Genetic variation in haemolytic activity in Atlantic salmon (Salmo salar L.)

Spontaneous and antibody-dependent haemolytic activity against sheep red blood cells (SRBC) has been studied in serum from Atlantic salmon, Salmo salar L. Considerable increase in haemolytic activity was detected when SRBC were sensitized with haemolysin produced in Atlantic salmon. Haemolytic activity was sensitive both to divalent cations and to heat treatment. Significant reduction in haemolytic activity was detected after absorption with SRBC, indicating the presence of natural antibody against SRBC in Atlantic salmon serum. Persistent haemolytic activity of unsensitized SRBC in serum absorbed to remove natural antibodies was found, which suggests the activation of the alternative complement pathway, while the observed increased haemolytic activity in the presence of specific antibody against SRBC suggests activation of the classical complement pathway. Spontaneous and antibody-dependent haemolytic activity were both analysed in absorbed and non-absorbed sera in a family material of Atlantic salmon. The material consisted of 574 fish belonging to 57 fullsib groups within 20 paternal halfsib groups. Fish with signs of sexual maturity generally showed reduced haemolytic activity. Statistically significant effect of sire on the spontaneous haemolytic activity of both absorbed and nonabsorbed serum, and on antibody-dependent activity, provides evidence of significant additive genetic variation in both the alternative and the classical complement activation in Atlantic salmon. Neither on a phenotypical nor on a family basis were the two traits statistically correlated. The estimated heritabilities were 0.2–0.3 with a standard error of approximately 0.1.     

Electron microscopic examination of antigen uptake by salmonid gill cells after bath immunization with a bacterin

Atlantic salmon, Salmo salar , were given 2-min bath immunization with Yersinia ruckeri O-antigen bacterins at doses of 10, 100, and 1000 μg ml⁻¹. The uptake of the antigen was followed by light and electron microscopy of samples taken immediately and periodically after immunization, and the immune response monitored by the passive haemolytic plaque assay. The particulate antigen was observed in the gill mucus, adhering to and within the pavement cells covering the gill filaments, and in mononuclear phagocytes below the epidermal gill cells. There was a direct doseresponse correlation in the observed immune response according to the numbers of splenic antibody-producing cells 14 days after immunization. The cells involved in the recognition and uptake of a bacterin are initial important steps in the immune response, and these studies may aid in the immunopotentiation of fish vaccines and bacterins.     

An alloy of zinc and innate immunity: Galvanising host defence against infection

Innate immune cells such as macrophages and neutrophils initiate protective inflammatory responses and engage antimicrobial responses to provide frontline defence against invading pathogens. These cells can both restrict the availability of certain transition metals that are essential for microbial growth and direct toxic concentrations of metals towards pathogens as antimicrobial responses. Zinc is important for the structure and function of many proteins, however excess zinc can be cytotoxic. In recent years, several studies have revealed that innate immune cells can deliver toxic concentrations of zinc to intracellular pathogens. In this review, we discuss the importance of zinc status during infectious disease and the evidence for zinc intoxication as an innate immune antimicrobial response. Evidence for pathogen subversion of this response is also examined. The likely mechanisms, including the involvement of specific zinc transporters that facilitate delivery of zinc by innate immune cells for metal ion poisoning of pathogens are also considered. Precise mechanisms by which excess levels of zinc can be toxic to microorganisms are then discussed, particularly in the context of synergy with other antimicrobial responses. Finally, we highlight key unanswered questions in this emerging field, which may offer new opportunities for exploiting innate immune responses for anti‐infective development.     

Interferon‐inducible GTPases in cell autonomous and innate immunity

Detection and clearance of invading pathogens requires a coordinated response of the adaptive and innate immune system. Host cell, however, also features different mechanisms that restrict pathogen replication in a cell‐intrinsic manner, collectively referred to as cell‐autonomous immunity. In immune cells, the ability to unleash those mechanisms strongly depends on the activation state of the cell, which is controlled by cytokines or the detection of pathogen‐associated molecular patterns by pattern‐recognition receptors. The interferon (IFN) class of cytokines is one of the strongest inducers of antimicrobial effector mechanisms and acts against viral, bacterial and parasitic intracellular pathogens. This has been linked to the upregulation of several hundreds of IFN‐stimulated genes, among them the so‐called IFN‐inducible GTPases. Two subfamilies of IFN‐inducible GTPases, the immunity‐related GTPases (IRGs) and the guanylate‐binding proteins (GBPs), have gained attention due to their exceptional ability to specifically target intracellular vacuolar pathogens and restrict their replication by destroying their vacuolar compartment. Their repertoire has recently been expanded to the regulation of inflammasome complexes, which are cytosolic multi‐protein complexes that control an inflammatory cell death called pyroptosis and the release of cytokines like interleukin‐1β and interleukin‐18. Here we discuss recent advances in understanding the function, the targeting and regulation of IRG and GBP proteins during microbial infections.     

The fifth dimension of innate immunity

Innate immunity has evolved as a first line defense against invading pathogens. Cellular and humoral elements of the innate immune system detect infectious parasites, initiate inflammatory resistance reactions and finally contribute to the elimination of the invaders. Repeated attacks by pathogenic agents induce adaptive responses of the innate immune system. Typically, reapplication of pathogens provokes tolerance of the affected organism. However, also stimulatory effects of primary infections on subsequent innate immune responses have been observed. The present overview touches an undervalued aspect in the innate immune response: Its pronounced dependency on pathogen load. In addition to localization and timing of innate immune responses the pathogen dose dependency might be considered as a “fifth dimension of innate immunity”. Experimental results and literature data are presented proposing a hormetic reaction pattern of innate immune cells depending on the dose of pathogens.     

The kinetics of the hypoferraemic response and changes in levels of alternative complement activity in diploid and triploid Atlantic salmon, following injection of lipopolysaccharide.

To study any possible effects of triploidy on the kinetics of the response of two non-specific disease factors, full sibling diploid and triploid Atlantic salmon were injected intraperitoneally with either lipopolysaccharide (1 mg kg(-1) body weight) or saline. Individually marked fish were repetitively blood sampled for up to 19 days. Total serum protein concentrations remained constant throughout the experiment indicating that the sampling regime did not cause haemodilution. The alternative complement pathway activity (measured by the titre of haemolytic activity against rabbit erythrocytes) in the serum of saline injected fish remained constant but in LPS-injected fish it fell to barely detectable levels 2 days after injection, but recovered to pre-treatment levels by about day 5. Triploid fish took slightly longer to reach full recovery levels than diploids. All groups of fish showed a hypoferraemic response, suggesting that the sampling regime was at least partially responsible. However, the response was more rapid and pronounced in the LPS-injected fish. In the latter, serum iron concentrations decreased to very low levels by day 2 post-injection in the diploid fish and by day 3 in the triploid fish. Pre-treatment iron levels were re-established by about 15 days post-injection in all groups. The data show only slight differences between the diploid and triploid fish, but the longer time taken for the triploids to recover complement activity and the slower onset of the hypoferraemic response following injection of LPS, suggest that they may be at a disadvantage compared with their diploid siblings in their defence against bacterial infections.     

昆虫天然免疫反应分子机制研究进展

昆虫体内缺乏高等脊椎动物所具有的获得性免疫系统, 只能依赖发达的天然免疫系统抵抗细菌、 真菌、 病毒等外源病原物的侵染。本文概括了昆虫天然免疫反应发生和作用的分子机制相关进展, 重点阐述了重要免疫相关因子在昆虫天然免疫反应中的功能和作用机制。昆虫天然免疫反应分为体液免疫和细胞免疫两种, 二者共同作用完成对病原物的吞噬 (phagocytosis)、 集结 (nodulation)、 包囊 (encapsulation)、 凝结 (coagulation)和黑化（melanization）等。当昆虫受到外界病原物的侵染时, 首先通过体内的模式识别蛋白（pattern recognition proteins/receptor, PRPs）识别并结合病原物表面特有的模式分子（pathogen-associated molecular pattern, PAMPs）, 继而一系列包括丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂在内的级联激活反应被激活和调控, 产生抗菌肽、 黑色素等免疫效应分子, 清除或杀灭外源物。抗菌肽是一类小分子量的阳离子肽, 具有广谱抗菌活性, 针对不同类型的病原物, 抗菌肽的产生机制也不尽相同。昆虫体内存在着两种信号转导途径调节抗菌肽的产生： 一是由真菌和大部分革兰氏阳性菌激活的Toll途径; 二是由革兰氏阴性菌激活的Imd途径（immune deficiency pathway）。这两个途径通过激活不同转录因子调控不同抗菌肽基因的表达参与昆虫体内的天然免疫反应。     

Antimicrobial activity in serum of Atlantic cod Gadus morhua L.: comparison between cultured and wild fish

The present investigation determined the antibacterial activities in the sera of both cultured and wild Atlantic cod Gadus morhua . Serum samples from both groups of fish were quantified for total protein, and their effects against fish pathogens Listonella anguillarum , Aeromonas salmonicida and Yersinia ruckeri were determined using co-incubation assay. Total serum protein concentrations were not significantly different between farmed and wild Atlantic cod. Sera of cultured Atlantic cod significantly decreased growth of L. anguillarum by at least two-log₁₀ reductions in bacterial count, while those of the wild Atlantic cod were able to inhibit the growth of all three fish bacterial pathogens. The present study showed that sera from wild fish possess broader antibacterial activities than cultured Atlantic cod and that these could provide an explanation for the differences regarding their immunity to bacterial infections as well as their health status.     

Structural and functional characterization of CATH_BRALE,the defense molecule in the ancient salmonoid,Brachymystax lenok

Thick-lipped lenok, Brachymystax lenok is one of the ancient fish species in China and northeast Asia countries. Due to the overfishing, the population of lenok has been declined significantly. Cathelicidins are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. This report identifies and characterizes the salmonoid cathelicidin (CATH_BRALE) from this ancient fish. It consists of open reading frame (ORF) of 886 bp encoding the putative peptide of 199 amino acids. Sequence alignment with other representative salmonid cathelicidins displayed two distinctive features of current lenok cathelicidin: high level of arginine, resulting in high positive charge and glycine residues, which is significantly different from most acknowledged types of cathelicidins; and the six-amino-acid tandem repeated sequence of RPGGGS detected in a variable number of copies among fish cathelicidins, suggesting the existence of a genetically unstable region similar to that found in some mammalian cathelicidins. Expression of CATH_BRALE is predominantly found in gill, with lower levels in the gastrointestinal tract and spleen. The homology modeled structure of CATH_BRALE exhibits structural features of antiparallel β-sheets flanked by α-helices that are representative of small cationic cathelicidin family peptides. CATH_BRALE possesses much stronger antimicrobial activity against gram-negative bacteria than that of the human ortholog, LL-37. The growth of two typical fish bacterial pathogens, gram-negative bacterium of Aeromonas salmonicida and Aeromonas hydrophila was substantially inhibited by synthetic CATH_BRALE, with both MICs as low as 9.38 μM.     

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.     

Binding of bacterial secondary messenger molecule c di-GMP is a STING operation

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body' defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING^-/- mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.     

Molecular characterization of hepcidin AS-hepc2 and AS-hepc6 in black porgy (Acanthopagrus schlegelii): expression pattern responded to bacterial challenge and in vitro antimicrobial activity

There are more diversified isoforms of the hepcidin gene that exist in fishes than in mammals, and elucidating the differences between these isoforms should provide insight into the functioning of hepcidin in fishes. In our study, AS-hepc2 and AS-hepc6 hepcidin isoforms from black porgy were characterized for their in vivo expression patterns following bacterial challenge, and their in vitro antimicrobial activities against Gram-positive and Gram-negative bacteria as well as fungi. As a result, two isoforms were observed to be widely distributed in all the tissues tested. AS-hepc2 was a liver-expressed hepcidin peptide which was always highly more expressed in the liver than in the other tissues tested no matter whether this was before or after bacterial challenge. AS-hepc6 was detected mainly in the head kidney and trunk kidney of normal fish, but, in the challenged fish, its expression involved more tissues than just the kidneys. The mature peptides of AS-hepc2 and AS-hepc6 were modeled for 3D structure and then synthesized for antimicrobial assay. AS-hepc6 had a wider antimicrobial spectrum than AS-hepc2 and, in particular, had more potent antifungal activity. Our study indicated that the two hepcidin isoforms had different characteristics in terms of their expression patterns and antimicrobial activity, and they were assumed to play an overlapping role in the innate immune system of black porgy against invading pathogens.     

Stimulation of TLRs by LMW-HA induces self-defense mechanisms in vaginal epithelium

The innate immune system is present throughout the female reproductive tract and functions in synchrony with the adaptive immune system to provide protection in a way that enhances the chances for fetal survival, while protecting against potential pathogens. Recent data show that activation of Toll-like receptor (TLR)2 and 4 by low-molecular weight hyaluronic acid (LMW-HA) in the epidermis induces secretion of the antimicrobial peptide β-defensin 2. In the present work, we show that LMW-HA induces vaginal epithelial cells to release different antimicrobial peptides, via activation of TLR2 and TLR4. Further, we found that LMW-HA favors repair of vaginal epithelial injury, involving TLR2 and TLR4, and independently from its classical receptor CD44. This wound-healing activity of LMW-HA is dependent from an Akt/phosphatidylinositol 3 kinase pathway. Therefore, these findings suggest that the vaginal epithelium is more than a simple physical barrier to protect against invading pathogens: on the contrary, this surface acts as efficient player of innate host defense, which may modulate its antimicrobial properties and injury restitution activity, following LMW-HA stimulation; this activity may furnish an additional protective activity to this body compartment, highly and constantly exposed to microbiota, ameliorating the self-defense of the vaginal epithelium in both basal and pathological conditions.     

Molecular mechanisms of the shrimp clotting system

Shrimp, like other invertebrates, relies solely on its innate immune system, to combat invading pathogens. The invertebrate immune system has ancient origins that involve cellular and humoral responses. The clotting system of the humoral immune response is the first line of defense against pathogens and also serves to prevent blood loss during injury and wound healing. Tranglutaminase and clotting protein are molecules involved in the blood clotting system of crayfish and shrimp. Studies have shown that the shrimp clotting system is linked with the activation of antimicrobial peptides, similar to that of the horseshoe crab. Unlike the horseshoe crab and crayfish blood coagulation which are well studied systems, blood clotting in shrimp remains poorly understood. Here we review the shrimp clotting system and its involvement in innate immunity.     

IL-15 links TLR2/1-induced macrophage differentiation to the vitamin D-dependent antimicrobial pathway

An essential function of the innate immune system is to directly trigger antimicrobial mechanisms to defend against invading pathogens. In humans, one such pathway involves activation by TLR2/1L leading to the vitamin D-dependent induction of antimicrobial peptides. In this study, we found that TLR2/1-induced IL-15 was required for induction of CYP27b1, the VDR and the downstream antimicrobial peptide cathelicidin. Although both IL-15 and IL-4 triggered macrophage differentiation, only IL-15 was sufficient by itself to induce CYP27b1 and subsequent bioconversion of 25-hydroxyvitamin D3 (25D3) into bioactive 1,25D3, leading to VDR activation and induction of cathelicidin. Finally, IL-15-differentiated macrophages could be triggered by 25D3 to induce an antimicrobial activity against intracellular Mycobacterium tuberculosis. Therefore, IL-15 links TLR2/1-induced macrophage differentiation to the vitamin D-dependent antimicrobial pathway.     

Large scale comparison of innate responses to viral and bacterial pathogens in mouse and macaque

Viral and bacterial infections of the lower respiratory tract are major causes of morbidity and mortality worldwide. Alveolar macrophages line the alveolar spaces and are the first cells of the immune system to respond to invading pathogens. To determine the similarities and differences between the responses of mice and macaques to invading pathogens we profiled alveolar macrophages from these species following infection with two viral (PR8 and Fuj/02 influenza A) and two bacterial (Mycobacterium tuberculosis and Francisella tularensis Schu S4) pathogens. Cells were collected at 6 time points following each infection and expression profiles were compared across and between species. Our analyses identified a core set of genes, activated in both species and across all pathogens that were predominantly part of the interferon response pathway. In addition, we identified similarities across species in the way innate immune cells respond to lethal versus non-lethal pathogens. On the other hand we also found several species and pathogen specific response patterns. These results provide new insights into mechanisms by which the innate immune system responds to, and interacts with, invading pathogens.