Pleiotropic Role of Rainbow Trout CXCRs in Response to Disease and Environment: Insights from Transcriptional Signatures and Structure Analysis

Chemokines are cytokines with chemoattractant capacities that exert their physiological functions through the binding of chemokine receptors. Thus, chemokine and receptor complexes exert important roles in regulating development and homeostasis during routine immune surveillance and inflammation. Compared to mammals, the physiology and structure of chemokine receptors in fish have not been systematically studied. Furthermore, the salmonid-specific whole genome duplication has significantly increased the number of functional paralogs of chemokine receptors. In this context, in the current study, trout exhibited 17 cxcr genes, including 12 newly identified and 5 previously identified receptors. Interestingly, gene expression of brain cxcr1 and cxcr4, kidney cxcr3 and cxcr4, and spleen cxcr3, cxcr4, and cxcr5 subtypes were altered by bacterial infection, whereas brain cxcr1, kidney cxcr1 and cxcr7, and liver cxcr2, cxcr3, and cxcr4 subtypes were changed in response to environmental changes. Based on protein structures predicted by ColabFold, the conserved amino acids in binding pockets between trout CXCR4.1 subtypes and human CXCR4 were also analyzed. Our study is valuable from a comparative point of view, providing new insights into the identification and physiology of salmonid chemokine receptors.


Introduction
Chemokines are small (8-15 kDa) proteins belonging to the cytokine family [1].Chemokines bind to G protein-coupled receptors (GPCRs), and complexes of chemokine and receptor regulate cell movement and activation [2].Based on the number and position of highly conserved N-terminal cysteines, chemokines are divided into four groups: CXC, CC, C, and CX3C (C indicates cysteine, and X/X3 indicates one or three non-cysteine amino acids) [1,2].For example, CCL2 represents a chemokine ligand of the CC subfamily, number 2, and CCR2 represents the receptor of CCL2 [2,3].It is well known that the physiological function of chemokines is to modulate cell migration, which gives them their name (from 'chemotactic cytokines') [4].Chemokines play an important role in regulating cellular migration during routine immune surveillance, inflammation, and development [2].Based on their physiological functions, chemokines can also be divided into two groups: inflammatory and homeostatic chemokines [5].Inflammatory chemokines are induced directly by inflammatory stimuli or related cells [4,5].Homeostatic chemokines, on the other hand, are involved in cell migration, organogenesis, and development, and they are constitutively expressed in discrete tissues or cells [4,5].

Ethics Statement
Our experiments were approved by the Institutional Review Board at Ocean University of China (permit number: 20141201) and performed in accordance with the U.K. Animal Scientific Procedures (Act, 1986) and associated guidelines, the EU Directive 2010/63/EU for animal experiments and the National Institutes of Health Guide for the Care and Use of Laboratory Animals use of laboratory animals (NIH Publications No. 8023, revised 1978).This study did not involve endangered or protected animals.

Genome-Wide Identification and Sequence Analyses
To identify the CXCR genes of rainbow trout, we searched the whole genome of rainbow trout obtained from NCBI (http://www.ncbi.nlm.nih.gov/,accessed on 24 January 2024) and performed tblastn analysis using all available CXCR sequences in the genome databases of human (Homo sapiens), mouse (Mus musculus), zebrafish (Danio rerio), Atlantic salmon (Salmo salar), fugu (Takifugu rubripes), Northern pike (Esox lucius), and channel catfish (Ictalurus punctatus) available in the NCBI (http://www.ncbi.nlm.nih.gov/),Ensembl (http://www.ensembl.org,accessed on 24 January 2024), and Uniport (http: //www.uniprot.org/,accessed on 24 January 2024) as queries with e-values of 1 × 10 −5 .To remove redundant sequences, we used ClustalW for multiple alignments.Tandem arrangement genes were identified by their locations in the reference genome.The coding sequences were predicted using ORF (opening reading frames) finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html,accessed on 24 January 2024), which were further validated by BLASTP against NCBI nonredundant (nr) protein database.In addition, we used the online ProtParam tool to characterize the molecular weight (MW) and theoretical isoelectric point (pI).
Based on the amino acid sequences of CXC chemokine receptors of humans, mice, zebrafish, Atlantic salmon, medaka, fugu, Northern pike, and channel catfish, a phylogenetic analysis was conducted with MEGA 7, using the neighbor-joining method, with a set of 1000 bootstrap replicates [38].

Gene Structure, Conserved Domains, and Motif Analysis of the CXCR
Gene exon-intron structures were analyzed using the Gene Structure Display Server (GSDS2.0)by comparing the codon sequences and genomic sequences of the 17 CXCR members.The transmembrane (TM) domains were predicted by the TMHMM Server v. 2.0 (http://services.healthtech.dtu.dk/service.php?TMHMM-2.0,accessed on 24 January 2024), comparing the results of previous studies in human and zebrafish GPCRs.Motif analyses were performed with Multiple EM for Motif Elicitation (MEME, version 4.11.4), with the limitation of ten motifs and optimum widths of motifs of 6-50 amino acids [39].

Expression Analysis Using Available RNA-Seq Datasets
Using our available RNA-Seq datasets, we analyzed the cxcr expression levels of rainbow trout in response to bacterial infection (phenotype/timeline-specific expressions).The RNA-Seq datasets were retrieved from our previous studies described above: 1.

4.
Liver samples from rainbow trout cultured in different stocking densities (unpublished data and count data are shown in Supplementary Materials).Rainbow trout were cultured in saltwater with initial densities at 9.15 kg/m 3 (low density (LD)), 13.65 kg/m 3 (moderate density (MD)), and 27.31 kg/m 3 (high density (HD)) for 84 days.The final densities were 22.00 (LD), 32.05 (MD), and 52.24 (HD) kg/m 3 , respectively.Liver samples were collected from LD, MD, and HD on day 84.

Structural Analysis of Trout CXCR4.1 Subtypes
ColabFold (ColabFold v1.5.5) was used to predict the protein structures by combining MMseqs2 with AlphaFold2 or RoseTTAFold [45].Compared to the ORF sequences, we showed amino acid sequences associated with TM, extracellular (ECL), and intracellular (ICL) loops with high confidence values.The amino acid sequences for structure prediction are shown in Supplementary Materials Text S1.The human CXCR4 (PDB ID: 4RWS) was used as a template.Comparison of the domains between trout and human CXCR4 and the cartoon, stick, and sphere structures of the proteins were generated by PyMOL software (PyMOL-2.5.4) [46,47].

Statistical Analysis
The RNA-Seq data (counts) were normalized with the Bioconductor DESeq2 Package [48,49].In order to obtain the belt data (Poisson) distribution for further statistical analysis, data of RNA-Seq were normalized by log transformation [50].After that, the normalized data were analyzed by an online R software Package (https://omicsforum.ca/, accessed on 24 January 2024) for multivariate analyses [51,52].Based on previous studies in the fishery and biomedical studies [53,54], we evaluated the whole profile of the cxcr expressions by performing the heatmap, principal components analysis (PCA), correlation coefficients, and variable importance in projection (VIP).Gene expression analyses were performed with GraphPad Prism 8.0.The results were evaluated by one-way analysis of variance (ANOVA) followed by a Tukey multiple range test, with p < 0.05 set to assign significant differences.Student's t-test was used for comparisons between two groups, with significance established when p < 0.05.Results were presented as mean ± standard error of the mean (SEM).

Identification and Annotation of cxcr Genes in Rainbow Trout
In our study, a total of 17 cxcr genes (12 newly identified and 5 previously identified receptors) were identified in the rainbow trout, with the predicted protein sequences ranging from 309 to 461 amino acids, the molecular weights ranging from 33.92 to 50.85 kDa, and the pIs ranging between 5.88 and 9.24 (Table 1).Based on the sequence information of identified receptors in humans, mice, and zebrafish, on sequence similarities among the trout receptors, and on the conserved seven transmembrane domains and DRY motif, the 17 cxcr genes were divided into seven families.Chromosomal locations of cxcr genes were also studied.In brief, the trout cxcr genes were distributed in eight different chromosomes (Chr2, 3, 8, 16, 18, 22, 24, and 28), including three genes on Chr2, five genes on Chr3, and four genes on Chr22.Copy numbers of the cxcr genes in rainbow trout were compared with those of human, mouse, chicken, zebrafish, and several teleost species (Table 2).Expanded copies of cxcr1, cxcr2, cxcr3, cxcr4, and cxcr7 genes were identified in rainbow trout (Table 2).

Phylogenetic Analysis and Gene Structure Analyses
Amino acid sequences of CXCR in rainbow trout and other species were used to construct a phylogenetic tree.The phylogenetic tree exerted a total of six subgroups, including the CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7 subgroups (Figure 1).All these CXCR proteins contained seven transmembrane domains, which are typically observed in GPCRs (Figure 2).

Phylogenetic Analysis and Gene Structure Analyses
Amino acid sequences of CXCR in rainbow trout and other species were used to construct a phylogenetic tree.The phylogenetic tree exerted a total of six subgroups, including the CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7 subgroups (Figure 1).All these CXCR proteins contained seven transmembrane domains, which are typically observed in GPCRs (Figure 2).

A. salmonicida
The heatmap indicated the overall expression profiles of cxcr genes in the brain and kidney between control and infected trout (Figure 4A).Expressions of representative genes (cxcr3, cxcr5, cxcr7.1a,and cxcr7.1b)are also shown (Figure 4B-E).

A. salmonicida
The heatmap indicated the overall expression profiles of cxcr genes in the brain and kidney between control and infected trout (Figure 4A).Expressions of representative genes (cxcr3, cxcr5, cxcr7.1a,and cxcr7.1b)are also shown (Figure 4B-E).

Transcriptional Profiles of cxcr in Trout in Response to Salinity Change and High Stocking Density
The overall transcriptional profiles of trout cxcr in the brain, kidney, and liver are shown in heatmaps (Figure 5A,B).Compared to DS, TS showed a significantly downregulated expression of brain cxcr1.1,up-regulated expression of kidney cxcr1.1, and down-regulated expression of kidney cxcr7.1b(Figure 5C-E).Compared to TF, TS showed a significantly down-regulated expression of liver cxcr3 and up-regulated expression of liver cxcr4.2b(Figure 5F,G).The overall hepatic cxcr expressions were clustered in a heatmap (Figure 5H).High stocking density significantly increased cxcr2.1 expressions when comparing HD to MD and LD (Figure 5I).

Transcriptional Profiles of cxcr in Trout in Response to Salinity Change and High Stocking Density
The overall transcriptional profiles of trout cxcr in the brain, kidney, and liver are shown in heatmaps (Figure 5A,B).Compared to DS, TS showed a significantly down-regulated expression of brain cxcr1.1,up-regulated expression of kidney cxcr1.1, and downregulated expression of kidney cxcr7.1b(Figure 5C-E).Compared to TF, TS showed a significantly down-regulated expression of liver cxcr3 and up-regulated expression of liver cxcr4.2b(Figure 5F,G).The overall hepatic cxcr expressions were clustered in a heatmap (Figure 5H).High stocking density significantly increased cxcr2.1 expressions when comparing HD to MD and LD (Figure 5I).

Characterization of cxcr Genes
Whole genome duplication occurred in teleost ancestors, resulting in increased paralogs of cxcr genes.Hence, previous studies have identified eight cxcr genes in a typical 3R teleost species, such as channel catfish [15].Atlantic salmon is an important 4R salmonid species, and 19 cxcr genes have been identified in Atlantic salmon [61].In the current study, a total of 17 cxcr genes were identified in rainbow trout based on available genomic information and our RNA-seq datasets (Figures 1 and 2), being consistent with previous studies that showed an expansion of chemokine systems in teleosts [62,63].Thus, the faster evolvement of chemokines and the fish-specific whole genome duplication have resulted in the expansion of both teleost chemokine and receptor genes [10,14,[64][65][66].In this study, we showed duplications of trout cxcr1, cxcr2, cxcr3, cxcr4, and cxcr7 due to whole genome duplication and lineage-specific tandem gene duplications (Figures 1 and 2).For example, cxcr3 and cxcr3.1awere localized quite close to each other on chromosome 2, and a large cluster of cxcr genes were located on chromosome 3 and 22, all of this suggesting a rapid evolution through tandem duplications [67], suggesting that gene duplication of CXC chemokines and receptors might acts as a predominant evolutionary mechanism for environment adaptation in fish [68].Compared to 3R teleosts, the 4R salmonid species exerted more cxcr paralogs, which is consistent with previous studies showing that genes (such as igf and igfbp genes, for example) involved in immunomodulation were further expanded in salmonid species [26,27].
The phylogenetic analysis showed explicit annotations of trout CXCR proteins, with most trout CXCR proteins clustered with their teleost counterparts (Figure 1).The seven transmembrane domains, a conserved and typical structure of GPCRs [69], were observed in all trout CXCR members, as well as a DRY motif in TM3 and an NPxxY motif in TM7, revealing sequence and structure similarities between trout CXCRs and mammalian family A GPCRs.

Physiological Functions of cxcr Genes
The chemokine system plays an important role in modulating the development of the immune system homeostasis during routine immune surveillance and inflammation [2,6,67].Recent studies reported the involvement of chemokine systems in regulating immunomodulation in teleosts, including catfish, trout, croaker, and bream [15,59,[70][71][72][73].However, most of these studies focused on studying CXCR-regulated immunomodulation in peripheral immune tissues rather than in the central nervous system.Therefore, we evaluated cxcr transcription levels in both brain and peripheral tissues in trout in response to V. anguillarum or A. salmonicida infections.
In humans and rodents, CXCR1 has been reported to be widely expressed in the brain and to play an important role in modulating neuroinflammation [74,75].In teleosts, CXCR1 regulates immune defense against pathogen infections [59,76,77].For example, peripheral cxcr1 was up-regulated by viral and bacterial infections in trout [59].Two cxcr1 subtypes have been identified in some teleost species as a consequence of the additional WGD [61,77], which is consistent with our results.In Asian swamp eel (Monopterus albus), a previous study showed different gene expressions between cxcr1.1 and cxcr1.2after pathogen infection [77].In this study, brain cxcr1.1 and cxcr1.2showed different transcriptional regulation in response to bacterial infections (Figure 3).A recent study showed cxcrs exhibited tissuespecific and time-dependent regulation of transcription in the head kidney, liver, and gill after A. salmonicida infection in turbot (Scophthalmus maximus) and black rockfish (Sebastes schlegelii) [68,78].These results suggest that cxcr1 subtypes might be differently involved in the response to disease in both brain and peripheral tissues.
Biomedical studies indicate that CXCR3 is involved in directing lymphocytes into inflammation areas and regulating the inflammatory state of both the CNS and peripheral tissues [79,80].A previous study in grass carp (Ctenopharyngodon idella) showed that cxcr3 is widely expressed in the brain and protects the brain from pathogen infection [81].We observed that brain cxcr3 was significantly up-regulated by A. salmonicida infection (Figure 4).Consistently, transcriptional profiles of cxcr3 subtypes were significantly altered by a bacterial and viral infection, LPS or polyI:C stimulation in teleost species, including rainbow trout, turbot, largemouth bass (Micropterus salmoides), and black rockfish [57,68,78,82].In rainbow trout, cxcr3 subtypes were differently induced by inflammatory stimulants and cytokines in head kidney cells and macrophages [59,76,83].Our results also showed that cxcr3 exhibited tissue-specific and subtype-dependent transcriptional regulation in peripheral tissues in response to pathogen infection.Our results further supported the involvement of CXCR3 in the neuro-immune network in fish [81].Human studies showed that CXCR3A and CXCR3B exert opposite functions in regulating cell growth, with the "Survival" and "Death" signals derived from CXCR3A and CXCR3B, respectively [84,85].In this study, we observed up-regulated cxcr3 subtypes in asymptomatic trout compared to symptomatic trout (Figure 3).Our results suggested trout cxcr3 subtypes might be functional orthologs of human CXCR3A.Further studies should investigate the regulatory mechanism(s) of the cxcr3-regulated "Survival" signals.
CXCR4 is one of the most well-studied chemokine receptors due to its important role in regulating the development of the immune system and also immunomodulation.In biomedical studies, CXCR4 serves as the therapeutic target of cancer metastasis and HIV-1 infection [86][87][88][89].There is growing evidence that CXCR4 is involved in infection defense, neuron pathophysiology, and response to stress in teleosts ( [90,91], Reviewed in [76]).For example, rainbow trout, grouper, and channel catfish showed up-regulated cxcr4 after viral and bacterial infections [15,82,90].CXCR4 was also widely expressed in the CNS, where it seems to be involved in neuron pathology [76,90].Our results showed brain cxcr4.1band cxcr4.2bexpression were significantly altered due to V. anguillarum infection, in correlation with a previous study that showed that nervous necrosis virus infection led to significantly upregulated cxcr4 expression in orange-spotted grouper [90].Alterations of environmental nitrate also induce cxcr4b expression in Wuchang bream (Megalobrama amblycephala) [91].Likewise, our results showed that both bacterial infection and environmental changes altered the expression of cxcr4 subtypes (Figures 3 and 5).CXCR5 serves as the homeostatic regulator for immune responses and neuron regeneration ([92,93], reviewed in [76]).In fish, cxcr5 was shown to be highly expressed in lymphoid tissues, including the kidney and spleen in grass carp [92].Consistent with previous studies showing cxcr5 expression is modulated by a range of immune stimulants and pathogen infection [68,92], in this study, symptomatic trout also showed down-regulated spleen cxcr5 in response to V. anguillarum infection (Figure 4).
Meanwhile, it is important to compare the extent of changes between chemokine receptors to the overall statistical pattern of changes in the RNA-Seq data and other genes that are not directly related to immunomodulation.In this study, we selected per1b (period circadian clock 1b).The per1b is widely expressed in both brain and peripheral tissues in humans (https://www.genecards.org/,accessed on 24 January 2024) and rainbow trout.The per1b gene regulates circadian rhythms of locomotion, metabolism, and behavior.In the brain, average fold-changes of all up-regulated genes between groups were ~1.59 (C/S) and 1.48 (C/A), and all down-regulated genes between groups were ~0.68 (C/S) and 0.75 (C/A).The fold-changes of per1b gene expression between groups were ~1.51 (C/S) and 0.94 (C/A), while fold-changes of cxcr1.2gene expression between groups were ~0.21 (C/S) and 0.57 (C/A) (Figure 3).In the kidney, average fold-changes of all up-regulated genes between groups were ~2.51 (C/S) and 1.76 (C/A), and all down-regulated genes between groups were ~0.59 (C/S) and 0.63 (C/A).The fold-changes of per1b gene expression between groups were ~0.84 (C/S) and 0.82 (C/A), while fold-changes of cxcr3 gene expression between groups were ~3.97 (C/S) and 1.21 (C/A) (Figure 3).In the spleen, average foldchanges of all up-regulated genes between groups were ~4.35 (C/S) and 2.71 (C/A), and all down-regulated genes between groups were ~0.45 (C/S) and 0.60 (C/A).The foldchanges of per1b gene expression between groups were ~0.95 (C/S) and 1.39 (C/A), while fold-changes of cxcr3 gene expression between groups were ~8.89 (C/S) and 0.64 (C/A) (Figure 3).These results suggested that infection or the fish response to infection are directly provoking alterations in trout cxcr transcription levels, especially between groups of control trout and symptomatic trout.
Finally, we showed that the amino acids of the binding pocket of IT1t (a small molecule) in trout CXCR4.1a/b were conserved to those of human CXCR4 [94] (Figure 6).Compared to endogenous ligands, these small molecule ligands (drugs) are stable and orally bioavailable [95].Therefore, small molecular ligands of human chemokine receptors could be used in the future as immunomodulators targeting fish CXCRs in the aquaculture industry.

Conclusions
In this study, we have identified 17 cxcr genes in rainbow trout with duplicated copies of cxcr1, cxcr2, cxcr3, cxcr4, and cxcr7.Gene expression analyses showed trout cxcr genes exhibited conserved functions with human orthologs.Transcription levels of cxcr genes were altered by bacterial infection and environmental changes, suggesting a pleiotropic role in regulating homeostasis and immune response.Trout CXCR4.1a(b)showed conserved residues for the binding pocket of IT1t (a small molecule ligand) with human CXCR4.Our results contribute to a better understanding of the immune role of CXCRs and their potential ligands in an important teleost species.This information could be used in the future to modulate immune responses to infectious diseases and adaptation to environmental changes.

Figure 1 .
Figure 1.Phylogenetic tree of CXCRs.The CXCR sequences were obtained from rainbow trout, Atlantic salmon, zebrafish, human, mouse, Northern pike, Channel catfish, and fugu.The number of nodes shows the bootstrapping values, and the black stars indicate trout CXCRs.

Figure 1 .
Figure 1.Phylogenetic tree of CXCRs.The CXCR sequences were obtained from rainbow trout, Atlantic salmon, zebrafish, human, mouse, Northern pike, Channel catfish, and fugu.The number of nodes shows the bootstrapping values, and the black stars indicate trout CXCRs.

Figure 2 .
Figure 2. Alignment of CXCR proteins.Residues of transmembrane domains (TMs), DRY motif (a highly conserved motif in family A GPCRs), and (semi)conserved cysteines are shaded in grey, red, and orange.TMs and (semi)conserved cysteines are defined from previous studies of salmon and teleost CXCRs [61,62].The highly conserved NPxxY motif in family A GPCRs is observed in TM 7.

Figure 2 .
Figure 2. Alignment of CXCR proteins.Residues of transmembrane domains (TMs), DRY motif (a highly conserved motif in family A GPCRs), and (semi)conserved cysteines are shaded in grey, red, and orange.TMs and (semi)conserved cysteines are defined from previous studies of salmon and teleost CXCRs [61,62].The highly conserved NPxxY motif in family A GPCRs is observed in TM 7.

Figure 4 .
Figure 4. Transcriptional profiles of cxcr in trout after A. salmonicida infection.(A): the heatmap of cxcr transcriptional profiles.(B-E): expression of the representative genes (with significant differences among groups).Asterisks indicate significant differences (one-way analysis ANOVA followed by Tukey's multiple comparison test with p < 0.05).Abbreviations: CB-control trout brain tissue; CK-control trout kidney tissue; IB-infected trout brain tissue; IK-infected trout kidney tissue.

Figure 4 . 19 Figure 5 .
Figure 4. Transcriptional profiles of cxcr in trout after A. salmonicida infection.(A): the heatmap of cxcr transcriptional profiles.(B-E): expression of the representative genes (with significant differences among groups).Asterisks indicate significant differences (one-way analysis ANOVA followed by Tukey's multiple comparison test with p < 0.05).Abbreviations: CB-control trout brain tissue; CK-control trout kidney tissue; IB-infected trout brain tissue; IK-infected trout kidney tissue.Biomolecules 2024, 14, x FOR PEER REVIEW 11 of 19

Figure 5 .
Figure 5. Transcriptional profiles of cxcr in trout after salinity and density changes.(A,B): principal component analysis (PCA) of cxcr transcriptional profiles in the brain (A) and liver and kidney (B).

Table 1 .
Summary of 17 cxcr genes in rainbow trout.

Table 2 .
Comparison of cxcr gene copies among mammals and teleosts.