Interaction of immunostimulants and stress on innate defence mechanisms of rainbow trout, Oncorhynchus mykiss

This study investigated the use of non-specific immunostimulants to alleviate stress-mediated suppression of defence mechanisms and subsequent susceptibility to bacterial pathogens in rainbow trout (Oncorhynchus mykiss). One yeast (1-3),)1-6)-β-glucan and a bacterial peptidoglycan were selected as immunostimulants from a panel of test substances on the basis of enhanced intracellular superoxide generation by kidney macrophages stimulated in vitro. Kidney macrophage effector activity was not affected after 1, 2, 3 or 4 weeks of in-feed treatment with 0.05% or 5% of glucan or peptidoglycan. However, production of bactericidal superoxide by inflammatory peritoneal macrophages did increase significantly after four weeks of oral treatment with 0.05% peptidoglycan. Although a single confinement of fish (93% reduction of water volume for five minutes) caused a physiological stress response, as indicated by hyperglycaemia in plasma, kidney and inflammatory macrophage activities were only affected after six daily confinements. Phagocytosis, intracellular superoxide production and killing of Aeromonas salmonicida in vitro by kidney macrophages were significantly reduced. Conversely, production of extracellular superoxide, which may be associated with damage to self, was enhanced. Peritoneal macrophages displayed a similar but less marked respiratory burst response after repeated confinement. Some of the alterations in macrophage function caused by daily confinement were prevented by feeding 0.05% peptidoglycan four weeks before the first confinement. The increase in kidney macrophage extracellular superoxide production caused by repeated confinement was significantly alleviated by in-feed peptidoglycan. Similarly, the decrease in intracellular production by peritoneal macrophages caused by repeated confinement was prevented by in-feed treatment with peptidoglycan. Neither peptidoglycan nor repetitive confinement had any effect on complement lytic activity. These results indicate that dietary peptidoglycan was able to reduce, by regulating macrophage function, the impact of stress on certain bactericidal defences and potential damage to self. However, there was no significant difference in the persistence of viable A. salmonicida in the spleen or blood of infected fish in any of the experimental treatments

A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells

Dendritic cells (DC) are known to support the activation of natural killer (NK) cells. However, little is known about the role for DC in NK-cell homeostasis. In order to investigate this question, a novel bacterial artificial chromosome transgenic mouse model was generated in which the diphtheria toxin receptor is expressed under the CD11c promoter. In these mice efficient DC depletion can be achieved over prolonged periods of time by multiple injections of diphtheria toxin. We show here that NK cells require DC for full acquisition of effector function in vivo in response to the bacterial-derived TLR ligand CpG. Importantly, DC were found to play an instrumental role for maintaining normal homeostasis of NK cells. This is achieved by IL-15 production by DC, which supports the homeostatic proliferation of NK cells. . There is much known about the molecular mechanisms of NK-cell functions, but the factors influencing NK-cell numbers are only beginning to be elucidated. Mature NK cells were typically thought to be a terminally differentiated population, with a very limited selfrenewal capacity. However, it was recently shown that a small percentage of NK cells actively proliferate in the steady state, resulting in a half life of the NK-cell population of about 17 days SHORT COMMUNICATION Ã These authors contributed equally to this study. Correspondence: Dr. Natalio Garbi e-mail: n. 2776 Importantly, we observe a previously unrecognized role for DC in optimal homeostatic proliferation of NK cells in lymphopenic conditions. In this process, DC-derived IL-15 appears to play an important role. Our data indicate that not only T lymphocytes, but also NK cells require DC for homeostatic proliferation. Results CD11c.DOG mice allow long-term ablation of DC in vivo without toxicity effects In order to study the effect of in vivo interaction between DC and NK cells, a system that permits long-term depletion of DC is required. The diphtheria toxin/diphtheria toxin receptor (DT/DTR) system introduced by Saito et al. Injection of 8 ng/gram body weight (gbw) DT induced depletion of DC (CD11c high MHC class II 1 ) in spleen, lymph nodes, thymus and bone marrow, albeit efficiency of depletion varied in the different organs tested The data shown in Although mice tolerated well 8 ng/gbw for prolonged periods of time, DC depletion was limited to a period of 11-12 days In order to confirm faithful expression of the BAC, we thoroughly examined CD11c expression and depletion of B cells, T cells, NK cells and NKT cells in spleens after 8 ng/gbw DT administration. The minor CD11c hi subpopulations of these cell types were effectively depleted, whereas CD11c int/lo cells were only partially depleted (Supporting Information In conclusion, the new BAC transgenic CD11c.DOG mouse model introduced here allows effective long-term depletion of CD11c 1 cells. DC are required for optimal NK-cell activation and homeostatic proliferation in vivo of NK cells. NK cells were activated by administration of the TLR-9 ligand CpG and the in vivo lytic activity determined by injection of CFSE-labelled tapasin-deficient splenocytes that served as NK targets. CpG administration in mice with a normal DC compartment lead to increased NK cytotoxicity in vivo ( The homeostasis of T cells has been well studied and found to require several factors including the presence of DC, self MHC molecules and the cytokine IL-7 for proliferation and survival ) in DC-depleted CD11c.DOG mice compared with DC-sufficient B6 mice ( DC-derived IL-15 contributes to lymphopenia-induced proliferation of NK cells The cytokine IL-15 has been reported to be crucial for activation and survival of NK cells Concluding remarks In summary, this study introduces a new BAC transgenic CD11c.DTR mouse model for prolonged in vivo depletion of DC by application of DT, without any apparent signs of toxicity. Experiments using this mouse line demonstrated a previously unrecognized role for DC in the homeostasis of NK cells, and showed that local production of IL-15 by DC is required for the maintenance of the NK-cell compartment. Thus, the present study adds an additional function to the long list of DC functions, which so far encompass antigen presentation to and activation of CD4 and CD8 T cells, deletion of T cells and induction of regulatory 1 ) NK cells were quantified 7 days after transfer into irradiated chimeras. Three mice were used in each group. The experiment was repeated three times with similar results. Ã po0.05, Student's t-test

Innate type 2 immunity in helminth infection is induced redundantly and acts autonomously following CD11c+ cell depletion

Infection with gastrointestinal helminths generates a dominant type 2 response among both adaptive (Th2) and innate (macrophage, eosinophil, and innate lymphoid) immune cell types. Two additional innate cell types, CD11chigh dendritic cells (DCs) and basophils, have been implicated in the genesis of type 2 immunity. Investigating the type 2 response to intestinal nematode parasites, including Heligmosomoides polygyrus and Nippostrongylus brasiliensis, we first confirmed the requirement for DCs in stimulating Th2 adaptive immunity against these helminths through depletion of CD11chigh cells by administration of diphtheria toxin to CD11c.DOG mice. In contrast, responsiveness was intact in mice depleted of basophils by antibody treatment. Th2 responses can be induced by adoptive transfer of DCs, but not basophils, exposed to soluble excretory-secretory products from these helminths. However, innate type 2 responses arose equally strongly in the presence or absence of CD11chigh cells or basophils; thus, in CD11c.DOG mice, the alternative activation of macrophages, as measured by expression of arginase-1, RELM-α, and Ym-1 (Chi3L3) in the intestine following H. polygyrus infection or in the lung following N. brasiliensis infection, was unaltered by depletion of CD11c-expressing DCs and alveolar macrophages or by antibody-mediated basophil depletion. Similarly, goblet cell-associated RELM-β in lung and intestinal tissues, lung eosinophilia, and expansion of innate lymphoid (“nuocyte”) populations all proceeded irrespective of depletion of CD11chigh cells or basophils. Thus, while CD11chigh DCs initiate helminth-specific adaptive immunity, innate type 2 cells are able to mount an autonomous response to the challenge of parasite infection

Charcot-Leyden Crystals activate the NLRP3 inflammasome and cause IL-1β inflammation [preprint]

Charcot-Leyden crystals (CLCs) are Galectin-10 protein crystals that can form after eosinophils degranule. CLCs can appear and persist in tissues from patients with eosinophilic disorders, such as asthma, allergic reactions, fungal, and helminthic infections. Despite abundant reports of their occurrence in human disease, the inflammatory potential of CLCs has remained unknown. Here we show that CLCs induce IL-1β release upon their uptake by primary human macrophages in vitro, and that they induce inflammation in vivo in mouse models of acute peritonitis and bronchitis. CLC-induced IL-1β was dependent on NLRP3 and caspase-1, and their instillation in inflammasome reporter mice promoted the assembly of ASC complexes and IL-1β secretion in the lungs. Our findings reveal that CLCs are recognized by the NLRP3 inflammasome, which may sustain inflammation that follows eosinophilic inflammatory processes

Ácidos grasos como marcadores de las relaciones tróficas entre el sestón, el zooplancton crustáceo y el sifonóforo Nanomia cara en Georges Basin y el cañón Oceanographer (NO Atlántico)

[EN] Fatty acid concentrations expressed as percentages of total fatty acid pools in seston, stage V copepodites of Calanus finmarchicus, adults of the euphausiid Meganyctiphanes norvegica, and the physonect siphonophore Nanomia cara were used to elucidate trophic links in Georges Basin and Oceanographer Canyon in September 2003. Seston at both locations was refractory and comprised mainly of saturated fatty acids. Phytoplankton did not contribute significantly to the fatty acid composition of seston or higher trophic levels. Only four fatty acids, i.e. 14:0, 16:0, 16:1 (n–7) and 18:1 (n–7), were transferred from seston to C. finmarchicus or M. norvegica, which suggested weak trophic interactions. Fatty acids transferred from the two species of crustaceans to N. cara included the same four fatty acids, along with three polyunsaturated fatty acids found in relatively high concentrations in both crustaceans, i.e. 20:3 (n–6), 20:5 (n–3) and 22:6 (n–3). In addition, 18:1 (n–9), which occurred in relatively high concentrations only in M. norvegica, and 18:0 and 18:2 (n–6), which were found in low concentrations in both crustaceans, also appeared to be transferred to N. cara. Overall, fatty acid trophic markers proved useful for identifying trophic links to N. cara[ES] En este estudio se utilizaron las concentraciones de ácidos grasos (expresadas como porcentajes) para identificar posibles relaciones tróficas entre el seston, el estadio V (copepoditos) de Calanus finmarchicus, los adultos del eufáusido Meganyctiphanes norvegica, y el sifonóforo fisonecto Nanomia cara en Georges Basin y el cañón submarino Oceanographer durante Septiembre de 2003. En ambos lugares el seston era muy refractario y compuesto básicamente por ácidos grasos saturados. El fitoplancton no contribuyó de forma significativa a la composición de ácidos grasos del seston o de niveles tróficos superiores. Sólo cuatro ácidos grasos [14:0, 16:0, 16:1 (n–7) y 18:1 (n–7)] se transfirieron potencialmente del seston a C. finmarchicus o M. norvegica, lo que sugiere una débil conexión trófica entre estos eslabones de la cadena. Los ácidos grasos transferidos de las dos especies de zooplancton crustáceo a N. cara incluyen los mismos descritos más arriba y otros tres ácidos grasos poliinsaturados [20:3 (n–6), 20:5 (n–3) y 22:6 (n–3)] encontrados en concentraciones relativamente elevadas en ambos crustáceos. Además, tanto el 18:1 (n–9) (encontrado en elevadas concentraciones en M. norvegica) y los 18:0 y 18:2 (n–6) (encontrados en bajas concentraciones en ambas especies de crustáceos) se transfieren a N. cara. Los ácidos grasos demuestran ser una herramienta útil para identificar conexiones tróficas en N. caraA grant to MJY from the National Science Foundation (NSF-0002493), the European Project EUROGEL, and USDA CRIS Project FLA-FAS-03978 supported this workPeer reviewe

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo

Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells

Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro–differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste