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. 2016 Nov 3;6:36048.
doi: 10.1038/srep36048.

Fish Gut-Liver Immunity During Homeostasis or Inflammation Revealed by Integrative Transcriptome and Proteome Studies

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Free PMC article

Fish Gut-Liver Immunity During Homeostasis or Inflammation Revealed by Integrative Transcriptome and Proteome Studies

Nan Wu et al. Sci Rep. .
Free PMC article

Abstract

The gut-associated lymphoid tissue, connected with liver via bile and blood, constructs a local immune environment of both defense and tolerance. The gut-liver immunity has been well-studied in mammals, yet in fish remains largely unknown, even though enteritis as well as liver and gallbladder syndrome emerged as a limitation in aquaculture. In this study, we performed integrative bioinformatic analysis for both transcriptomic (gut and liver) and proteomic (intestinal mucus and bile) data, in both healthy and infected tilapias. We found more categories of immune transcripts in gut than liver, as well as more adaptive immune in gut meanwhile more innate in liver. Interestingly reduced differential immune transcripts between gut and liver upon inflammation were also revealed. In addition, more immune proteins in bile than intestinal mucus were identified. And bile probably providing immune effectors to intestinal mucus upon inflammation was deduced. Specifically, many key immune transcripts in gut or liver as well as key immune proteins in mucus or bile were demonstrated. Accordingly, we proposed a hypothesized profile of fish gut-liver immunity, during either homeostasis or inflammation. Current data suggested that fish gut and liver may collaborate immunologically while keep homeostasis using own strategies, including potential unique mechanisms.

Figures

Figure 1
Figure 1. Strategy for identification of regulated immune genes in fish gut and liver by integrative analysis of both DGE transcriptomic and iTRAQ-based quantitative proteomic data.
(A) Tilapias were infected intraperitoneally with live S. agalactiae, and then sampling for DGE profiling of gut or liver was done at 0 h (just before challenge), together with 12 h and 36 h post challenge, meanwhile sampling for iTRAQ analysis of intestinal mucus or bile was done at 0 h and 36 h, from both healthy and infected fish; (B) Immune related annotation was found out via bioinformation analysis of both DGE and iTRAQ results using public database (including GO, KEGG, and InterPro); (C) On one hand, the comparison was done in both inter-tissue/fluid data (gut vs liver, or intestinal mucus vs bile) and intra-tissue/fluid data (gut, liver, mucus or bile), then Venn-regional analysis was applied to screened out immune homeostasis-related transcripts and proteins; on the other hand, regulated gut-liver immune transcripts or proteins were revealed by intra-tissue (same tissue at different time-points) or intra-fluid (same fluid at different time-points) comparison.
Figure 2
Figure 2. Classification of fish immune transcripts of gut or liver advantage by two levels of the tilapia immune gene library.
(A) Major immune processes (at the first level) involved in gut and liver; (B) Immune gene categories (at the second level) involved in gut; (C) Immune gene categories (at the second level) involved in liver. The gut advantage transcripts were significantly involved in “pattern recognition” (gut vs liver, 156:45 at 0 h, 128:42 at 12 h, and 148:46 at 36 h), “antigen processing and regulators” (77:15 at 0 h, 69:18 at 12 h, and 76:15 at 36 h), “inflammatory cytokines and receptors” (107: 46 at 0 h, 121: 41 at 12 h, and 121: 53 at 36 h), “adapters, effectors and signal transducers” (49:20 at 0 h, 29:20 at 12 h, and 42:21 at 36 h), “innate immune cells related” (15:4 at 0 h, 11:5 at 12 h, and 13:5 at 36 h), “T/B cell antigen activation” (126:20 at 0 h, 113:24 at 12 h, and 130:23 at 36 h), and “other genes related to immune cell response” (114:63 at 0 h, 122:60 at 12 h, and 115:65 at 36 h), while compared to gut, liver advantage transcripts were significantly involved in “acute phase reactions” (liver vs gut, 26:4 at 0 h, 25:3 at 12 h, and 25:3 at 36 h) and “complement system” (30:10 at 0 h, 29:12 at 12 h, and 32:9 at 36 h).
Figure 3
Figure 3. Classification of fish immune proteins of intestinal mucus or bile advantage by two levels of the tilapia immune gene library.
(A) Major immune processes (at the first level) involved in intestinal mucus and bile; (B) Immune gene categories (at the second level) in intestinal mucus; (C) Immune gene categories (at the second level) in bile. Mucus specific advantage proteins were mainly involved in “inflammatory cytokines and receptors” (mucus vs bile, 4:2 at 0 h, and 2:2 at 36 h) and “other genes related to immune cell response” (15:10 at 0 h, and 7:2 at 36 h). While, the bile specific ones were mainly involved in “acute phase reactions” (bile vs mucus, 23:0 at 0 h, and 15:0 at 36 h), “complement system” (22:0 at 0 h, and 4:0 at 36 h), “innate immune cells related” (1:0 at 0 h, and 1:0 at 36 h). The common involved immune processes were mainly in “pattern recognition” (bile vs mucus, 16:9 at 0 h, and 11:7 at 36 h) and T/B cell antigen activation (5:1 at 0 h, and 0:1 at 36 h).
Figure 4
Figure 4. Venn-regional analysis of gut or liver advantage transcripts.
(A) Venn analysis among data of 0 h, 12 h, and 36 h, for both the total and immune differential transcripts between gut and liver, and the percentage of immune transcripts vs total transcripts was labeled in each region. The total No. of differentially expressed genes between gut and liver was 5788, 4702 and 5952 at 0 h, 12 h and 36 h respectively, and after filtered by the immune gene library the numbers decreased to 927, 872, and 942. And the dashed red lined regions, including region a, b, e and d, indicated data at 0 h, in another word in homeostasis status, whereas the dashed green lined regions (f, c and g) specifically indicated data upon inflammational states (12 h and 36 h). (B) Transcripts No. involved in major immune processes for each region. For gut advantage transcripts, in regions a, b and c, most of them were involved in “pattern recognition”, “T/B cell activation”, “other genes related to immune response”, “inflammatory cytokines and receptors”, as well as “antigen processing and regulators”; in regions d and e, except for the above mentioned, most of them were also involved in “adapters, effectors and signal transducers”; in regions f and g, most of them were involved in “pattern recognition”, “inflammatory cytokines and receptors”, “T/B cell antigen activation”, as well as “other genes related to immune response”. On the other hand, for liver advantage ones, in region a, most were involved in “other genes related to immune response”, “complement system”, “pattern recognition”, as well as “acute phase reactions”. In regions d and g, most were involved in “other genes related to immune response”, “inflammatory cytokines and receptors”, as well as “pattern recognition”, while in region f, also in “antigen processing and regulators”, as well as “T/B cell activation”, except for the above mentioned. In other regions (b, c and e), the liver advantage genes were much fewer.
Figure 5
Figure 5. Venn-regional analysis of intestinal mucus or bile advantage proteins.
(A) Venn analysis between data of steady (0 h) and inflammational (36 h) for both the total and immune differential proteins between intestinal mucus and bile, and the percentage of immune proteins vs total proteins was labeled in each region. The total No. of differentially expressed genes between intestinal mucus and bile was 1057 and 789 at 0 h and 36 h respectively, and after filtered by immune gene library the numbers decreased to 117 and 60. The dashed red lined regions, including region a and b, indicated data at 0 h, in another word in homeostasis status, whereas the dashed green lined region (c) specifically indicated data upon inflammation (36 h). (B) Protein No. involved in major immune processes for each region. In region a, the most enriched mucus advantage proteins were involved in “pattern recognition”, “adapters, effectors and signal transducers”, as well as “other proteins related to immune response”; meanwhile, for bile advantage ones, in “acute phase reactions”, “pattern recognition”, and “complement system”. In region b, the most enriched mucus advantage ones were involved in “other proteins related to immune response” and “inflammatory cytokines and receptors”; meanwhile, for bile advantage ones, in “complement system”, “other proteins related to immune response”, “acute phase reactions”, “pattern recognition”, and “T/B cell antigen activation”. In region c, there were much fewer immune proteins, with the most enriched mucus advantage ones in “adapters, effectors and signal transducers” as well as in “pattern recognition” for bile.
Figure 6
Figure 6. Analysis of common genes between liver advantage transcripts and bile advantage proteins.
(A) Venn analysis between genes of both liver advantage transcripts and bile advantage proteins, with percentages of common components. (B) The KEGG pathway graphic of complement cascades in bile advantage proteins at steady state (0 h). Three complement activating pathways as well as inhibitory factors were revealed.
Figure 7
Figure 7. Hypothesized fish gut-liver immune mechanisms involved in either homeostasis or inflammation.
The hypothesized portrait of fish gut-liver immunity was drawn accordingly to the discussion of current revealed immune genes in tilapias’ gut and liver. Besides immune components at both intestinal mucosal barrier and liver reticulo-endothelial system, intestinal mucus and bile also contain many immune modulators or activators. During immune homeostasis, on one hand, in gut, genes of possible immunomodulatory role, including innate immune molecules, such as galectin and c-type lectin, as well as regulatory T/B cell related ones, together with genes responsible immune-suppression, such as IL1R, were found with abundance. On the other hand, in liver, in addition to immunomodulatory and immune suppression genes, innate immune molecules, such as acute phase proteins, complement components and anti-microbial peptides, which could be delivered from bile to intestinal mucus, were found of great importance for basic function. In addition, molecules (chemokines and integrins, with some members different from mammals) related to migration of lymphocytes between gut and liver were also inferred particular at steady state (termed homing). While upon inflammation, in gut, the immune genes, responsible for immune activation, including both innate ones, such as fish-egg lection (fish-specific), CFD and C1q, and adaptive ones, mainly T cell response (CD8+T, Th1 and Th17) related, were prevailing. At the same time in liver, genes for activation of immune responses, mainly including innate immune molecules and cells, together with relative lower T cell response, were found. Innate immune factors could be also transported from bile to intestinal mucus upon inflammation. Genes labeled or related to immune cells in the diagram were all highlighted in Tables S6 and S7.

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