D-dopachrome tautomerase from Japanese sea bass (Lateolabrax japonicus) is a chemokine-like cytokine and functional homolog of macrophage migration inhibitory factor
D-多巴色素互变异构酶（DDT）是巨噬细胞迁移抑制因子（MIF）蛋白超家族的一员，它是一种具有趋化因子样特征的新型细胞因子。然而，目前关于鱼类DDT的研究鲜有报道。在本研究中，我们从花鲈（Lateolabrax japonicus）中鉴定出一个DDT同源物（LjDDT）。序列分析表明，LjDDT具有已知DDT和MIF同源物的典型特征，且与条石鲷（Oplegnathus fasciatus）DDT进化相关性最高。LjDDT基因mRNA在健康花鲈所有检测组织中均有表达，其中肝脏中的表达量最高。花鲈在感染哈维氏弧菌后，检测到肝、脾和头肾组织中的LjDDT转录水平显著下调。首先，我们制备了重组LjDDT（rLjDDT）及相应的抗体（anti-rLjDDT）。注射100 μg/g anti-rLjDDT对感染哈维氏弧菌的花鲈存活具有显著的保护作用。rLjDDT在体内外均能诱导花鲈单核/巨噬细胞（MO/MФ）和淋巴细胞迁移，但对中性粒细胞迁移无明显作用。rLjDDT对脂多糖（LPS）诱导产生的M1型MO/MФ有体外趋化作用，而对cAMP诱导产生的M2型MO/MФ无体外趋化作用。其次，我们通过RNA干扰（RNAi）分别敲低花鲈MO/MФ上的CD74（LjCD74）和CXCR4（LjCXCR4）表达，发现LjCD74敲低导致rLjDDT增强的MO/MФ的迁移作用被抑制，rLjMIF抑制的MO/MФ迁移作用被解除，但LjCXCR4敲低对两者无明显影响，揭示LjCD74可能是LjDDT和LjMIF在花鲈MO/MФ上的主要趋化受体。另外，同时加入rLjDDT和rLjMIF对MsiRNA、LjCD74si或LjCXCR4si处理后的花鲈MO/MФ迁移无明显影响，揭示rLjDDT和rLjMIF两者可能存在拮抗作用。综上所述，我们的研究首次表明，DDT可能作为一种MIF的拮抗剂，通过CD74介导MO/MФ趋化募集，在鱼类抗细菌感染的免疫应答中发挥作用。Abstract:
D-dopachrome tautomerase (DDT), a member of the macrophage migration inhibitory factor (MIF) protein superfamily, is a newly described cytokine with chemokine-like characteristics. However, research on fish DDT remains limited. In this study, we identified a DDT homolog (LjDDT) from the Japanese sea bass, Lateolabrax japonicus. Sequence analysis showed that LjDDT had typical sequence features of known DDT and MIF homologs and was most closely related to DDT of rock bream (Oplegnathus fasciatus). LjDDT transcripts were detected in all tested tissues of healthy Japanese sea bass, with the highest expression found in the liver. Upon infection with Vibrio harveyi, LjDDT transcripts were significantly down-regulated in the three tested tissues, including the liver, spleen, and head kidney. Recombinant LjDDT (rLjDDT) and the corresponding antibody (anti-rLjDDT) were subsequently prepared. The administration of 100 μg/g anti-rLjDDT had a statistically significant protective effect on the survival of V. harveyi-infected fish. Moreover, rLjDDT was able to induce the migration of monocytes/macrophages (MO/MФ) and lymphocytes both in vitro and in vivo, but without significant influence on the migration of neutrophils. rLjDDT exhibited chemotactic activity for lipopolysaccharide (LPS) -stimulated M1-type MO/ MΦ in vitro, but not for cAMP-stimulated M2-type MO/MΦ. Furthermore, the knockdown of LjCD74, but not LjCXCR4, significantly down-regulated the rLjDDT-enhanced migration of MO/MΦ and relieved the rLjMIF-inhibited migration of MO/MΦ. These results indicate that LjCD74 may be the major chemotactic receptor of LjDDT and LjMIF in Japanese sea bass MO/MΦ. Combined rLjDDT+ rLjMIF treatment had no significant effect on the migration of MsiRNA, LjCD74si-, or LjCXCR4sitreated MO/MΦ compared to the control group, suggesting that the roles of LjDDT and LjMIF may be antagonistic. In conclusion, our study demonstrates for the first time that DDT may play a role in the immune responses of fish against bacterial infection through chemotactic recruitment of MO/MΦ via mediation of CD74 as an antagonist of MIF.
Figure 1. Multiple alignments of amino acid sequences of LjDDT with other DDT homologs (A) or LjMIF (B)
Threshold for shading was >60%, with similar residues shaded gray and identical residues shaded black. LjDDT: Japanese sea bass DDT; LcDDT: Large yellow croaker DDT; FhDDT: Mummichog DDT; OmDDT: Rainbow smelt DDT; ElDDT: Northern pike DDT; OlDDT: Japanese ricefish DDT; SsDDT: Atlantic salmon DDT; OmyDDT: Rainbow trout DDT; TrDDT: Tiger puffer DDT; OfDDT: Rock bream DDT; BpDDT: Mudskipper DDT; OnDDT: Nile tilapia DDT; DrDDT: Zebrafish DDT; AmDDT: Mexican tetra DDT; MmDDT: Mouse DDT; LjMIF: Japanese sea bass MIF. GenBank accession Nos. of sequences used are listed in Supplementary Table S1. Active site residues Pro, Lys, and Ile are marked with“*”. “CXXC”motif is denoted with dotted box.
Figure 2. Phylogenetic tree of DDT nucleotide using neighbor-joining method (1 000 bootstrap replicates; maximum composite likelihood model) in MEGA v7
Site of Japanese sea bass DDT is marked with“◆”. Values at forks indicate percentage of trees in which this grouping occurred after bootstrapping (1 000 replicates; shown only when >60%). Scale bar shows number of substitutions per base. GenBank accession Nos. of sequences used are listed in Supplementary Table S1.
Figure 3. mRNA expression analysis of LjDDT in tissues of healthy (A) and V. harveyi-infected Japanese sea bass (B–D)
A: LjDDT mRNA expression level relative to that of Lj18S rRNA, calculated using 2–ΔCT method. B–D: Tissues were collected at different time points after bacterial infection. LjDDT mRNA expression levels relative to that of Lj18S rRNA were calculated using 2-ΔΔCT method. Data are expressed as mean±SEM of results from four fish. Values denoted by different letters are significantly different when compared by ANOVA (P<0.05).
Figure 4. Prokaryotic expression and Western blot analysis of LjDDT
A: 12% SDS-PAGE analysis of bacterial lysates and purified rLjDDT. Lane M: protein marker; Lane 1: pET-28a-LjDDT/BL21 before IPTG induction; Lane 2: pET-28a-LjDDT/BL21 after IPTG induction; Lane 3: Purified rLjDDT. B: Western blot analysis of rLjDDT and native LjDDT in liver of Japanese sea bass. Lane 4: pET28a-LjDDT/BL21 before IPTG induction, negative control; Lane 5: Purified rLjDDT; Lane 6: Japanese sea bass serum; Lane 7: Japanese sea bass liver lysates.
Figure 5. Effect of LjDDT on survival rate of V. harveyi-infected Japanese sea bass
Fish were ip-injected with equal volumes of rLjDDT, IsoIgG, or anti-rLjDDT, respectively, 30 min after V. harveyi infection (1×104 CFU/fish) or 1 h before V. harveyi infection (1×104 CFU/fish). Control group received an equal volume of PBS. Fish mortality was monitored daily for 9 d. n=30.
Figure 6. In vitro effect of rLjDDT and rLjMIF on migration of MO/MФ (A), lymphocytes (B), and neutrophils (C) at different concentrations (0, 1.0, and 10.0 μg/mL, respectively)
Cells were counted under a light microscope at 400× magnification. Data are expressed as mean±SEM. n=4. Values denoted by different letters are significantly different when compared by ANOVA (P< 0.05).
Figure 7. In vivo effect of rLjDDT and rLjMIF administration on MO/MФ (A), lymphocyte (B), and neutrophil (C) numbers in abdominal cavity of Japanese sea bass at different concentrations (0, 1.0, and 10.0 μg/g respectively)
Cells were counted under a light microscope at 400× magnification 24 h after administration of rLjDDT and rLjMIF. Data are expressed as mean± SEM. n=4. Values denoted by different letters are significantly different when compared by ANOVA (P<0.05).
Figure 8. Effect of rLjDDT on migration of polarized Japanese sea bass MO/MΦ
LPS and cAMP were used to induce M1 and M2 polarization of MO/MΦ, respectively. Activities of iNOS (A) and arginase (B) were determined. After incubation with rLjDDT for 4 h, migration percentage of LPS- (C) or cAMP- (D) stimulated MO/MΦ was determined. Non-stimulated resting MO/MΦ were used as negative control (NC). Data are expressed as mean±SEM. n=4; Values denoted by different letters are significantly different when compared by ANOVA (P<0.05).
Figure 9. Effect of LjCD74 and LjCXCR4 knockdown on rLjDDT and rLjMIF-induced migration of MO/MΦ, respectively
Histogram displays effect of LjCD74 (A) and LjCXCR4 (B) siRNA transfection on knockdown of MO/MΦ LjCD74 and LjCXCR4 mRNA expression by RT-qPCR analysis. C: Migration percentage of Japanese sea bass MO/MФ was examined in a Transwell chamber in presence or absence of 10.0 μg/mL rLjDDT, rLjMIF, or rLjDDT+rLjMIF combined. Each bar represents mean±SEM, n=4. Values denoted by different letters are significantly different when compared by ANOVA (P<0.05).
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ZoolRes-41-1-39-Supplementary Tables and Figures.pdf