulation of tgf-β1 gene expression was also observed in response to rVP1 immunization. ...
In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-β1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1⁺ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-β1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.
... The pleiotropic regulatory cytokine IL10 is considered one of the most important anti-inflammatory cytokines responsible for limiting and terminating inflammation, in order to prevent damage and help to the formation of long-lived memory cells against future threats. This cytokine has been extensively studied in mammals and fish with no apparent major functional differences [71,72]. One of its main activities is the inhibition of the expression of pro-inflammatory cytokines by phagocytes and T lymphocytes, among which, il1β is one of the main affected [73]. ...
... IL10 also increases the cytotoxic activity of mammalian NK cells [74] and stimulates B cell proliferation, maturation and antibody secretion both in mammals and fish [73]. The up-regulation of il10 during long-lasting parasitic infections of carp with T. borreli and T. carassii actually coincided with the appearance of higher concentrations of specific antibodies against the parasites and the concomitant downregulation of pro-inflammatory genes [71,75]. In the current study, the higher expression of il10 in AI of SUR fish is also parallel to lower expression of il1β, higher expression of immunoglobulins (particularly the soluble forms) and a higher expression of the cytotoxic enzyme gzma. ...
... Nowadays, IL-10 and its two receptor genes have been studied in several species of teleost fish (Piazzon et al., 2016). IL-10 has been identified in model fish, such as fugu (Fugu rubripes) (Zou et al., 2003) and zebrafish (Danio rerio) (Zhang et al., 2005), and also in several economically important species, such as common carp (Cyprinus carpio) (Piazzon et al., 2015;Ram et al., 2003), goldfish (Carassius auratus) (Grayfer et al., 2011), grass carp (Ctenopharyngodon idella) (Wei et al., 2013), rainbow trout (Oncorhynchus mykiss) (Inoue et al., 2005), sea bass (Dicentrarchus labrax) (Pinto et al., 2007) and Atlantic cod (Gadus morhua) (Seppola et al., 2008). ...
... Despite the detailed functional understanding of IL-10 in mammals (Moore et al., 2001;Sabat et al., 2010), and the ligand-receptor characterization and some inhibitory effect of IL-10 in different species of teleost fish (Grayfer et al., 2011;Piazzon et al., 2015Piazzon et al., , 2016, the conservation of IL-10 and its receptor complex, IL-10R1, IL-10R2, and its immunosuppressive effect on IL-6, IL-1β, IL-8, TNF-α expression and on reactive oxygen radical production were characterized in a perciform species, the mandarin fish, or the so-called Chinese perch S. chuatsi. ...
Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1β, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.
... Our work demonstrates that K. pneumoniae-controlled STAT6 activation was dependent on type I IFN and IL10 following the activation of TLR4governed signalling pathway. Type I IFNs, IL10 and TLR4 also appeared early during the evolution of vertebrates (Piazzon et al, 2016;Liu et al, 2020). Therefore, K. pneumoniae has evolved to manipulate an innate axis conserved during evolution formed by TLR4-type I IFN-IL10-STAT6 to rewire macrophages. ...
The strategies deployed by antibiotic-resistant bacteria to counteract host defences are poorly understood. Here, we elucidate a novel host-pathogen interaction resulting in skewing lung macrophage polarisation by the human pathogen Klebsiella pneumoniae. We identify interstitial macrophages (IMs) as the main population of lung macrophages associated with Klebsiella. Single-cell transcriptomics and trajectory analysis of cells reveal type I IFN and IL10 signalling, and macrophage polarisation are characteristic of infected IMs, whereas Toll-like receptor (TLR) and Nod-like receptor signalling are features of infected alveolar macrophages. Klebsiella-induced macrophage polarisation is a singular M2-type we termed M(Kp). To rewire macrophages, Klebsiella hijacks a TLR-type I IFN-IL10-STAT6 axis. Absence of STAT6 limits Klebsiella intracellular survival and facilitates the clearance of the pathogen in vivo. Glycolysis characterises M(Kp) metabolism, and inhibition of glycolysis results in clearance of intracellular Klebsiella. Capsule polysaccharide governs M(Kp). Klebsiella also skews human macrophage polarisation towards M(Kp) in a type I IFN-IL10-STAT6-dependent manner. Klebsiella induction of M(Kp) represents a novel strategy to overcome host restriction, and identifies STAT6 as target to boost defences against Klebsiella.
... Thus, systemic inflammation can limit availability of tryptophan for serotonin synthesis. However, SCFAs in systemic circulation are known to lower the proinflammatory cytokines (TNF-α, IL-1β, IL-6) and elevate anti-inflammatory and regulatory cytokines such as IL-10 which may indirectly increase availability of tryptophan for serotonin synthesis by balancing the cytokines (Liu et al., 2012;Piazzon et al., 2016). ...
The chicken gastrointestinal tract has a diverse microbial community. There is increasing evidence for how this gut microbiome affects specific molecular pathways and the overall physiology, nervous system and behavior of the chicken host organism due to a growing number of studies investigating conditions such as host diet, antibiotics, probiotics, and germ-free and germ-reduced models. Systems-level investigations have revealed a network of microbiome-related interactions between the gut and state of health and behavior in chickens and other animals. While some microbial symbionts are crucial for maintaining stability and normal host physiology, there can also be dysbiosis, disruptions to nutrient flow, and other outcomes of dysregulation and disease. Likewise, alteration of the gut microbiome is found for chickens exhibiting differences in feather pecking (FP) behavior and this alteration is suspected to be responsible for behavioral change. In chickens and other organisms, serotonin is a chief neuromodulator that links gut microbes to the host brain as microbes modulate the serotonin secreted by the host’s own intestinal enterochromaffin cells which can stimulate the central nervous system via the vagus nerve. A substantial part of the serotonergic network is conserved across birds and mammals. Broader investigations of multiple species and subsequent cross-comparisons may help to explore general functionality of this ancient system and its increasingly apparent central role in the gut-brain axis of vertebrates. Dysfunctional behavioral phenotypes from the serotonergic system moreover occur in both birds and mammals with, for example, FP in chickens and depression in humans. Recent studies of the intestine as a major site of serotonin synthesis have been identifying routes by which gut microbial metabolites regulate the chicken serotonergic system. This review in particular highlights the influence of gut microbial metabolite short chain fatty acids (SCFAs) on the serotonergic system. The role of SCFAs in physiological and brain disorders may be considerable because of their ability to cross intestinal as well as the blood-brain barriers, leading to influences on the serotonergic system via binding to receptors and epigenetic modulations. Examinations of these mechanisms may translate into a more general understanding of serotonergic system development within chickens and other avians.
... A variety of different immune cell types of both innate and adaptive immunity are able to produce and/or respond to IL-10 (Piazzon et al., 2016). B cells and most types of innate cells produce IL-10. ...
Interleukin (IL)-10 is considered a prototypical anti-inflammatory cytokine, significantly contributing to the maintenance and reestablishment of immune homeostasis. Accordingly, it has been shown in the intestine that IL-10 produced by Tregs can act on effector T cells, thereby limiting inflammation. Herein, we investigate whether this role also applies to IL-10 produced by T cells during central nervous system (CNS) inflammation. During neuroinflammation, both CNS-resident and -infiltrating cells produce IL-10; yet, as IL-10 has a pleotropic function, the exact contribution of the different cellular sources is not fully understood. We find that T-cell-derived IL-10, but not other relevant IL-10 sources, can promote inflammation in experimental autoimmune encephalomyelitis. Furthermore, in the CNS, T-cell-derived IL-10 acts on effector T cells, promoting their survival and thereby enhancing inflammation and CNS autoimmunity. Our data indicate a pro-inflammatory role of T-cell-derived IL-10 in the CNS.
... Regarding the MMe function, we confirm that although they produce pro-inflammatory cytokines such as IL-6 (14), they do so to a lesser extent than classic M1 macrophages in subjects with dyslipidemia. Interestingly, we also found that these MMe produce IL-10, a cytokine with potent anti-inflammatory and regulatory activities [34], and that this production is higher in MMe compared to M1. Regarding the polarization to classic M1 and alternative M2 macrophages, we found no differences between healthy people and those with dyslipidemia; in this sense, Baardman et al. [35] have shown that hypercholesterolaemia alters macrophage metabolism and phenotype, attenuating the inflammatory phenotype in M1 macrophages. ...
Macrophages are mediators of inflammatory having an important role in the pathogenesis of cardiovascular diseases. Recently, a pro-inflammatory subpopulation, known as metabolically activated macrophages (MMe), has been described in conditions of obesity and metabolic syndrome where they are known to release cytokines which can promote insulin resistance. Dyslipidemia represents an important feature in metabolic syndrome and corresponds to one of the main modifiable risk factors for the development of cardiovascular diseases. Circulating monocytes can differentiate into macrophages under certain conditions. They correspond to a heterogeneous population, which include inflammatory and anti-inflammatory subsets; however, there is a wide spectrum of phenotypes. Therefore, we decided to investigate whether the metabolic activated monocyte (MoMe) subpopulation is already present under dyslipidemia conditions. Secondly, we assessed if different levels of cholesterol and triglycerides, play a role in the polarization towards the metabolic phenotype (MMe) of macrophages. Our results indicate that MoMe cells are found in both healthy and dyslipidemia patients, with cells displaying the following metabolic phenotype: CD14 varCD36+ABCA1+PLIN2+.
Furthermore, the percentages of CD14 ++CD68 +CD80 + pro-inflammatory monocytes are higher in dyslipidemia than in healthy subjects. When analysing macrophage differentiation, we observed that MMe percentages were higher in dyslipidemia group than in healthy subjects. These MMe have the ability to produce high levels of IL-6 and the anti-inflammatory cytokine IL-10. Furthermore, ABCA1 expression in MMe correlates with LDL serum levels. Our study highlights the dynamic contributions of metabolically activated macrophages in dyslipidemia, which may have a complex participation in low-grade inflammation due to their pro- and anti-inflammatory function.
... IL-10 is a pleiotropic anti-inflammatory cytokine that limits and terminates the immune response induced by viral infection and thus alleviates the inflammatory damage caused by the host immune system and restores homeostasis [52]. The significant upregulation of IL-10 found in FAdV-4-infected chickens might contribute to the anti-inflammatory activity in the primary target organ of FAdV-4. ...
Hepatitis-hydropericardium syndrome (HHS), caused by fowl adenovirus serotype 4 (FAdV-4), has spread on chicken farms worldwide, causing huge economic losses. Currently, the exact mechanism of pathogenesis of FAdV-4 remains unknown. Despite the severe inflammatory damage observed in chickens infected with pathogenic FAdV-4, few studies have focused on the host immune system-virus interactions and cytokine secretion. Host immunity acts as one of the most robust defense mechanisms against infection by pathogens, and cytokines are important in their elimination. However, excessive inflammatory cytokine secretion could contribute to the pathogenesis of FAdV-4. Understanding of the roles of cytokines produced during FAdV-4 infection is important for the study of pathogenicity and for developing strategies to control FAdV-4. Several previous studies have addressed the immune responses to FAdV-4 infection, but there has not been a systematic review of this work. The present review provides a detailed summary of the current findings on cytokine production induced by FAdV-4 infection to accelerate our understanding of FAdV-4 pathogenesis.
... The IL6 gene have essential functions in hematopoiesis, inflammation, immune response, and the acute phase response (Schaper and Rose-John, 2015). The IL10RB gene encodes a protein that, together with the protein encoded by the IL10RA, is essential for the IL10-induced signal transduction, which exhibits important anti-inflammatory and regulatory activities in immune processes during the development of infection and establishment of the disease (Piazzon et al., 2016;Fang and Zhu, 2020). ...
This study aimed to identify structural variations in the form of runs of homozygosity (ROH) and copy number variants (CNVs) in the genome of the Brazilian Senepol cattle and to better explore the potential biological functions of the genes located within such regions. A total of 140 animals were genotyped with the GeneSeek® Genomic Profiler™ Bovine 50K. Autosomal ROH and CNVs were detected after appropriated quality control. A total of 5,531 ROH were identified, with an average number of 40.37 per animal and an average length of 5.09 Mb. BTA1 had the greatest number of ROH per chromosome (n=458), while BTA20 displayed the greatest fraction of the chromosome covered with ROH (22.06%). A total of 181 CNVs were identified, with an average length size of 184.67 kb, with values ranging from 26.61 to 334.48 kb. A total of 16 CNV regions (CNVRs) were seen distributed among eleven autosomes, and the largest number of CNVRs (n=4) was described on BTA12. Autozygosity islands were identified using an outlier approach, and none of them overlapped with CNVRs. Our study revealed that the limited genetic basis together with the narrow number of imported animals to disseminate the breed might have strongly contributed to the low effective population size and the high genomic autozygosity proportion described in this population. The functional enrichment analysis revealed several significant terms (p<0.05) within the autozygosity islands and CNVRs closely linked to molecular and immune response mechanisms. The average FROH of different lengths were low to in the studied population, however, the autozygotic proportion in the genome indicates moderate to high inbreeding levels. The results exposed the need of implementing mating programs to control the increase of inbreeding and coancestry in Brazilian Senepol cattle.
In these sense, Senepol breeders should apply selection and mating strategies to minimize the occurrence of long ROH in the offspring, as well as import of new genetic material to avoid the loss of genetic diversity. This study revealed the existence of overlapped regions between CNVRs and ROH throughout the genome of the Senepol cattle, that contributes to a better understanding of the functional role of genomic structural variations in taurine cattle adapted to tropical regions.
... While IL-10 can inhibit phagocyte function, antigen presentation, co-stimulatory molecule expression, T cell proliferation, IL-2 and IFNγ production, it can also promote NK cell activity, B cell activation and isotype switching [16]. The immunoregulatory role of IL-10 at mucosal tissues is well conserved among mammalian and non-mammalian vertebrates and defects in IL-10 signaling result in inflammatory bowel disease in mouse and man [17]. This suggests that IL-10 is employed to promote tolerance against microbiota and dietary antigens and to protect the integrity of epithelial barriers [18]. ...
Tregs are the immune system’s in-house combatants against pathological immune activation. Because they are vital to maintenance of peripheral tolerance, it is important to understand how they perform their functions. To this end, various mechanisms have been proposed for Treg-mediated immune inhibition. A major group of mechanisms picture Tregs as skilled thieves stealing a plethora of molecules that would otherwise promote immune effector functions. This suggests that several million years of evolution have endowed Tregs with efficient ways to deprive immune effectors of activating stimuli to prevent immunopathology for survival of the host. Although we are still long way from deciphering their complete set of tricks, this review will focus on the types of “crimes” committed by these master thieves in both secondary lymphoid organs and non-lymphoid tissue.
... We also examined the potential of two (recombinant) cytokines, Il10b and Ifnγ2, to modulate such proliferative response. Interleukin10 can have multiple effects on B and T lymphocytes, including regulation of proliferation and differentiation [as reviewed in Ref. (81)], and carp Il10b was shown to promote survival and enhance proliferation of antigenspecific B and T cells (47). Ifnγ2 has multiple effects and in carp was found to enhance antigen specific responses during in vitro stimulation of carp leukocytes and phagocytes (48). ...
Although spring viremia of carp virus (SVCV) can cause high mortalities in common carp, a commercial vaccine is not available for worldwide use. Here, we report a DNA vaccine based on the expression of the SVCV glycoprotein (G) which, when injected in the muscle even at a single low dose of 0.1 µg DNA/g of fish, confers up to 100% protection against a subsequent bath challenge with SVCV. Importantly, to best validate vaccine efficacy, we also optimized a reliable bath challenge model closely mimicking a natural infection, based on a prolonged exposure of carp to SVCV at 15°C. Using this optimized bath challenge, we showed a strong age-dependent susceptibility of carp to SVCV, with high susceptibility at young age (3 months) and a full resistance at 9 months. We visualized local expression of the G protein and associated early inflammatory response by immunohistochemistry and described changes in the gene expression of pro-inflammatory cytokines, chemokines, and antiviral genes in the muscle of vaccinated fish. Adaptive immune responses were investigated by analyzing neutralizing titers against SVCV in the serum of vaccinated fish and the in vitro proliferation capacity of peripheral SVCV-specific T cells. We show significantly higher serum neutralizing titers and the presence of SVCV-specific T cells in the blood of vaccinated fish, which proliferated upon stimulation with SVCV. Altogether, this is the first study reporting on a protective DNA vaccine against SVCV in carp and the first to provide a detailed characterization of local innate as well as systemic adaptive immune responses elicited upon DNA vaccination that suggest a role not only of B cells but also of T cells in the protection conferred by the SVCV-G DNA vaccine.
... Among the characterized activities of teleost Il10 is the ability to strongly inhibit pro-inflammatory gene expression, respiratory burst and nitrogen radical production by macrophages and neutrophils, to trigger B cell proliferation and antibody production as well as to promote memory T cell proliferation. Altogether, this underlines the crucial role played by IL10 in the regulation of the immune system of mammals as well as teleost fish (Piazzon et al., 2016). ...
We recently reported on the functional characterization of carp Il10. We showed that carp Il10 is able to downregulate proinflammatory activities by carp phagocytes and promote B cell proliferation, differentiation and antibody production as well as proliferation of memory T cells. Taking advantage of the recent annotation of the carp genome, we completed the sequence of a second il10 paralogue, named il10b, the presence of which was expected owing to the recent (8 million years ago) fourth round of whole genome duplication that occurred in common carp. In the present study we closely compared the two Il10 paralogues and show that Il10a and Il10b have almost identical gene structure, synteny, protein sequence as well as bioactivity on phagocytes. Although the two il10 paralogues show a large overlap in tissue expression, il10b has a low constitutive expression and is highly upregulated upon infection, whereas il10a is higher expressed under basal conditions but its gene expression remains constant during viral and parasitic infections. This differential regulation is most likely due to the observed differences in their promoter regions. Altogether our results demonstrate that gene duplication in carp, although recent, led to sub-functionalization and expression divergence rather than functional redundancy of the Il10 paralogues, yet with very similar protein sequences.
The immune system evolves under the ever-changing selective pressure of pathogens and its effective functioning involves not only immune-related cells and mediators. It also requires neuroendocrine-immune interaction, including regulation of specific organismal behaviour. Mechanisms of the behavioural immune system result in a change of psychological behaviour, comprising sickness behaviour: fever, decreased appetite, as well as depressed locomotor, social and exploratory activities. Behavioural changes are induced by inflammatory mediators released upon infection, which predominantly activate glial cells such as astrocytes and microglia. This phenomenon is evolutionarily conserved and can be observed in all vertebrates. In the oldest and most numerous vertebrate group, teleost fish, inflammatory cytokines (e.g. IL-1β, TNF-α and type I IFNs) induce behavioural fever and several other behavioural changes. Pathogens developed several mechanisms to reach the brain, including passive diffusion through injured endothelia, infection of endothelial cells or a Trojan horse tactic in phagocytes. Therefore local infection in fish is often connected with neuroinflammation and microglia activation. Among vertebrates, the zebrafish model provides a useful tool for in vitro and in vivo studies of microglia at a single-cell level. The advantages of this model are its small size, rapid external development and embryo transparency, which create ample possibilities for genetic manipulation.
T and B lymphocytes (T and B cells) are immune effector cells that play critical roles in adaptive immunity and defend against external pathogens in most vertebrates, including teleost fish. In mammals, the development and immune response of T and B cells is associated with cytokines including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors during pathogenic invasion or immunization. Given that teleost fish have evolved a similar adaptive immune system to mammals with T and B cells bearing unique receptors (B-cell receptors (BCRs) and T-cell receptors (TCRs)) and that cytokines in general have been identified, whether the regulatory roles of cytokines in T and B cell-mediated immunity are evolutionarily conserved between mammalians and teleost fish is a fascinating question. Thus, the purpose of this review is to summarize the current knowledge of teleost cytokines and T and B cells as well as the regulatory roles of cytokines on these two types of lymphocytes. This may provide important information on the parallelisms and dissimilarities of the functions of cytokines in bony fish versus higher vertebrates, which may aid in the evaluation and development of adaptive immunity-based vaccines or immunostimulants.
Megalocytivirus (MCV) is emerging as a major viral pathogen in aquaculture and has caused substantial economic losses due to its wide infectious spectrum. As the spotted knifejaw (Oplegnathus punctatus) is one of the most susceptible fish to MCV, the MCV infection in spotted knifejaw caused massive mortality, therefore significantly hampers the farming industry. Despite of the significant economic importance, the immune response of spotted knifejaw during MCV infection is still unclear, which limits the breeding of resistant fish lines and becomes a research priority for aquaculture worldwide. In this study, we challenged spotted knifejaw with a spotted knifejaw MCV strain (SKIV-SD) and performed a transcriptomic analysis using spleen samples from five different time points (days 0, 1, 4, 7, and 10). Fifteen samples (three for each time point) were employed to construct the RNA libraries. A total of over 100 Gb of clean bases was generated, and the clean reads per library ranged from 39.37 M to 60.87 M. A total of 23,365 genes were annotated, and 32,542 new transcripts were identified. Through a comparative transcriptomic analysis of the five groups, the nucleotide binding oligomerization domain-like (NOD-like) receptor signalling pathway, haematopoietic cell lineage, and cytosolic DNA-sensing pathway were closely related to MCV challenge, and the gene expression levels in these pathways were validated by qPCR. Our data provide a genetic resource and shed light on the immune response to MCV infection in spotted knifejaw.
For a long time, how anti-inflammatory factors evolved was largely unknown. In this study, we chose a marine invertebrate, Litopenaeus vannamei, as a model and identified that shrimp mesencephalic astrocyte-derived neurotrophic factor (MANF) was an LPS-induced plasma protein, which exerted its anti-inflammatory roles on shrimp hemocytes by suppressing ERK phosphorylation and Dorsal expression. In addition, we demonstrated that shrimp MANF could be associated with a receptor protein tyrosine phosphatase (RPTP) to mediate negative regulation of ERK activation and Dorsal expression. More interestingly, shrimp RPTP-S overexpression in 293T cells could switch shrimp and human MANF-mediated ERK pathway activation to inhibition. In general, our results indicate that this conserved RPTP is the key component for extracellular MANF-mediated ERK pathway inhibition, which gives a possible explanation about why this neurotropic factor could both protect neuron cells from apoptosis and inhibit immune cell M1 activation in various species.
The teleost head kidney is a highly relevant immune organ, and myeloid cells play a major role in this organ’s innate and adaptive immune responses. Because of their complexity, the early phases of the innate immune reaction of fish against bacteria are still poorly understood. In this study, naïve rainbow trout were stimulated with inactivated A. salmonicida and sampled at 12 h, 24 h and 7 d poststimulation. Cells from the head kidney were magnetically sorted with a monoclonal antibody mAB21 to obtain one (MAb21-positive) fraction enriched with myeloid cells and one (MAb21-negative) fraction enriched with lymphocytes and thrombocytes. The gene expression pattern of the resulting cell subpopulations was analysed using a panel of 43 immune-related genes. The results show an overall downregulation of the complement pathway and cytokine production at the considered time points. Some of the selected genes may be considered as parameters for diagnosing bacterial furunculosis of rainbow trout.
CD4⁺ helper T cells play key and diverse roles in inducing adaptive immune responses in vertebrates. The CD4 molecule, which is found on the surfaces of CD4⁺ helper T cells, can be used to distinguish subsets of helper T cells. Teleosts are the oldest living species with bona-fide CD4 coreceptors. Although some components of immune systems of teleosts and mammals appear to be similar, many physiological differences are represented between them. Previous studies have shown that two CD4 paralogs are present in teleosts, whereas only one is present in mammals. Therefore, in this review, the CD4 molecular structure, expression profiles, subpopulations, and biological functions of teleost CD4⁺ helper T cells were summarized and compared with those of their mammalian counterparts to understand the differences in CD4 molecules between teleosts and mammals. This review provides suggestions for further studies on the CD4 molecular function and regulatory mechanism of CD4⁺ helper T cells in teleost fish and will help establish therapeutic strategies to control fish diseases in the future.
Background:
The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified.
Methods:
We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues.
Results:
We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfβ in target tissues.
Conclusion:
These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.
The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis. :
Interleukin (IL)-10, an immune-regulatory cytokine, exerts various biological functions through interaction with IL-10 receptors. In teleost, very limited functional studies on IL-10 receptors have been documented. In this study, we reported the expression patterns of IL-10 receptor 1 (CsIL-10R1) and receptor 2 (CsIL-10R2) of tongue sole (Cynoglossus semilaevis) and examined their biological properties. The expression of CsIL-10R1 and CsIL-10R2 occurred in multiple tissues and were regulated by bacterial challenge. In vitro binding studies showed that recombinant extracellular region of CsIL-10R1 (rCsIL-10R1ex) rather than rCsIL-10R2ex could bind with rCsIL-10. Cellular study showed that both CsIL-10R1 and CsIL-10R2 were expressed on peripheral blood leukocytes (PBLs), and blockade of CsIL-10R1 or CsIL-10R2 by antibody could reduce inhibitory effect of CsIL-10 on ROS production of PBLs. When injected in vivo, anti-rCsIL-10R1 or anti-rCsIL-10R2 antibody dramatically promoted the expression of proinflammatory cytokines and suppressed bacterial dissemination in tongue sole tissues. Consistently, the overexpression of CsIL-10R1 or CsIL-10R2 significantly enhanced bacterial dissemination, and the overexpression of CsIL-10R1M bearing STAT3 site mutation reduced bacterial dissemination. Overall, these results demonstrate for the first time teleost IL-10 receptors play a negative role in antibacterial immunity and add insight into the function of CsIL-10 receptors.
Philasterides dicentrarchi is a free-living ciliate that causes high mortality in marine cultured fish, particularly flatfish, and in fish kept in aquaria. At present, there is still no clear picture of what makes this ciliate a fish pathogen and what makes fish resistant to this ciliate. In the present study, we used transcriptomic techniques to evaluate the interactions between P. dicentrarchi and turbot leucocytes during the early stages of infection. The findings enabled us to identify some parasite genes/proteins that may be involved in virulence and host resistance, some of which may be good candidates for inclusion in fish vaccines. Infected fish responded to infection by generating a very potent inflammatory response, indicating that the fish use all of the protective mechanisms available to prevent entry of the parasite. The findings also provide some valuable insight into how the acute inflammatory response occurs in fish. Abstract: The present study analyses the interactions between Philasterides dicentrarchi (a ciliate parasite that causes high mortalities in cultured flatfish) and the peritoneal cells of the turbot Scophthalmus maximus during an experimental infection. The transcriptomic response was evaluated in the parasites and in the fish peritoneal cells, at 1, 2 and 4 h post-infection (hpi) in turbot injected intraperitoneally (ip) with 10^7 ciliates and at 12 and 48 hpi in turbot injected ip with 10^5 ciliates. Numerous genes were differentially expressed (DE) in P. dicentrarchi, relative to their expression in control ciliates (0 hpi): 407 (369 were up-regulated) at 1 hpi, 769 (415 were up-regulated) at 2 hpi and 507 (119 were up-regulated) at 4 hpi. Gene ontology (GO) analysis of the DE genes showed that the most representative categories of biological processes affected at 1, 2 and 4 hpi were biosynthetic processes, catabolic processes, biogenesis, proteolysis and transmembrane transport. Twelve genes of the ABC transporter family and eight genes of the leishmanolysin family were DE at 1, 2 and 4 hpi. Most of these genes were strongly up-regulated (UR), suggesting that they are involved in P. dicentrarchi infection. A third group of UR genes included several genes related to ribosome biogenesis, DNA transcription and RNA translation. However, expression of tubulins and tubulin associated proteins, such as kinesins or dyneins, which play key roles in ciliate division and movement, was down-regulated (DR). Similarly, genes that coded for lysosomal proteins or that participate in the cell cycle mitotic control, glycolysis, the Krebs cycle and/or in the electron transport chain were also DR. The transcriptomic analysis also revealed that in contrast to many Biology 2020, 9, 337 2 of 25 parasites, which passively evade the host immune system, P. dicentrarchi strongly stimulated turbot peritoneal cells. Many genes related to inflammation were DE in peritoneal cells at 1, 2 and 4 hpi. However, the response was much lower at 12 hpi and almost disappeared completely at 48 hpi in fish that were able to kill P. dicentrarchi during the first few hpi. The genes that were DE at 1, 2 and 4 hpi were mainly related to the apoptotic process, the immune response, the Fc-epsilon receptor signalling pathway, the innate immune response, cell adhesion, cell surface receptors, the NF-kappaB signalling pathway and the MAPK cascade. Expression of toll-like receptors 2, 5 and 13 and of several components of NF-κB, MAPK and JAK/STAT signalling pathways was UR in the turbot peritoneal cells. Genes expressing chemokines and chemokine receptors, genes involved in prostaglandin and leukotriene synthesis, prostaglandins, leukotriene receptors, proinflammatory cytokines and genes involved in apoptosis were strongly UR during the first four hours of infection. However, expression of anti-inflammatory cytokines such as Il-10 and lipoxygenases with anti-inflammatory activity (i.e., arachidonate 15-lipoxygenase) were only UR at 12 and/or 48 hpi, indicating an anti-inflammatory state in these groups of fish. In conclusion, the present study shows the regulation of several genes in P. dicentrarchi during the early stages of infection, some of which probably play important roles in this process. The infection induced a potent acute inflammatory response, and many inflammatory genes were regulated in peritoneal cells, showing that the turbot uses all the protective mechanisms it has available to prevent the entry of the parasite.
Iridoviridae is an important virus family in aquaculture that is characterized by its wide spectrum of infection in aquatic animals. Recently, spotted knifejaw iridovirus (SKIV) has attracted particular attention because it causes a high death rate and severe economic loss. The ribosomal protein L10 gene (rpl10) is widely reported to participate in the antiviral process in animals and plants. To illustrate its immune roles in spotted knifejaw, we cloned rpl10 and studied its expression patterns in this study. The 3,463 bp rpl10 genomic sequence consisted of 6 exons and 5 introns, with a 1,473 bp transcript encoding a 215 amino acid protein. Tissue expression analysis revealed a wide distribution of rpl10, while higher expression was observed in immune‐related tissues (liver and kidney). Time‐course expression patterns were further examined in three tissues (spleen, liver and kidney) after SKIV infection. Significant upregulation was observed in the spleen and liver at 4 days post infection, while in the kidney, the upregulation occurred at 6 hr post infection. This study would facilitate further investigation into the functionality of rpl10 in the antiviral defence of spotted knifejaw.
Interleukin (IL)-10 is considered a prototypical anti-inflammatory cytokine, which significantly contributes to the maintenance and reestablishment of immune homeostasis. However, this classical view fails to fully describe the pleiotropic roles of IL-10. Indeed, IL-10 can also promote immune responses, e.g. by supporting B-cell and CD8+ T-cell activation. The reasons for these seemingly opposing functions are unclear to a large extent. Recent and previous studies suggest that the cellular source and the microenvironment impact the function of IL-10. However, studies addressing the mechanisms which determine whether IL-10 promotes inflammation or controls it have just begun. This review first summarizes the recent findings on the heterogeneity of IL-10 producing T cells and their impact on the target cells. Finally, we will propose two possible explanations for the dual functions of IL-10.
Interleukin-10 (IL-10) is a pleiotropic cytokine and plays a crucial role in immunity. In the current study, we examined the expression patterns and biological functions of tongue sole Cynoglossus semilaevis IL-10 (CsIL-10). CsIL-10 is composed of 186 amino acid residues and shares 46.3%-71.7% identities with other teleost IL-10. Csil-10 expression occurred in multiple tissues and was regulated by bacterial infection. Recombinant CsIL-10 (rCsIL-10) in the form of a dimer bound to a wide range of bacterial species but did not affect bacterial growth. rCsIL-10 could interact with peripheral blood leukocytes (PBL) and significantly reduce the phagocytic activity, ROS production, and apoptosis of PBL. When injected in vivo, rCsIL-10 significantly suppressed the expression of proinflammatory cytokines and promoted bacterial dissemination in tongue sole tissues. Consistently, knockdown of Csil-10 significantly inhibited bacterial infection in tongue sole. Taken together, these results indicate that CsIL-10 plays a negative regulatory role in the immune response against bacterial infection.
Tuberculosis ranks as one of the world's deadliest infectious diseases causing more than a million casualties annually. IL10 inhibits the function of Th1 type cells, and IL10 deficiency has been associated with an improved resistance against Mycobacterium tuberculosis infection in a mouse model. Here, we utilized M. marinum infection in the zebrafish (Danio rerio) as a model for studying Il10 in the host response against mycobacteria. Unchallenged, nonsense il10e46/e46 mutant zebrafish were fertile and phenotypically normal. Following a chronic mycobacterial infection, il10e46/e46 mutants showed enhanced survival compared to the controls. This was associated with an increased expression of the Th cell marker cd4-1 and a shift towards a Th1 type immune response, which was demonstrated by the upregulated expression of tbx21 and ifng1, as well as the down-regulation of gata3. In addition, at 8 weeks post infection il10e46/e46 mutant zebrafish had reduced expression levels of proinflammatory cytokines tnfb and il1b, presumably indicating slower progress of the infection. Altogether, our data show that Il10 can weaken the immune defense against M. marinum infection in zebrafish by restricting ifng1 response. Importantly, our findings support the relevance of M. marinum infection in zebrafish as a model for tuberculosis.
In mammals, the inducible cytokine interleukin 10 is a feedback negative regulator of inflammation. To
determine the extent to which this function is conserved in birds, recombinant chicken IL-10 was
expressed as a secreted human Ig Fc fusion protein (chIL-10-Fc) and used to immunise mice. Five
monoclonal antibodies (mAb) which specifically recognise chicken IL-10 were generated and characterised.
Two capture ELISA assays were developed which detected native chIL-10 secreted from chicken
bone marrow-derived macrophages (chBMMs) stimulated with lipopolysaccharide (LPS). Three of the
mAbs detected intracellular IL-10. This was detected in only a subset of the same LPS-stimulated
chBMMs. The ELISA assay also detected massive increases in circulating IL-10 in chickens challenged
with the coccidial parasite, Eimeria tenella. The same mAbs neutralised the bioactivity of recombinant
chIL-10. The role of IL-10 in feedback control was tested in vitro. The neutralising antibodies prevented IL-
10-induced inhibition of IFN-g synthesis by mitogen-activated lymphocytes and increased nitric oxide
production in LPS-stimulated chBMMs. The results confirm that IL-10 is an inducible feedback regulator
of immune response in chickens, and could be the target for improved vaccine efficacy or breeding
strategies.
Zoonosis is the leading cause of emerging infectious diseases. In a recent article, R. S. Shabman et al. (mSphere 1[1]:e00070-15, 2016,
http://dx.doi.org/10.1128/mSphere.00070-15
) report the identification of a novel gammaherpesvirus in a cell line derived from the microbat
Myotis velifer incautus.
This is the first report on a replicating, infectious gammaherpesvirus from bats. The new virus is named bat gammaherpesvirus 8 (BGHV8), also known as
Myotis
gammaherpesvirus 8, and is able to infect multiple cell lines, including those of human origin. Using next-generation sequencing technology, the authors constructed a full-length annotated genomic map of BGHV8. Phylogenetic analysis of several genes from BGHV8 revealed similarity to several mammalian gammaherpesviruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV).
Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease of carp and encodes for an Il10 homolog (ORF134). Our previous studies with a recombinant ORF134-deleted strain and the derived revertant strain suggested that cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]) is not essential for viral replication in vitro, or virulence in vivo. In apparent contrast, cyhv3Il10 is one of the most abundant proteins of the CyHV-3 secretome and is structurally very similar to carp Il10 and also human IL10. To date, studies addressing the biological activity of cyhv3Il10 on cells of its natural host have not been performed. To address the apparent contradiction between the presence of a structurally conserved Il10 homolog in the genome of CyHV-3 and the lack of a clear phenotype in vivo using recombinant cyhv3Il10-deleted viruses, we used an in vitro approach to investigate in detail whether cyhv3Il10 exerts any biological activity on carp cells. In this study, we provide direct evidence that cyhv3Il10 is biologically active and, similarly to carp Il10, signals via a conserved Stat3 pathway modulating immune cells of its natural host, carp. In vitro, cyhv3Il10 deactivates phagocytes with a prominent effect on macrophages, while also promoting proliferation of Igm+ B cells and memory T cells. Collectively, this study demonstrates a clear biological activity of cyhv3Il10 on cells of its natural host and indicates that cyhv3Il10 is a true viral ortholog of carp Il10. Furthermore, to our knowledge, this is the first report on biological activities of a nonmammalian viral Il10 homolog.
Importance:
Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome, and used this information to take steps towards developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are: (i) TeHV-3 has a novel genome structure; (ii) its closest relative is a turtle herpesvirus; (iii) it contains interleukin-10 and semaphorin genes, the first time these have been reported in an alphaherpesvirus; (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo; and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.
Subsequent to the two rounds of whole-genome duplication that occurred in the common ancestor of vertebrates, a third genome duplication occurred in the stem lineage of teleost fishes. This teleost-specific genome duplication (TGD) is thought to have provided genetic raw materials for the physiological, morphological, and behavioral diversification of this highly speciose group. The extreme physiological versatility of teleost fish is manifest in their diversity of blood-gas transport traits, which reflects the myriad solutions that have evolved to maintain tissue O(2) delivery in the face of changing metabolic demands and environmental O(2) availability during different ontogenetic stages. During the course of development, regulatory changes in blood-O(2) transport are mediated by the expression of multiple, functionally distinct hemoglobin (Hb) isoforms that meet the particular O(2)-transport challenges encountered by the developing embryo or fetus (in viviparous or oviparous species) and in free-swimming larvae and adults. The main objective of the present study was to assess the relative contributions of whole-genome duplication, large-scale segmental duplication, and small-scale gene duplication in producing the extraordinary functional diversity of teleost Hbs. To accomplish this, we integrated phylogenetic reconstructions with analyses of conserved synteny to characterize the genomic organization and evolutionary history of the globin gene clusters of teleosts. These results were then integrated with available experimental data on functional properties and developmental patterns of stage-specific gene expression. Our results indicate that multiple alpha- and beta-globin genes were present in the common ancestor of gars (order Lepisoteiformes) and teleosts. The comparative genomic analysis revealed that teleosts possess a dual set of TGD-derived globin gene clusters, each of which has undergone lineage-specific changes in gene content via repeated duplication and deletion events. Phylogenetic reconstructions revealed that paralogous genes convergently evolved similar functional properties in different teleost lineages. Consistent with other recent studies of globin gene family evolution in vertebrates, our results revealed evidence for repeated evolutionary transitions in the developmental regulation of Hb synthesis
We resequenced the genome of equid herpesvirus 2 (EHV2) strain 86/67 and sequenced the genomes of EHV2 strain G9/92 and equid
herpesvirus 5 (EHV5) strain 2-141/67. The most prominent genetic differences are the dissimilar locations of the interleukin-10
(IL-10)-like genes and the presence of an OX-2-like gene in EHV5 only.
Interleukin-10 (IL-10) is a pleiotropic cytokine that plays an important role in immune system. In the present study, the IL-10 gene of African clawed frog (Xenopus tropicalis) was first cloned, and its expression pattern and 3D structure were also analyzed. The frog IL-10 mRNA encoded 172 amino acids which possessed several conserved features found in IL-10s from other species, including five-exon/four-intron genomic structure, conserved four cysteine residues, IL-10 family motif, and six α-helices. Real-time PCR showed that frog IL-10 mRNA was ubiquitous expressed in all examined tissues, highly in some immune related tissues including kidney, spleen, and intestine and lowly in heart, stomach, and liver. The frog IL-10 mRNA was upregulated at 24 h after LPS stimulation, indicating that it plays a part in the host immune response to bacterial infection. Another IL, termed as IL-20, was identified from the frog IL-10 locus, which might be the homologue of mammalian IL-19/20 according to the analysis results of the phylogenetic tree and the sequence identities.
In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8(+) memory T cells while downregulating CD4(+) memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM(+) B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM(+) B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.
Copyright © 2014 by The American Association of Immunologists, Inc.
Mammalian type I interferons (IFNs) signal through a receptor composed of the IFNAR1 and IFNAR2 chains. In zebrafish two-cysteine IFNs utilize a receptor composed of CRFB1 and CRFB5, while four-cysteine IFNs signal through a receptor formed by CRFB2 and CRFB5. In the present work two CRFB clusters were identified in different chromosomes of Atlantic salmon. Genes of three CRFB5s, one CRFB1, one CRFB2 and the novel CRFB5x were identified, cloned and studied functionally. All CRFBs were expressed in 10 different organs, but the relative expression of CRFBs varied. Mx-reporter assay was used to study which CRFBs that might be involved in receptors for salmon IFNa, IFNb and IFNc. The results of Mx-reporter assays suggest that IFNa signals through a receptor composed of CRFB1a as the long chain and either CRFB5a, CRFB5b or CRFB5c as the short chain; IFNc signals through a receptor with CRFB5a or CRFB5c as the short chain while IFNb may signal through a receptor with CRFB5x as a short chain. Taken together, the present work demonstrates that Atlantic salmon has a more diverse repertoire of type I IFN receptors compared to zebrafish or mammals.
Despite efforts to generate new vaccines and antibiotics for tuberculosis, the disease remains a public health problem worldwide. The zebrafish Danio rerio has emerged as a useful model to investigate mycobacterial pathogenesis and treatment. Infection of zebrafish with Mycobacterium marinum, the closest relative of the Mycobacterium tuberculosis complex, recapitulates many aspects of human tuberculosis. The zebrafish model affords optical transparency, abundant genetic tools and in vivo imaging of the progression of infection. Here, we review how the zebrafish-M. marinum system has been deployed to make novel observations about the role of innate immunity, the tuberculous granuloma, and crucial host and bacterial genes. Finally, we assess how these findings relate to human disease and provide a framework for novel strategies to treat tuberculosis.
Campylobacter jejuni is the leading cause of bacterial food-borne infection; chicken meat is its main source. C. jejuni is considered commensal in chickens based on experimental models unrepresentative of commercial production. Here we show that the paradigm of Campylobacter commensalism in the chicken is flawed. Through experimental infection of four commercial breeds of broiler chickens, we show that breed has a significant effect on C. jejuni infection and the immune response of the animals, although these factors have limited impact on the number of bacteria in chicken ceca. All breeds mounted an innate immune response. In some breeds, this response declined when interleukin-10 was expressed, consistent with regulation of the intestinal inflammatory response, and these birds remained healthy. In another breed, there was a prolonged inflammatory response, evidence of damage to gut mucosa, and diarrhea. We show that bird type has a major impact on infection biology of C. jejuni. In some breeds, infection leads to disease, and the bacterium cannot be considered a harmless commensal. These findings have implications for the welfare of chickens in commercial production where C. jejuni infection is a persistent problem.
Studying macrophage biology in the context of a whole living organism provides unique possibilities to understand the contribution of this extremely dynamic cell subset in the reaction to infections, and has revealed the relevance of cellular and molecular processes that are fundamental to the cell-mediated innate immune response. In particular, various recently established zebrafish infectious disease models are contributing substantially to our understanding of the mechanisms by which different pathogens interact with macrophages and evade host innate immunity. Transgenic zebrafish lines with fluorescently labeled macrophages and other leukocyte populations enable non-invasive imaging at the optically transparent early life stages. Furthermore, there is a continuously expanding availability of vital reporters for subcellular compartments and for probing activation of immune defense mechanisms. These are powerful tools to visualize the activity of phagocytic cells in real time and shed light on the intriguing paradoxical roles of these cells in both limiting infection and supporting the dissemination of intracellular pathogens. This Review will discuss how several bacterial and fungal infection models in zebrafish embryos have led to new insights into the dynamic molecular and cellular mechanisms at play when pathogens encounter host macrophages. We also describe how these insights are inspiring novel therapeutic strategies for infectious disease treatment.
Clonal evolution is the process by which genetic and epigenetic diversity is created within malignant tumor cells. This process culminates in a heterogeneous tumor, consisting of multiple subpopulations of cancer cells that often do not contain the same underlying mutations. Continuous selective pressure permits outgrowth of clones that harbor lesions that are capable of enhancing disease progression, including those that contribute to therapy resistance, metastasis and relapse. Clonal evolution and the resulting intratumoral heterogeneity pose a substantial challenge to biomarker identification, personalized cancer therapies and the discovery of underlying driver mutations in cancer. The purpose of this Review is to highlight the unique strengths of zebrafish cancer models in assessing the roles that intratumoral heterogeneity and clonal evolution play in cancer, including transgenesis, imaging technologies, high-throughput cell transplantation approaches and in vivo single-cell functional assays.
Background
Two novel avipoxviruses from South Africa have been sequenced, one from a Feral Pigeon (Columba livia) (FeP2) and the other from an African penguin (Spheniscus demersus) (PEPV). We present a purpose-designed bioinformatics pipeline for analysis of next generation sequence data of avian poxviruses and compare the different avipoxviruses sequenced to date with specific emphasis on their evolution and gene content.
Results
The FeP2 (282 kbp) and PEPV (306 kbp) genomes encode 271 and 284 open reading frames respectively and are more closely related to one another (94.4%) than to either fowlpox virus (FWPV) (85.3% and 84.0% respectively) or Canarypox virus (CNPV) (62.0% and 63.4% respectively). Overall, FeP2, PEPV and FWPV have syntenic gene arrangements; however, major differences exist throughout their genomes. The most striking difference between FeP2 and the FWPV-like avipoxviruses is a large deletion of ~16 kbp from the central region of the genome of FeP2 deleting a cc-chemokine-like gene, two Variola virus B22R orthologues, an N1R/p28-like gene and a V-type Ig domain family gene. FeP2 and PEPV both encode orthologues of vaccinia virus C7L and Interleukin 10. PEPV contains a 77 amino acid long orthologue of Ubiquitin sharing 97% amino acid identity to human ubiquitin.
Conclusions
The genome sequences of FeP2 and PEPV have greatly added to the limited repository of genomic information available for the Avipoxvirus genus. In the comparison of FeP2 and PEPV to existing sequences, FWPV and CNPV, we have established insights into African avipoxvirus evolution. Our data supports the independent evolution of these South African avipoxviruses from a common ancestral virus to FWPV and CNPV.
Vertebrate evolution has been shaped by several rounds of whole-genome duplications (WGDs) that are often suggested to be associated with adaptive radiations and evolutionary innovations. Due to an additional round of WGD, the rainbow trout genome offers a unique opportunity to investigate the early evolutionary fate of a duplicated vertebrate genome. Here we show that after 100 million years of evolution the two ancestral subgenomes have remained extremely collinear, despite the loss of half of the duplicated protein-coding genes, mostly through pseudogenization. In striking contrast is the fate of miRNA genes that have almost all been retained as duplicated copies. The slow and stepwise rediploidization process characterized here challenges the current hypothesis that WGD is followed by massive and rapid genomic reorganizations and gene deletions.
The zebrafish research community is celebrating! The zebrafish genome has recently been sequenced, the Zebrafish Mutation Project (launched by the Wellcome Trust Sanger Institute) has published the results of its first large-scale ethylnitrosourea (ENU) mutagenesis screen, and a host of new techniques, such as the genome editing technologies TALEN and CRISPR-Cas, are enabling specific mutations to be created in model organisms and investigated in vivo. The zebrafish truly seems to be coming of age. These powerful resources invoke the question of whether zebrafish can be increasingly used to model human disease, particularly common, chronic diseases of metabolism such as obesity and type 2 diabetes. In recent years, there has been considerable success, mainly from genomic approaches, in identifying genetic variants that are associated with these conditions in humans; however, mechanistic insights into the role of implicated disease loci are lacking. In this Review, we highlight some of the advantages and disadvantages of zebrafish to address the organism's utility as a model system for human metabolic diseases.
Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae, is the causative agent of a lethal disease in common and koi carp. CyHV-3 ORF134 encodes an interleukin-10 (IL-10) homologue. The present study was devoted to this ORF. Transcriptomic analyses revealed that ORF134 is expressed as a spliced gene belonging to the early-late class. Proteomic analyses of CyHV-3 infected cell supernatant demonstrated that the ORF134 expression product is one of the most abundant proteins of the CyHV-3 secretome. To investigate the role of ORF134 in viral replication in vitro and in virulence in vivo, a deleted strain and a derived revertant strain were produced using BAC cloning technologies. The recombinant ORF134 deleted strain replicated in vitro comparably to the parental and the revertant strains. Infection of fish by immersion in water containing the virus induced comparable CyHV-3 disease for the three virus genotypes tested (wild type, deleted and revertant). Quantification of viral DNA by real time TaqMan PCR (in the gills and the kidney) and analysis of carp cytokine expression (in the spleen) by RT-qPCR at different times post-infection did not revealed any significant difference between the groups of fish infected with the three virus genotypes. Similarly, histological examination of the gills and the kidney of infected fish revealed no significant differences between fish infected with ORF134 deleted virus versus fish infected with the control parental or revertant strains. All together, the results of the present study demonstrate that the IL-10 homologue encoded by CyHV-3 is essential neither for viral replication in vitro nor for virulence in common carp.
The recent ban on the use of antibiotics as a feed additive has led to the search for alternative sources of antibiotics in the feed industry. Presently, probiotics are considered as a potential substitute for antibiotic as a live biotherapeutic agent to improve animal health and performance. Accordingly, study was focused on evaluating the effect of Saccharomyces boulardii (Sb) and Bacillus subtilis B10 (Bs) on ultrastructure modulation and mucosal immunity development in broiler chickens. A total of three hundred 1-d-old Sanhuang broilers (a Chinese cross breed) were randomized into 3 groups, each group with 5 replications (n = 20). The control group (Ctr) was fed a basal diet containing an antibiotic (virginiamycin, 20 mg/kg). Meanwhile, broilers in experimental groups received Sb and Bs (1 × 10 cfu/kg of feed) in addition to the basal diet for 72 d. The results of the experimental groups revealed a significant improvement in live BW and relative weight of bursa of Fabricius and thymus. Also, intestinal villus height, width, and number of goblet cells increased in the Sb and Bs groups. Meanwhile, modulation in the intestinal ultrastructure and increased mRNA expression levels of occluding, cloudin2, and cloudin3 (P < 0.05) were observed in the Sb and Bs groups. Moreover, IgA-positive cells significantly increased in the jejunum of Sb- and Bs-supplemented groups (P < 0.05). Intestinal cytokines interleukin-6, tumor necrosis factor-α, interleukin-10, transforming growth factor-β, and secretory IgA concentrations were (P < 0.05) improved in the probiotic groups; however, Sb induced inflammatory and antiinflammatory cytokines (P < 0.05) in comparison with the Ctr group. The present findings conclusively revealed that Sb and Bs increased IgA-positive cells in the lumen of the intestinal villus and revealed that Sb and Bs could modulate intestinal ultrastructure through increasing occluding, cloudin2, and cloudin3 mRNA expression levels in broiler intestine.
Koi herpesvirus (KHV) (species Cyprinid herpesvirus 3) ORF134 was shown to transcribe a spliced transcript encoding a 179-amino-acid (aa) interleukin-10 (IL-10) homolog (khvIL-10) in koi fin (KF-1) cells. Pairwise sequence alignment indicated that the expressed product shares 25% identity with carp IL-10, 22 to 24% identity with mammalian (including primate) IL-10s, and 19.1% identity with European eel herpesvirus IL-10 (ahvIL-10). In phylogenetic analyses, khvIL-10 fell in a divergent position from all host IL-10 sequences, indicating extensive structural divergence following capture from the host. In KHV-infected fish, khvIL-10 transcripts were observed to be highly expressed during the acute and reactivation phases but to be expressed at very low levels during low-temperature-induced persistence. Similarly, KHV early (helicase [Hel] and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes were also expressed at high levels during the acute and reactivation phases, but only low-level expression of the ITP gene was detected during the persistent phase. Injection of khvIL-10 mRNA into zebrafish (Danio rerio) embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10. Downregulation of the IL-10 receptor long chain (IL-10R1) using a specific morpholino abrogated the response to both khvIL-10 and zebrafish IL-10 transcripts, indicating that, despite the structural divergence, khvIL-10 functions via this receptor. This is the first report describing the characteristics of a functional viral IL-10 gene in the Alloherpesviridae.
The presence of activated intratumoral T cells correlates clinically with better prognosis in patients with cancer. Although tumor vaccines can increase the number of tumor-specific CD8(+) T cells in systemic circulation, they frequently fail to increase the number of active and tumor reactive T cells within the tumor. Here we show that treatment with the pleiotropic cytokine interleukin-10 (IL-10) induces specific activation of tumor-resident CD8(+) T cells as well as their intratumoral expansion in several mouse tumor models. We found that inhibition of T-cell trafficking from lymphoid organs did not impair IL-10-induced tumor rejection or the activation of tumor-resident CD8(+) T cells. Tumor-resident CD8(+) T cells expressed elevated levels of the IL-10 receptor and were directly activated by IL-10, resulting in prominent phosphorylation of STAT3 and STAT1. Although CD4(+) T cells, regulatory T cells, NK cells, and dendritic cells have been reported as prominent targets of IL-10 in the tumor microenvironment, we found that expression of the IL-10R was required only on CD8(+) T cells to facilitate IL-10-induced tumor rejection as well as in situ expansion and proliferation of tumor-resident CD8 T cells. Together, our findings indicate that IL-10 activates CD8(+) T-cell-mediated tumor control and suggest that IL-10 may represent a potential tumor immunotherapy in human patients with cancer.
Interferons (IFNs) play a major role in orchestrating the innate immune response toward viruses in vertebrates, and their defining characteristic is their ability to induce an antiviral state in responsive cells. Interferons have been reported in a multitude of species, from bony fish to mammals. However, our current knowledge about the molecular function of fish IFNs as well as their evolutionary relationship to tetrapod IFNs is limited. Here we establish the three-dimensional (3D) structure of zebrafish IFNϕ1 and IFNϕ2 by crystallography. These high-resolution structures offer the first structural insight into fish cytokines. Tetrapods possess two types of IFNs that play an immediate antiviral role: type I IFNs (e.g., alpha interferon [IFN-α] and beta interferon [IFN-β]) and type III IFNs (lambda interferon [IFN-λ]), and each type is characterized by its specific receptor usage. Similarly, two groups of antiviral IFNs with distinct receptors exist in fish, including zebrafish. IFNϕ1 and IFNϕ2 represent group I and group II IFNs, respectively. Nevertheless, both structures reported here reveal a characteristic type I IFN architecture with a straight F helix, as opposed to the remaining class II cytokines, including IFN-λ, where helix F contains a characteristic bend. Phylogenetic trees derived from structure-guided multiple alignments confirmed that both groups of fish IFNs are evolutionarily closer to type I than to type III tetrapod IFNs. Thus, these fish IFNs belong to the type I IFN family. Our results also imply that a dual antiviral IFN system has arisen twice during vertebrate evolution.
IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Thl T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-y)-induced production of 1L-1, IL-6, and TNF-< proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS-and IFN-r-induced expression of IL-la, IL-6, or TNF-ft mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-7 was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-7-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.
The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.
Aggad, D., C. Stein, D. Sieger, M. Mazel, P. Boudinot, P. Herbomel, J.-P. Levraud, G. Lutfalla, and M. Leptin. 2010. In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish. J. Immunol . 185: [6774–6782][1].
Two errors appeared in the published morpholino sequences (Table II).
In the
Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process.
The interleukin (IL)-10 cytokine family includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26 and the lambda/type III interferons. They are highly pleiotropic and mediate a variety of activities, including immune suppression and antibacterial immunity. To exert their functions they signal through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptors; IL-10R1, IL-20R1 or IL-22R1) and a subunit with a short intracellular domain (R2 type receptors; IL-10R2 or IL-20R2). In this study we report the identification of three R1 type receptors (named IL-10R1/CRFB7, IL-20R1a/CRFB8a and IL-20R1b/CRFB8b) and one R2 type receptor (named IL-10R2/CRFB4) in rainbow trout. The nomenclature of the receptors was supported by homology analysis, conserved motifs and phylogenetic tree analysis, confirming they belong to the piscine class 2 cytokine receptor family. For instance, they all displayed the presence of characteristic features, such as conserved fibronectin type-III domains. Expression analysis in tissues collected from healthy fish revealed different patterns of expression for each receptor, suggesting their potential involvement in different types of immune responses. When studying the modulation of the genes in cell lines and primary cultures, a greater effect was observed in the cell lines, where the expression of most receptors was affected by incubation with microbial mimics (LPS and PolyI:C) or the pro-inflammatory cytokine rIFN-γ. In addition, expression of the four receptors was modulated by viral infection, suggesting a potential involvement of such receptors and their ligands in antiviral defence.
The goal of this work was to identify interleukin (IL)-related genes in the gilthead sea bream (GSB) (Sparus aurata L.) and how they are modulated by the parasite Enteromyxum leei, a myxozoan that causes severe enteritis with a strong inflammatory response. A Blast-X search of our transcriptomic GSB database (www.nutrigroup-iats.org/seabreamdb) identified 16 new sequences encompassing seven ILs (IL-7, IL-8, IL-10, IL-12β, IL-15, IL-18, and IL-34), the interleukin enhancer-binding factor 2 (ILF2), and eight IL receptors (IL-R); IL-R1, IL-6RA, IL-6RB, IL-8RA, IL-10RA, IL-10RB, IL-18R1, and IL-22R). Except for ILF2, their expression, plus that of IL-1β, IL-1R2, IL-6, and TNF-α (from public repositories), were analysed by 96-well PCR array of samples of blood, spleen, head kidney, and intestine of GSB that were anally intubated with E. leei (recipient group, RCPT). Only the expression profile of the intestine of RCPT fish showed significant difference as compared to samples from PBS-inoculated fish. At 17 days post inoculation (dpi), the expression of key pro-inflammatory ILs, such as IL-8, IL-8R, IL-12β, and TNFα was significantly up-regulated, whereas at 64 dpi, anti-inflammatory IL expression (IL-6, IL-6RB, IL-7, IL-10, IL-10RA, and IL-15) was predominant. These results indicate a modification of the IL expression at late times post infection, probably to protect the fish intestine from the parasite and damage inflicted by an excessive inflammatory response. Furthermore, the response is mainly mediated at the local level as no significant changes were detected in blood, spleen and head kidney.
Many viruses have evolved strategies to deregulate the host immune system. These strategies include mechanisms to subvert or recruit the host cytokine network. Interleukin-10 (IL-10) is a pleiotropic cytokine that has both immunostimulatory and immunosuppressive properties. However, its key features relate mainly to its capacity to exert potent immunosuppressive effects. Several viruses have been shown to up regulate the expression of cellular IL 10 (cIL-10), with, in some cases, enhancement of infection by suppression of immune functions. Other viruses encode functional orthologues of cIL-10, called viral IL-10s (vIL-10s). The present review is devoted to these virokines. To date, vIL-10 orthologues have been reported for 12 members of the family Herpesviridae, two members of the family Alloherpesviridae, and seven members of the family Poxviridae. Study of vIL-10s demonstrated several interesting aspects on the origin and the evolution of these viral genes; such as for example, the existence of multiple (potentially up to 9) independent gene acquisition events at different times during evolution, viral gene acquisition resulting from recombination with cellular genomic DNA or cDNA derived from cellular mRNA, and the evolution of cellular sequence in the viral genome to restrict the biological activities of the viral orthologues to those beneficial for the virus life cycle. In this review, various aspects of the vIL 10s described to date are reviewed, including their genetic organization, protein structure, origin, evolution, biological properties and potential in applied research.
Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T cells, monocytes, and macrophages, is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T cell, the T regulatory cell, which may figure prominently in control of immune responses and tolerance in vivo. Uniquely among hemopoietic cytokines, IL-10 has closely related homologs in several virus genomes, which testify to its crucial role in regulating immune and inflammatory responses. This review highlights findings that have advanced our understanding of IL-10 and its receptor, as well as its in vivo function in health and disease.
Analysis of gene polymorphisms and disease association is essential for assessing putative candidate genes affecting susceptibility or resistance to disease. In this paper, we report the results of an association analysis between single nucleotide polymorphisms (SNPs) in common carp (Cyprinus carpio) innate immune response genes and resistance to cyprinid herpesvirus-3 (CyHV-3) infection. Twenty-three SNPs from 14 genes were genotyped in the progeny of a full-sib family, which was subjected to a CyHV-3 challenge. The challenge was carried out by cohabitation of naïve fish with CyHV-3-infected fish and the experimental fish subsequently were scored as susceptible or resistant. Fisher exact probability and chi-square tests were used to assess the association of SNP genotypes or alleles with CyHV-3 resistance. No association between SNPs in common carp immune response genes and resistance to CyHV-3 was found, except for SNPs in IL-10a. For IL-10a, both heterozygous and homozygous fish carrying the GCG haplotype were more susceptible to CyHV-3 infection (P
The interleukin-10 receptor 2 (IL-10R2, IL-10Rβ) is required for the signalling of the class 2 cytokines IL-10, IL-22, IL-26 and IFN-λ(1-3) . Here, we describe the identification of the Pekin duck IL-10R2 (duIL-10R2) common chain and its gene structure. The duIL-10R2 cDNA encodes a 343 amino acid protein that has an amino acid identity of 76% and 42% with chicken and human IL-10R2, respectively. Binding residues of human IL-10R2 for IL-10 and IL-22 were mostly conserved in the avian IL-10R2 proteins within loops L3 and L5, but not within loops L2 and L6. Homology modelling of the duIL-10R2 extracellular domain structure using soluble human IL-10R2 (shIL-10R2, PDB ID: 3LQM) as a template revealed a protruding loop L5 and two distinct clefts between loops L2/L3 and L3/L5, similar to shIL-10R2. However, in contrast to the three amino acid β-hairpin loop L2 of shIL-10R2, loop L2 of duIL-10R2 is five residues longer. Residues within a putative Tyk2 binding site were highly conserved across all vertebrate IL-10R2 proteins examined. The duIL-10R2 gene shares a seven exon-six intron structure with chicken and human IL-10R2 genes, but avian genes are more compact. DuIL-10R2 mRNA was constitutively expressed in all tissues. Mitogen stimulation of duck peripheral blood mononuclear cells (PBMC) did not alter transcript levels. Our observations suggest that genomic organization and structural features implicated in multiple cytokine-binding properties of human IL-10R2 are conserved in duck IL-10R2, but the evolutionary changes that appear to have lead to low-affinity cytokine interaction within loop L2 are distinct to mammalian species.
Cellular interleukin-10 (IL-10) gene from the peripheral blood mononuclear cells of the healthy Dromedary camel (Camelus dromedarius) and viral IL-10 (vIL-10) from the skin scabs of the Dromedary camels infected with contagious ecthyma (a parapoxviral infection in the camels) were amplified by polymerase chain reaction, cloned and characterized. Sequence analysis revealed that the open reading frame (ORF) of dromedarian camel IL-10 is 537bp in length, encoding 178 amino acid polypeptide while open reading frame of vIL-10 from camel is 561bp, encoding 187 amino acid polypeptide. The Dromedary camel IL-10 exhibited 62.6% and 68.5% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from camel. Sequence analysis also revealed that the Dromedary camel IL-10 shared 99.4% and 98.3% identity at the nucleotide and amino acid level, respectively, with the Bactrian camel (Camelus bactrianus). But vIL-10 from camel shared 84.7% and 83.4% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from reindeer (Rangifer tarandus), which is a ruminant species belonging to the order Artiodactyla. The present study was conducted to evaluate the evolutionary origin of the camel parapoxvirus with parapoxviruses of cattle and sheep and the resultant sequence analysis revealed that camel parapoxvirus is closely related to cattle parapoxvirus than sheep parapoxvirus (Orf virus).
In Th1 and Th2 memory lymphocytes, the genes for the cytokines interleukin (IL)-4 and interferon- (IFN-) are imprinted for expression upon restimulation. This cytokine memory is based on expression of the transcription factors T-bet for IFN-, and GATA-3 for IL-4, and epigenetic modification of the cytokine genes. In Th2 cells, expression of the cytokine IL-10 is also induced by GATA-3. Here, we show that this induction is initially not accompanied by epigenetic modification of the IL-10 gene. Only after repeated restimulation of a memory Th2 cell in the presence of IL-4, extensive histone acetylation of the IL-10 gene is detectable. This epigenetic imprinting correlates with the development of a memory for IL-10 in repeatedly restimulated Th2 cells. In Th1 cells, IL-10 expression is induced by IL-12, but the IL-10 gene lacks detectable histone acetylation. Accordingly, IL-10 expression in restimulated memory Th1 cells remains conditional on the presence of IL-12. This finding defines a potential anti-inflammatory role for IL-12 in Th1 recall responses. While in primary Th1 responses IL-12 is required to induce expression of the pro-inflammatory cytokine IFN-, in secondary Th1 responses IFN- re-expression is independent of IL-12, which still is able to induce expression of the anti-inflammatory cytokine IL-10.
Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases.
Here we describe the cloning and expression of Pekin duck IL-10 (duIL-10) and a six exon-5 intron structure of an IL-10 gene. Two transcripts encoding duIL-10 with an alternatively spliced 3'UTR, and a transcript lacking exon 5 with a novel coding sequence for its C-terminus (duIL-10ΔE5) were isolated from splenocytes. The duIL-10 protein has an amino acid identity of 79% and 47% with chicken and human IL-10, respectively. The duck IL-10 gene shares a similar structure of the respective exons 1-5 with the IL-10 genes of other vertebrates but has an alternative exon. The duIL-10 3D structure by homology modeling was similar to that of the human IL-10 monomer, whereas the predicted duIL-10ΔE5 protein lacks helix F. DuIL-10 and duIL-10ΔE5 transcripts were most abundant in primary and secondary immune organs and lung. Recombinant duIL-10 suppressed duck IL-2 transcripts in mitogen-activated PBMCs. Our observation suggests evolutionary conservation of structure and function of the duIL-10 protein but the roles of the novel IL-10 splice variants in the regulation of duck immune responses and evolution of vertebrate immunity remain to be elucidated.
Macrophages play a central role in infections, as a target for pathogens and in activation of the immune system. Interleukin-10 (IL-10), a cytokine produced by macrophages, is a potent immunosuppressive factor. Some intracellular pathogens specifically target macrophages for infection and use IL-10 to dampen the host immune response and stall their elimination from the host. Certain viruses induce production of cellular IL-10 by macrophages, whereas other viruses encode their own viral IL-10 homologs. Additionally, specific bacteria, including several Mycobacteria spp. and Listeria monocytogenes, can survive and replicate in macrophages while inducing cellular IL-10, highlighting a potential role for IL-10 of macrophage origin in the immunosuppressive etiology of these pathogens. Thus, the exploitation of IL-10 appears to be a common mechanism of immunosuppression by a diverse group of intracellular pathogens that can infect macrophages.
Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection.
Interleukin-10 (IL-10) is a multifunctional cytokine and plays an important role in diseases. In this study, IL-10 gene was cloned and characterized from catla (Catla catla), which is a highly commercially important fish species in the Indian subcontinent. The result indicated that the full-length catla IL-10 (cIL-10) gene had five exons and four introns with an open reading frame of 540 nucleotides encoding a polypeptide of 179 amino acids. A phylogenetic analysis of cIL-10 gene sequence showed that cIL-10 clustered with freshwater carps group as expected. Quantitative real-time polymerase chain reaction analysis showed that cIL-10 was expressed in gill, liver, kidney, intestine, skin and heart and its expression profile was up-regulated in bacterial infection and LPS treatment. A close relationship of high cIL-10 expression and low pro-inflammatory cytokine IL-1 beta expression was observed in the treated group of fish, which might reveal the role of cIL-10 as an anti-inflammatory cytokine. Mechanism of cIL-10 induction was investigated by blocking nuclear factor (NF)-?B -signalling with BAY 11-7082 in catla kidney cell culture. Blocking NF-?B suppressed IL-10 induction by LPS, and thus it revealed that cIL-10 was induced through NF-?B signalling. These data could be helpful to understand the function of IL-10 in fish in response to vaccinations, probiotics and various diseases.
Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune system for various conserved pathogen-associated molecular motifs. Chicken TLR3 and TLR21 (avian equivalent to mammalian TLR9) recognize poly I:C (double-stranded RNA) and CpG-ODN (a CpG-motif containing oligodeoxydinucleotide), respectively. Interaction between TLR3 and TLR21 agonists poly I:C and CpG-ODN has been reported to synergize in expression of proinflammatory cytokines and chemokines and the production of nitric oxide in chicken monocytes. However, the interaction between poly I:C and CpG-ODN on the expression of interferons (IFNs) and Th1/Th2 cytokines remains unknown. The objective of the present study was to investigate the effect of the interaction between poly I:C and CpG-ODN on the mRNA expression levels of IFN-α and IFN-β, Th1 cytokines IFN-γ and IL-12, Th2 cytokine IL-4, and regulatory IL-10 in chicken monocytes. When stimulated with either agonist alone, CpG-ODN significantly up-regulated the expression of INF-γ, IL-10, and IL-12p40, but not IFN-α and IFN-β; whereas poly I:C induced the expression of INF-γ, IFN-α, IFN-β, and IL-10; but not IL-12p40. However, stimulation with a combinatory CpG-ODN and poly I:C further synergistically increased the expression of IFN-γ and IL-10 mRNA. Our results provide strong evidence supporting the critical role of TLR3 and TLR21 in avian innate immunity against both viral and bacterial infections; and the synergistic interaction between the TLR3 and TLR21 pathways produces a stronger Th1-biased immune response in chicken monocytes. Our result also suggest a potential use of poly I:C and CpG-ODN together as a more efficient adjuvant for poultry vaccine development.
Interleukin-10 (IL-10) mediates its broad anti-inflammatory and immunoregulatory effects through two cell surface receptors by which binding to the IL-10 receptor 1 (IL-10R1) is the initial step that leads to recruitment of IL-10R2 and initiation of the ternary complex signal transduction cascade. The duck IL-10R1 (duIL-10R1) cDNA was obtained by using RT-PCR and 5'RACE. The deduced 574 amino acid protein has an amino acid identity of 62%, 27% and 28% with chicken, mouse and human IL-10R1, respectively. Comparison of the duIL-10R1 cDNA with duck genomic sequences revealed a seven exon-six intron structure of the duck IL-10R1 gene that shares a similar size with the respective exons 1-7 of the chicken and human IL-10R1 genes, but the avian genes are more compact. Promoter analysis identified putative binding sites for regulatory elements such as CCAAT enhancer binding protein-α, specificity protein 1 (Sp1), nuclear factor 1 (NF1), transcriptional regulatory protein Oct-1, nuclear factor (NF) κB and interferon-stimulated gene factor-3 (ISGF-3). A canonical TATA box was absent in proximity of the transcription initiation site, but a CpG island was present. Sequence analysis of the predicted duIL-10R1 protein revealed characteristic features of class-II cytokine receptors (CFR2) family members and a considerable degree of conservation of residues implicated in ligand binding across higher vertebrates. The predicted secondary structure of the duIL-10R1 extracellular domain is compatible with the two-subdomain structure of the human IL-10R1 protein established by its crystal structure. The 3D model structure shows conservation of the positions of conserved contact residues within four of the five ligand-binding loops. Within the cytoplasmic domain, residues implicated in signal transduction were conserved including two redundant peptide motifs GYXXQ essential for recruitment and activation of STAT3. DuIL-10R1 mRNA expression was most abundant in spleen, thymus, peripheral blood mononuclear cells (PBMCs) and lung. Mitogen stimulation of PBMCs transiently increased duIL-10R1 mRNA expression. Our observations suggest significant evolutionary conservation of the IL-10R1 genomic organization, protein structure and receptor function through the JAK/STAT signalling pathway across higher vertebrates.
Viral interleukin 10 (IL-10) like open reading frames have been identified in several pox- and herpesviruses, including the fish herpesviruses Anguillid herpesvirus 1 (AngHV-1) and Cyprinid herpesvirus 3 (CyHV-3). European eel (Anguilla anguilla) IL-10 was sequenced, in order to compare European eel and common carp (Cyprinus carpio) IL-10 with their alloherpesviral counterparts. Homology between the virus and host IL-10 amino acid sequences is low, which is confirmed by phylogenetic analysis. However, the three dimensional structures of the fish and alloherpesviral IL-10 proteins as predicted by modeling are highly similar to human IL-10. Closely related AngHV-1 and CyHV-3 are expected to have obtained their viral IL-10 genes independently in the course of coexistence with their respective hosts. The presence and structural conservation of these alloherpesviral IL-10 genes suggest that they might play an important role in the evolution of pathogenesis.
This is the first report of the identification and molecular characterization of an interleukin-10 receptor 1 in bony fish. By gene synteny analysis, we identified the zebrafish interleukin-10 receptor 1 (IL10R1) and using this IL10R1 sequence, we cloned the goldfish IL10R1 cDNA transcript. The identified fish IL10R1 protein sequences had a putative JAK1 binding site, only one of the two STAT3 binding sites, that are present in all other vertebrates IL10R1 proteins as well as C-terminal serine rich areas, believed to be responsible for the anti-inflammatory properties of IL10R1. Phylogenetically, the fish IL10R1 proteins grouped independently of the amphibian, avian and mammalian IL10R1s. Quantitative gene expression analysis of the IL10R1 of zebrafish and goldfish revealed highest mRNA levels in the spleen tissues. High mRNA levels were also observed in the zebrafish muscle in contrast to low mRNA levels in the muscle of the goldfish. Moderate IL10R1 mRNA levels were seen in most other tissues examined and lowest gene expression was in the liver of both fish species. Goldfish monocytes stimulated with a recombinant goldfish interleukin-10 (rgIL-10) or with heat killed fish pathogens, Aeromonas salmonicida or Trypanosoma carassii, exhibited significantly reduced mRNA levels of the IL10R1. Furthermore, we produced a recombinant form of the goldfish IL10R1 (rgIL10R1) and using in vitro binding studies, demonstrated that the dimerized rgIL-10 specifically interacted with rgIL10R1. Our results suggest that interleukin-10 system has been highly conserved throughout evolution.
The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1β and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.
Interleukin-10 (IL-10) is a pleiotropic immune-regulatory cytokine that is expressed in various species of fish and higher vertebrates, and is activated during infection. In spite of its important role, IL-10 has not been well characterized either functionally or structurally in fish. To analyze its properties and function, we constructed a 3D model of IL-10 in the Indian major carp, the catla (Catla catla), which is a highly preferred fish species and the most commercially important one in the Indian subcontinent. The catla IL-10 model was constructed by comparative modeling using human IL-10 (2ILK) as the template, and a 5 ns molecular dynamics (MD) simulation was carried out to characterize its structural and dynamical features, which was validated by the SAVES, WHAT IF and MolProbity servers. Analysis using the VAST server revealed a comparatively low level of homology between catla and human IL-10 amino acids at the N-terminal (22.7%) compared to the C-terminal (38.29%). Six conserved domains (A-F) were predicted in catla that threaded well with human IL-10, but their putative interaction sites varied significantly. The amino acid residues in helices A and F differed in length between catla and human IL-10, which may lead to the differences in the IL-10/IL-10R complexes of these two species. The existence of two highly conserved amino acid residues (Cys5 and Cys10) in fish IL-10 but not in higher vertebrate (including human) IL-10 was analyzed in this 3D model. CastP, cons-PPISP and InterProSurf server identified several binding pockets with various probe radii, but Cys5 and Cys10 did not form any significant bonds relating to structural stabilization or protein-protein interactions.
