Fish & Shellfish Immunology 58 (2016) 259e265 Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Short communication Identification and expressional analysis of NLRC5 inflammasome gene in smolting Atlantic salmon (Salmo salar) * Juan Pablo Pontigo a, b, , María Jose Agüero a, Patricio Sanchez c, Ricardo Oyarzún a, d, Carolina Vargas-Lagos a, e, Jorge Mancilla d, f, Hans Kossmann g, Francisco J. Morera h, ** *** Alejandro J. Yanez~ c, i, , Luis Vargas-Chacoff a, j, a Instituto de Ciencias Marinas y Limnologicas, Universidad Austral de Chile, Valdivia, Chile b Programa Doctorado en Ciencias, Mencion Biología Celular y Molecular, Escuela de Graduados, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile c Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile d Programa de Doctorado en Ciencias de la Acuicultura, Universidad Austral de Chile, Los Pinos s/n, Balneario Pelluco, Puerto Montt, Chile e Programa de Magister en Ciencias, Mencion Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile f Marine Harvest Chile, Ruta 226 Km8, Puerto Montt, Chile g Trapananda Asesorias, Cristobal Colon 486, Puerto Montt, Chile h Instituto de Farmacología y Morfofisiología, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile i Interdisciplinary Center for Aquaculture Research (INCAR), Concepcion, Chile j Centro Fondap de Investigacion de Altas Latitudes (IDEAL), Universidad Austral deChile, Casilla 567, Valdivia, Chile article info abstract Article history: The NOD-like receptors (NLRs) were recently identified as an intracellular pathogen recognition receptor Received 8 August 2016 family in vertebrates. While the immune system participation of NLRs has been characterized and Received in revised form analyzed in various mammalian models, few studies have considered NLRs in teleost species. Therefore, 13 September 2016 this study analyzed the Atlantic salmon (Salmo salar) NLRC5. Structurally, Atlantic salmon NLRC5 pre- Accepted 14 September 2016 sented leucine-rich repeat subfamily genes. Phylogenetically, NLRC5 was moderately conserved between Available online 15 September 2016 S. salar and other species. Real-time quantitative PCR revealed NLRC5 expression in almost all analyzed organs, with greatest expressions in the head kidney, spleen, and hindgut. Furthermore, NLRC5 gene Keywords: Innate immunity expression decreased during smolt stage. These data suggest that NLRC5 participates in the Atlantic fi Inflammation salmon immune response and is regulated, at least partly, by the smolti cation process, suggesting that Leucine-rich repeats there is a depression of immune system from parr at smolt stage. This is the first report on the NLRC5 NLR gene in salmonid smolts. NOD-like receptors © 2016 Elsevier Ltd. All rights reserved. Nucleotide binding Pathogen 1. Introduction vertebrates, which have poorly developed adaptive immunity [1]. The innate immune response rapidly recognizes pathogenic parti- Innate immunity is the first line of defense against pathogens in cle invasion and initiates defensive actions. In vertebrates, the innate immune system recognizes various pathogenic and non- pathogenic microorganisms via pattern recognition receptors able to recognize specific pathogen associated molecular patterns, Abbreviations: LRR, leucine rich repeat; NCBI, National Center for Biotechnology þ þ thereby initiating the immune response [2,3]. Information; NKA, Na /K -ATPase; NLRs, NOD-like receptors; NOD, nucleotide- binding oligomerization domain; SsNLRC5, Salmo salar NOD-like receptor C5. The immune response is comprised by different cells able to * Corresponding author. Instituto de Ciencias Marinas y Limnologicas, Uni- interact with and consequently respond to (e.g. via inflammation) versidad Austral de Chile, Valdivia, Chile. foreign particles. The inflammatory response is evidenced by ** Corresponding author. distinct mechanisms, such as leukocyte recruitment to the infection *** Corresponding author. site; antimicrobial effector system activation; and adaptive im- E-mail addresses: [email protected] (J.P. Pontigo), [email protected] (A.J. Yanez),~ [email protected] (L. Vargas-Chacoff). mune response stimulation [4]. Several pattern recognition http://dx.doi.org/10.1016/j.fsi.2016.09.031 1050-4648/© 2016 Elsevier Ltd. All rights reserved. 260 J.P. Pontigo et al. / Fish & Shellfish Immunology 58 (2016) 259e265 receptors are involved in pathogen elimination and control. These 2.2. Identification and sequencing of S. salar NLRC5 (SsNLRC5) include inducible retinoic acid receptors, Toll-like receptors, and through transcriptomic analyses nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs, alternatively termed intracellular nucleotide-binding Massive Illumina RNAseq assessments were performed for parr- domain, leucine rich repeat (LRR) containing receptors [1]. NLRs and smolt-stage Atlantic salmon head kidney tissues. The tran- particularly participate in innate defenses against bacteria. Specific scriptome was annotated by accessing the non-redundant National to NLR proteins is the presence of a nucleotide binding domain Center for Biotechnology Information (NCBI) database using a flanked by highly variable LRRs. These nucleotide binding-LRR dedicated Illumina MiSeq sequencing computer at the Austral- domains containing various receptors, including NLRs [5]. OMICS Center of the Faculty of Sciences, Universidad Austral de In farmed fish, pathogens can induce a systemic stress response, Chile. which is highly demanding on energy reserves [6,7]. The realloca- tion of metabolic energy negatively interferes with other physio- 2.3. Gene expression analyses logical processes, such as immunological capacity, thus influencing the ability of several immune system components to resist infection Total RNA was extracted with the TRIzol reagent (Invitrogen) [8e12]. For example, increased cortisol levels may decrease following the manufacturer's instructions, and the obtained sam- lymphocyte quantities, consequently inducing a selective sup- ples were treated with amplification grade DNase I (1 U mg1 RNA, pression of phagocytic and complementary immune activities in Invitrogen). The SuperScript III RNase H Reverse Transcriptase the head kidney and blood, as well as increasing infection suscep- platform (Invitrogen) synthesized first-strand cDNA from 1 mgof tibility in teleosts [13]. Fish are naturally exposed to stress, as is the total RNA using the oligo-dT18 primer at 50 C for 50 min. Quan- case for salmonids during changing environment. titative PCR analysis was carried out on the Mx3000p instrument In particular, Atlantic salmon (Salmo salar) undergo smoltifica- (Stratagene). Reaction mixtures were incubated for 10 min at 95 C, tion, a migratory process for growth [14] that is a critical stage for followed by 40 cycles of 15 s at 95 C, 1 min at 60 C, and finally 15 s successful aquaculture. Standard farming practices move smolting at 95 C, 1 min at 60 C, and 15 s at 95 C. The mRNA gene ex- fish from fresh to seawater. This transition is not simply a migration pressions were normalized to the Atlantic salmon ribosomal pro- from hypo-to hyperosmotic conditions; it also generates costs for tein S18 using the comparative DDCt method [18]. The primers used several biological functions, such as in the metabolic, endocrine, are listed in Table 1. In all cases, each qRT-PCR was performed with osmoregulatory and immune systems, been changes in amino acid triplicate samples and repeated with at least two independent levels, hormones as cortisol-growth hormone (increasing levels) or samples. þ prolactin (decreasing levels), and osmotic process (changes in Na , þ K -ATPase sub-units in gills) the more reported [9,10]. Conse- 2.4. Phylogenetic analysis quently, smolting can increase, at least temporarily, the suscepti- bility of fish to pathogenic infection, likely with significant A 10-sequence set was compiled to infer phylogenetic re- consequences. Therefore, active osmoregulation occurs during lationships among several species in a consensus tree. Sequences smoltification, with low osmolality inducing turgor in macrophages were manually trimmed by deleting alignment gaps. The sequence þ À and decreasing K and Cl concentrations [15e17]. These modu- set was then analyzed using Treefinder (v. March 2011) with a lations normally activate the inflammasome, but a detailed char- mixed model and 1000 bootstrap replications. Bayesian inference acterization is lacking for salmonids. analysis was computed using the MrBayes software (v. 3.2.6) with a Considering the above-detailed knowledge gap, the aims of this mixed substitution model for 105 generations and burn-in of 25%. study were to identify and analyze NLRC5 inflammasome gene þ þ expression during S. salar smoltification and to more fully under- 2.5. Gill Na ,K -ATPase activity (NKA) stand why S. salar smolts suffered increased pathogenic infections in the transition to seawater. Gill NKA activity was determined using the microassay method described by McCormick [19]. 2. Materials and methods 2.6. Statistical analyses 2.1. Animals All data are shown as the mean ± standard error (SE). As- sumptions of normality and homogeneity were tested for the Atlantic salmon (Salmo salar) from the parr and smolt stages detected variances. Differences were evaluated using one-way were provided by the Calabozo Fish Farm, Chile (Marine Harvest). ANOVA