- Our results bring to light the first indications that non-canonical cytoplasmic pattern recognition receptors (PRRs) such as NOD-like and RIG-I-like receptor homologs take part in the immune response of Ep. - Over-expression of several members of the lectin- complement pathways in parallel with novel transmembrane and Ig containing ficolins (CniFLs) suggest an active defense against the pathogen. - Conclusions: To our knowledge, this pioneering study is first to follow the kinetics of the innate immune response in a cnidarian during the onset of infection with a bacterial pathogen. - Full list of author information is available at the end of the article. - The current consensus is that inverte- brates lack the components of the well characterized vertebrate adaptive immune system. - Indeed, cnidarians, including corals and anemones, have co-evolved since the Paleozoic era (570–245 Mya) with a multitude of unicellular algae, bacteria and viruses, and possess an in- nate immune system with an extensive list of genes homologous to those of the innate immunity of verte- brates [1–3]. - Therefore, com- parative studies of the innate immune response in corals, Hydra, Nematostella, and Exaiptasia have the potential to help us understand how certain aspects of innate immunity evolved and may also point to alter- native paths to activation of well conserved key factors of immunity.. - Comparisons of the genomes of Acropora corals in symbiosis with algae, the aposymbiotic anemone Nema- tostella vectensis and the freshwater polyp Hydra vul- garis with those of other metazoans show increased complexity in gene families including Toll/Interleukin-1 Receptor- (TIR), NAIP/C2TA/HET-E/TP1- (NACHT), Tumor Necrosis Factor- (TNF) and Death-domains (DD) containing proteins [4–7]. - Although around twenty studies have investigated the innate immune response of cnidar- ian species to bacterial infection, only a few used the model Exaiptasia and none investigated the progress of the innate immune response over the onset of infection.. - [11] used quantitative real-time PCR to show that genes of the complement pathway posi- tively respond to both colonization with Symbiodinaceae and infection with Sm in aiptasia. - Here, RNA-seq sequencing on clones of the symbiotic Ep strain CC7 was used to identify the innate immune response of the host to bacterial infection. - On the other hand, environmental strains nat- urally occur in the mucus of coral species [39, 40], which make Vp a member of the rich bacterial community as- sociated with cnidarians. - Our results improve our under- standing of innate immunity through the measurement of gene activity in response to infection in one of the most basally ancestral tissue-forming animals and high- lights several fascinating gene targets for future func- tional studies.. - Under the microscope, Vp in contact with the host can be seen swarming all over the outward sur- face of the anemones, which shows signs of tissue dam- age and invasion at 12 and 24 h post-inoculation. - After 24 h in the infectious medium some mortality among Ep. - This alignment identi- fied 132,179 contigs (41% of the holobiont transcrip- tome) specific to Ep using BLASTX, which were then exclusively used in the rest of the analyses. - Our refer- ence transcriptome includes contigs corresponding to non-overlapping fragments of the same transcript. - Among these, 2761 belong to 82 different “immune_class” cat- egories of the CateGOrizer tool (Supplementary Figure 2).. - The data was analyzed using two methods: 1) a pairwise compari- son of gene expression levels between treated and con- trol anemones at each time point, and 2) a time series analysis of the changes in gene expression over time for each experimental group.. - The general trends primarily revealed from the pair- wise gene expression analyses between control and in- fected samples at each of the four time points show that:. - regulatory activity in response to the infection over 12 h ranges from ≤1 to 3% of the whole transcriptome.. - The proportion of the aiptasia-specific DECs within the holobiont (i.e. - the host and its symbionts) response ranges from 70 to 83% across up−/down-regulated DECs at the four time points of the experiment (Table 1). - The numbers of up- and down-regulated DECs are similar after 1-h post-infection, but the percentage of up- regulated DECs dramatically increased to 81% of the Ep gene activity after 3 h. - To complement the list of DECs potentially in- volved in the innate immune response of Ep, more per- missive lists were created to include genes associated with the recognition of the pathogen, the transduction of a defensive signal and the degradation of the invader.. - Thus, 2–8% of the aiptasia-specific up-regulated DECs represented the receptor activity, signal transduction and peptidase activity GO classes, while the same GO classes were represented by 2–6% of the down-regulated DECs.. - 2 only shows the results of enrichment analyses performed on up-regulated DECs and GO cat- egories related to different aspects of the immune re- sponse. - The results of the enrichment analyses strongly sup- port an innate immune response activity already detect- able after 1 h and involving up-regulated genes linked to. - As the immune response progresses, the ‘regulation of apoptotic process’ becomes evident after 3 h, and re- mains apparent through the rest of the experiment.. - 0.05) in the transcriptomes of Vp treated samples compared to controls after 1, 3, 6, and 12 h post-infection. - The evolution of the immune response through time can be followed across the four panels for the three general GO categories: BP: biological processes, MF: molecular functions, and CC: cellular components. - The role of the TNF pathway in the immune response of Ep gathers even more support at 3 and 6 h, with the enrichment of. - Expres- sion profiles diverge based on the changes with time but also the overall levels of expression of the DEC clusters.. - A full list of the DECs detected by the time series analysis and organized by cluster is given in Supplementary Table 3.. - effects of the infection on the transcriptome of Exaipta- sia are illustrated in Fig. - [41] in a relevant study that analyzed the transcriptomic response of the amphioxus - Branchiostoma belcheri - to Vp.. - Furthermore, li- gands and receptors of the TNF family suggest the induc- tion of potential cytokine dependent pathways possibly connecting to an apoptosis-like pathway reported below.. - DECs likely involved in the apoptotic process were already detected after 1-h post-infection and further pro- gression of the apoptotic signal was observed at each fol- lowing time point. - A homolog of the key apoptotic executor – CASP3 – is detected as the overall most up-regulated DEC of this analysis, with a fold change of 1250.5 after 6 h post- infection (Robust Exact Test - FDR = 3.06E − 17. - Table 7 suggest that both the extrinsic and intrinsic apoptotic pathways are playing a role in the anemone’s response. - In addition, the apoptotic initiator Apaf-1 is also represented in the expression profiles of clusters 2 and 7 (Fig. - 5), and neither TLR ho- mologs nor orthologs of the Hydra’s HyLRR-2 and HyTRR-1 genes [42] were identified in the Ep’s re- sponses to LPS or Vp (Supplementary Table 8 versus Supplementary Tables 4 and 5, respectively). - This time series RNA-seq experiment was conducted to investigate the molecular innate immune response of the symbiotic Exaiptasia pallida strain CC7 to Vibrio para- haemolyticus strain O3:K6 infection over 12 h. - Analyses focus on three main aspects of the innate immune re- sponse of the host: 1) the genes differentially expressed in infected anemones, 2) the gene expression changes. - Our observations of Ep’s physiological re- sponse to Vp also concur with the descriptions of the same species infected with the coral pathogens Vc and V. - Although darkening of the tissues during those infections was linked to melanin production, our search for evidence of melanin biosynthesis at the tran- script level failed to identify homologs in this dataset.. - Additional genes were identified as potential cytoplas- mic PRRs, including interferon-induced helicase C domain-containing protein 1 (IFIH1) and RIG-I-like re- ceptor 3 (RLR-3), which both contain the RIG domain of the RLRs and play a role in the intracellular recognition of viruses. - Involvement of the lectin-complement pathway. - In the response to Vp infection, we found that anemones up-regulated Ficolin-1 homologs containing TMD and Ig (unlike bilaterian ficolins [45. - at 3, 6, and 12 h (Supplementary Table 4), as well as several components of the lectin-complement pathway. - [11] used quantitative real-time PCR to show up-regulation of both Ep’s Bf-1 and MASP genes in response to 24-h exposure to low and high concen- trations of the pathogen Serratia marcescens, and con- cluded that concentration of microbes, and symbiotic state influence complement gene expression. - The co- expression of TMD/Ig-containing Ficolin-1 and genes of the lectin-complement pathways detected here sup- ports a potential role of CniFLs in the initial recogni- tion of pathogens [10], and highlights both the lectin and alternative pathways active roles in the defense of anthozoans against pathogenic bacteria. - Our results highlight the diversity of lectins potentially involved in the recognition, potential. - Here, neither homologs of canonical vertebrate TLR or IL-1R, nor orthologs of the Hydra’s HyTRR and HyLRR genes [42] were identified in the Ep transcriptomic re- sponses. - Aiptasia’s genome pos- sesses homologs of the major PRR types including NLRs, RLRs, and C-type lectin receptors (CLRs) but lack TLRs.. - Functional conservation of the TLR-to-NF-κB was confirmed in Nematostella [32] and O. - Indeed, the regulation of the ‘TNF-mediated signaling pathway’. - One of these pathways likely starts with the activation of the TNFR1, which is the receptor for the proinflammatory cytokine TNFα and the cytotoxic protein lymphotoxin- alpha in humans. - TNFAIP3 is an essential compo- nent of the ubiquitin-editing protein complex that ensures the transient nature of inflammatory signaling. - In the coral A. - Moreover, the conservation of the TNF-induced apop- totic response was demonstrated in the coral Acropora digitifera [52]. - Both pathways could en- gage the apoptotic executor - CASP3, which is up- regulated for the duration of the infection with a peak of 1250 in fold change at 6 h. - These results strongly suggest that apoptosis plays an important role in the innate im- mune response of Ep against bacterial pathogens. - As Ep is capable of recovering from a punctual exposure to Vp, it is probable that apoptosis plays a part in the removal of invading bacteria and the survival of the host. - Comparison between responses to Vp infection and LPS The genes of the TLR-to-NF-κB like pathway identified in the Ep’s response to Vp infection were not activated by LPS. - Hydra polyps exposed to LPS treatments acti- vate the expression of the antimicrobial peptides in a dose-dependent manner, suggesting that LPS is an in- ducer of immune defenses in cnidarians [35]. - Indeed, studies on corals have shown that LPS can trigger an im- mune response in several species and more specifically genes of the TLR-to-NF-κB pathway [34, 50].. - Overall, these results suggest that in cnidarians lacking TLR homologs, LPS can still trigger the produc- tion of cytokines and antimicrobial peptides independ- ently of the TLR-to-NF-κB pathway.. - Several of the genes similar to the potential novel acti- narian immune genes (NG1, NG2, and NG3) proposed by van der Burg et al. - have looked at the physiological and gene expression re- sponse of the coral Pocillopora damicornis (Pd) under temperature-dependent infection with Vc. - However, these genes show the opposite regulation under the viru- lent Vc treatment, leading the authors to conclude the exact opposite to what was found in our study, “the greater the stress, the greater the down-regulation”.. - Here, HSP70 and HSP90 DECs were up- regulated at 3, 6 and 12 h in the response to Vp (Supple- mentary Table 4). - To our knowledge, this is the first time series study of the transcriptomic response to bacterial infection in a cnidarian model. - Individual clones of the sea anemone Ep strain CC7 were acquired from the Pringle laboratory at Stanford University and kept at the Scientific Center of Monaco under controlled conditions in the Ecosystems and Immunity Laboratory of the Biomedical Department.. - FSW at 0.45 μm is renewed every week and anemones are fed twice a week with artemias, except on the week of the experiment during which they fast.. - After Vp or LPS added to treatment wells, anemones were placed in the Percival chamber at 27 °C under gen- tle orbital agitation of 500 rpm for the time of the ex- periment. - Photos were taken at each time point under bright light and the 0.8X magnification to monitor the overall appearance of the animals. - Further observations were recorded at 5X magnification to assess the progression of the infection and the integrity of the animal tissue. - New reference transcriptomes were assembled de novo using all of the Exaiptasia pallida libraries produced here and either guided by the Ep genome produced by the Pringle La- boratory (version 1.1) or genome-free using Trinity v2.6.5 release [65]. - Transmembrane domains were identified via the TMHMM (v.2.0) of the DTU Bioinformatics server.. - Transcript-level quantification was estimated for gene and isoform expression by allocat- ing multi-mapping reads among all transcripts of the reference transcriptome using the RSEM [66] and Bowtie-2 [67] packages.. - 0.05 for variable selection in the stepwise regression. - A cutoff value for the R-square of the regression model was set to 0.9. - Rodolphe Pontier-Bres for their help in the laboratory, and the Professors P. - designed and ran the experiment, performed the bulk of the computational analyses, interpreted the results and wrote the manuscript.. - supervised all aspects of the project. - Funding was entirely provided by the government of Monaco, which played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - The complex NOD-like receptor repertoire of the coral Acropora digitifera includes novel domain combinations. - Defining the origins of the NOD-like receptor system at the base of animal evolution. - Morphological revision of the genus Aiptasia and the family Aiptasiidae (Cnidaria, Actiniaria, Metridioidea). - The role of complement in cnidarian- Dinoflagellate Symbiosis and immune challenge in the sea Anemone Aiptasia pallida. - Regulation of cnidarian – dinoflagellate mutualisms: evidence that activation of a host TGF β innate immune pathway promotes tolerance of the symbiont. - Allorecognition maturation in the broadcast-spawning coral Acropora millepora. - A conserved toll-like receptor-to-NF- κ B signaling pathway in the endangered coral Orbicella faveolata. - Comparative RNA-Seq based dissection of the regulatory networks and environmental stimuli underlying Vibrio parahaemolyticus gene expression during infection.. - The role of seafood in foodborne diseases in the United States of America. - Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida. - Phylogenetic characterization of culturable bacterial diversity associated with the mucus and tissue of the coral Acropora digitifera from the Gulf of Mannar: Culturable bacterial diversity of A. - Precambrian origins of the TNFR superfamily.. - Physiological responses of the scleractinian coral Pocillopora damicornis to bacterial stress from Vibrio coralliilyticus
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