Toll-like receptors (TLRs) are known to detect a defined spectrum of microbial structures. However, the knowledge about the specificity of teleost Tlr factors for distinct pathogens is limited so far. We measured baseline expression profiles of 18 tlr genes and associated signaling factors in four immune-relevant tissues of rainbow trout Oncorhynchus mykiss. Intraperitoneal injection of a lethal dose of Aeromonas salmonicida subsp. salmonicida induced highly increased levels of cytokine mRNAs during a 72-hour postinfection (hpi) period. In contrast, only the fish-specific tlr22a2 and the downstream factor irak1 featured clearly increased transcript levels, while the mRNA concentrations of many other tlr genes decreased. Flow cytometry quantified cell trafficking after infection indicating a dramatic influx of myeloid cells into the peritoneum and a belated low level immigration of lymphoid cells. T and B lymphocytes were differentiated with RT-qPCR revealing that B lymphocytes emigrated from and T lymphocytes immigrated into head kidney. In conclusion, no specific TLR can be singled out as a dominant receptor for A. salmonicida. The recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is hence of key importance for mounting a first immune defense against invading A. salmonicida.

Pigs are anatomically, genetically and physiologically comparable to humans and represent a natural host for influenza A virus (IAV) infections. Thus, pigs may represent a relevant biomedical model for human IAV infections. We set out to investigate the systemic as well as the local immune response in pigs upon two subsequent intranasal infections with IAV H1N1pdm09. We detected decreasing numbers of peripheral blood lymphocytes after the first infection. The simultaneous increase in the frequencies of proliferating cells correlated with an increase in infiltrating leukocytes in the lung. Enhanced perforin expression in αβ and γδ T cells in the respiratory tract indicated a cytotoxic T cell response restricted to the route of virus entry such as the nose, the lung and the bronchoalveolar lavage. Simultaneously, increasing frequencies of CD8αα expressing αβ T cells were observed rapidly after the first infection, which may have inhibited uncontrolled inflammation in the respiratory tract. Taking together, the results of this study demonstrate that experimental IAV infection in pigs mimics major characteristics of human seasonal IAV infections.

In mammals, CCR7 is the chemokine receptor for the CCL19 and CCL21 chemokines, molecules with a major role in the recruitment of lymphocytes to lymph nodes and Peyer's patches in the intestinal mucosa, especially naïve T lymphocytes. In the current work, we have identified a CCR7 orthologue in rainbow trout (Oncorhynchus mykiss) that shares many of the conserved features of mammalian CCR7. The receptor is constitutively transcribed in the gills, hindgut, spleen, thymus and gonad. When leukocyte populations were isolated, IgM(+) cells, T cells and myeloid cells from head kidney transcribed the CCR7 gene. In blood, both IgM(+) and IgT(+) B cells and myeloid cells but not T lymphocytes were transcribing CCR7, whereas in the spleen, CCR7 mRNA expression was strongly detected in T lymphocytes. In response to infection with viral hemorrhagic septicemia virus (VHSV), CCR7 transcription was down-regulated in spleen and head kidney upon intraperitoneal infection, whereas upon bath infection, CCR7 was up-regulated in gills but remained undetected in the fin bases, the main site of virus entry. Concerning its regulation in the intestinal mucosa, the ex vivo stimulation of hindgut segments with Poly I:C or inactivated bacteria significantly increased CCR7 transcription, while in the context of an infection with Ceratomyxa shasta, the levels of transcription of CCR7 in both IgM(+) and IgT(+) cells from the gut were dramatically increased. All these data suggest that CCR7 plays an important role in lymphocyte trafficking during rainbow trout infections, in which CCR7 appears to be implicated in the recruitment of B lymphocytes into the gut.

Whirling disease (WD), caused by the myxozoan parasite Myxobolus cerebralis, is responsible for high mortalities in rainbow trout hatcheries and natural populations. To elucidate how resistant and susceptible rainbow trout strains respond to early invasion, a well-established model of WD was used to demonstrate the kinetics of local and systemic immune responses in two rainbow trout strains, the susceptible American Trout Lodge (TL) and the more resistant German Hofer strain (HO).

Non-retroviral integrated RNA viral sequences (NIRVs) potentially encoding ∼280 amino acid homologs to filovirus VP35 proteins are present across the Myotis genus of bats. These are estimated to have been maintained for ∼18 million years, indicating their co-option. To address the reasons for co-option, 16 Myotis VP35s were characterized in comparison to VP35s from the extant filoviruses Ebola virus and Marburg virus, in which VP35s play critical roles in immune evasion and RNA synthesis. The Myotis VP35s demonstrated a conserved suppression of innate immune signaling, albeit with reduced potency, in either human or Myotis cells. Their attenuation reflects a lack of dsRNA binding that in the filoviral VP35s correlates with potent suppression of interferon responses. Despite divergent function, evolution has preserved in Myotis the structure of the filoviral VP35s, indicating that this structure is critical for co-opted function, possibly as a regulator of innate immune signaling.

Adverse life circumstances evoke a common "conserved transcriptional response to adversity" (CTRA) in mammalian leukocytes. To investigate whether this pattern is preserved in lower vertebrates, maraena whitefish (Coregonus maraena) were exposed for 9 days to different stocking densities: ~10 kg/m3 (low density), ~33 kg/m3 (moderate), ~60 kg/m3 (elevated), and ~100 kg/m3 (high). Transcriptome profiling in the liver and kidney of individuals from each group suggested that crowding conditions activate stress-related signaling and effector pathways. Remarkably, about one-quarter of the genes differentially expressed under crowding conditions were involved in the activation of immune pathways such as acute-phase response and interleukin/TNF signaling attended by the simultaneous reduction of antiviral potency. Network analysis confirmed the complex interdigitation of immune- and stress-relevant pathways with interleukin-1 playing a central role. Antibody-based techniques revealed remarkable changes in the blood composition of whitefish and demonstrated the correlation between increasing stocking densities and elevated number of myeloid cells together with the increased phagocytic activity of peripheral blood leukocytes. In line with current studies in mammals, we conclude that crowding stress triggers in whitefish hallmarks of a CTRA, indicating that the stress-induced molecular mechanisms regulating the immune responses not only are conserved within mammals but were established earlier in evolution.

Expansions of gene families are predictive for ongoing genetic adaptation to environmental cues. We describe such an expansion of the carcinoembryonic antigen (CEA) gene family in certain bat families. Members of the CEA family in humans and mice are exploited as cellular receptors by a number of pathogens, possibly due to their function in immunity and reproduction. The CEA family is composed of CEA-related cell adhesion molecules (CEACAMs) and secreted pregnancy-specific glycoproteins (PSGs). PSGs are almost exclusively expressed by trophoblast cells at the maternal-fetal interface. The reason why PSGs exist only in a minority of mammals is still unknown.

Structure and function of factors contributing to the innate immune system of lower vertebrates, including fish are only sparsely characterized. We retrieved with RT-PCR cDNA copies of two closely related Toll-like receptors (TLR) from liver RNA of the rainbow trout (Oncorhynchus mykiss). The cDNA sequences are homologous to 95.6%. The phylogenetic analysis of their deduced amino acid sequences places these twin factors closely to other known TLRs from fish. The twin factors are equally expressed in all tissues analysed, most abundantly in spleen and head kidney and lowest in adipose tissue. Formalin-inactivated Aeromonas salmonicida pathogens induce their expression up to eight-fold in vitro in peripheral blood lymphocytes and in tissues from spleen and head kidney. Our sequence information will be useful to establish expression constructs for these factors necessary to analyse the pathogen specific signal transduction activating the innate immune defence in fish.

Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.

Four 'protein inhibitors of activated STAT' (PIAS) control STAT-dependent and NF-κB-dependent immune signalling in humans. The genome of rainbow trout (Oncorhynchus mykiss) contains eight pias genes, which encode at least 14 different pias transcripts that are differentially expressed in a tissue- and cell-specific manner. Pias1a2 was the most strongly expressed variant among the analysed pias genes in most tissues, while pias4a2 was commonly low or absent. Since the knock-out of Pias factors in salmonid CHSE cells using CRISPR/Cas9 technology failed, three structurally different Pias protein variants were selected for overexpression studies in CHSE-214 cells. All three factors quenched the basal activity of an NF-κB promoter in a dose-dependent fashion, while the activity of an Mx promoter remained unaffected. Nevertheless, all three overexpressed Pias variants from trout strongly reduced the transcript level of the antiviral Stat-dependent mx gene in ifnγ-expressing CHSE-214 cells. Unlike mx, the overexpressed Pias factors modulated the transcript levels of NF-κB-dependent immune genes (mainly il6, il10, ifna3, and stat4) in ifnγ-expressing CHSE-214 cells in different ways. This dissimilar modulation of expression may result from the physical cooperation of the Pias proteins from trout with differential sets of interacting factors bound to distinct nuclear structures, as reflected by the differential nuclear localisation of trout Pias factors. In conclusion, this study provides evidence for the multiplication of pias genes and their sub-functionalisation during salmonid evolution.