Fish-specific TLR18 in Nile tilapia (Oreochromis niloticus) recruits MyD88 and TRIF to induce expression of effectors in NF-κB and IFN pathways in melanomacrophages.

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18, to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α, interferon (IFN) d2.13, tilapia piscidin (TP)2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages.

Growth Performance and Disease Resistance against Vibrio parahaemolyticus of Whiteleg Shrimp (Litopenaeus vannamei) Fed Essential Oil Blend (Phyto AquaBiotic).

Acute Hepatopancreatic Necrosis Disease (AHPND) is a serious and emerging disease caused by a group of strains of Vibrio parahaemolyticus and affects farmed shrimp, particularly whiteleg shrimps (Liptopenaeus vannamei). The objective of this study is to assess the effect of dietary supplementation with two dosages of an essential oil mixture (Phyto AquaBiotic, abbreviated as PAB) on growth performance and mortality reduction after challenge against V. parahaemolyticus. PAB was mixed with basal diets at rates of 0, 1 and 2 g/kg and fed for 42 days. Each tank was stocked with 100 individuals with experimentation performed in triplicate. The results showed an improvement in growth performance in a dose-dependent manner, specifically regarding daily weight gain, specific growth rate and total biomass, which were significantly improved compared to control (p < 0.05). Further, PAB significantly reduced mortalities when challenged against Vibrio parahaemolyticus (p < 0.05) and decreased Vibrio spp. count in the hepatopancreas of infected shrimp. Overall, PAB was efficient in reducing mortalities in cases of disease outbreaks at a rate of 2 g/kg.

Sterile alpha and TIR motif-containing protein 1 is a negative regulator in the anti-bacterial immune responses in nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is one of the most important food fish in the world. However, the farming industry has encountered significant challenges, such as pathogen infections. Toll-like receptors (TLRs) play an essential role in the initiation of the innate immune system against pathogens. Sterile alpha and TIR motif-containing protein 1 (SARM1) is one of the most evolutionarily conserved TLR adaptors, and its orthologs are present in various species from worms to humans. SARM1 plays an important role in negatively regulating TIR domain-containing adaptor proteins inducing IFNß (TRIF)-dependent TLR signaling in mammals, but its immune function remains poorly understood in fish. In this study, O. niloticus SARM1 (OnSARM1) was cloned and its evolutionary status was verified using bioinformatic analyses. mRNA expression of OnSARM1 was found at a higher level in the trunk kidney and muscle in healthy fish. The examination of its subcellular location showed that the OnSARM1 was detected only in the cytoplasm of THK cells, and colocalized with OnMyD88, OnTRIF and OnTRIF in small speckle-like condensed granules. The transcript levels of OnMyD88, OnTIRAP, OnTRIF, and downstream effectors, including interleukin (IL)-1ß, IL-8, IL-12b and type I interferon (IFN)d2.13, were regulated conversely to the expression of OnSARM1 in the head kidney from Aeromonas hydrophila and Streptococcus agalactiae infected fish. Moreover, the treatment of THK cells with lysates from A. hydrophila and S. agalactiae enhanced the activity of the NF-κB promoter, but the effects were inhibited in the OnSARM1 overexpressed THK cells. Overexpression of OnSARM1 alone did not activate the NF-κB-luciferase reporter, but it suppressed OnMyD88- and OnTIRAP-mediated NF-κB promoter activity. Additionally, OnSARM1 inhibited the mRNA expression of proinflammatory cytokines and hepcidin in A. hydrophila lysate stimulated THK cells. Taken together, these findings suggest that OnSARM1 serves as a negative regulator by inhibiting NF-κB activity, thereby influencing the transcript level of proinflammatory cytokines and antimicrobial peptides in the antibacterial responses.

Expression, signal transduction, and function analysis of TIRAP and TRIF in Nile tilapia (Oreochromis niloticus).

Toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) and toll/interleukin 1 receptor-domain-containing adapter-inducing interferon-ß (TRIF) are crucial adaptors of signal transduction for the signaling pathways of toll-like receptors (TLRs). TIRAP and TRIF perform an essential function in an antimicrobial immune response; however, their function in Nile tilapia remains unknown. Herein, TIRAP and TRIF from Nile tilapia were identified and functionally characterized. Phylogenetic analysis showed that OnTIRAP and OnTRIF clustered with corresponding homologs from other fish species, with comparable gene structures to those of select vertebrate TIRAP and TRIF genes, respectively. The expression profiles of OnTIRAP and OnTRIF were broadly distributed in the ten tissues investigated, with high transcript levels noticed in immune organs. The transcription levels of OnTIRAP and OnTRIF were upregulated in response to bacterial and poly (I:C) challenges. GFP signals were only detected in the cytoplasmic region of fish cells transfected with OnTIRAP-GFP and OnTRIF-GFP expression plasmids. Moreover, overexpression of OnTIRAP and OnTRIF activated interferon-ß (IFN-ß) and activator protein 1 (AP1) reporters in HEK 293 cells. Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter was only observed in OnTRIF-overexpressing HEK 293 cells. Furthermore, the results of the co-immunoprecipitation analysis showed that OnTRIF, but not OnTIRAP, was recruited as an adaptor protein by OnTLR25. This study provides the first evidence on the functions of OnTIRAP and OnTRIF in the immune system of Nile tilapia against pathogens and may serve as the basis for further investigations on TLR signaling in fish.

Functional characterization of myeloid differentiation factor 88 in Nile tilapia (Oreochromis niloticus).

Myeloid differentiation factor 88 (MyD88) is an indispensable component of the toll-like receptor (TLR)- and interleukin 1 receptor-mediated signaling pathways and plays an important role in defensive immune responses. In this study, we cloned MyD88 from the Nile tilapia, Oreochromis niloticus (OnMyD88). Phylogenetic analysis showed that OnMyD88 is grouped with other teleost MyD88s and has the highest amino acid identity and similarity to fugu MyD88. The gene organization of OnMyD88 is similar to MyD88 in several other fish species, except for Atlantic salmon. Gene expression studies revealed that OnMyD88 is highly expressed in immune-related tissues, such as spleen and head kidney, and is up-regulated in spleen and head kidney tissues from fish infected by Streptococcus agalactiae. Functional studies showed that overexpressed OnMyD88 could activate NF-κB and AP-1 in human embryonic kidney (HEK) 293 and tilapia head kidney (THK) cell lines and induce the expression of proinflammatory cytokines such as IL-1ß and IL-12b and chemokine IL-8 in THK cells. The result of the co-immunoprecipitation demonstrated that OnMyD88 can be recruited by OnTLR25 as its adaptor. Taken together, our study indicates that OnMyD88 plays an essential role in the innate immune responses of Nile tilapia against bacterial infection.