Table 1.
Primers used for PCR cloning and real-time PCR analysis of gene expression
Fig 1.
Phylogenetic analysis of rainbow trout BAFF-R, BCMA and TACI.
The rooted phylogenetic tree shows the relationship between available sequences and those representing rainbow trout TNFR17 or BCMA, TNFR13B or TACI and TNFR13C or BAFF-R (in bold) with other fish species and with human and other available mammalian sequences. The tree was constructed based on a Clustal V multiple alignment of the complete sequences for each TNFR and was bootstrapped 1000 times as indicated. Accession numbers of the sequences used for the phylogenetic analysis are as follows: Homo sapiens TNFR13C [NP_443177], TNFR13B [NP_036584] and TNFR17 [NP_001183]; Mus musculus TNFR13C [NP_082351], TNFR13B [NP_067324] and TNFR17 [NP_035738]; Oryctolagus cuniculus TNFR13C [XP_008273381], TNFR13B [XP_008250513] and TNFR17 [XP_008255958]; Salmo salar TNFR13C [XP_014059270], TNFR13B [XP_014034414] and TNFR17 [XP_014034440]; Sinocyclocheilus rhinocerous TNFR13C [XP_016414908] and TNFR17 [XP_016397935]; Sinocyclocheilus anshuiensis TNFR13C [XP_016321777]; Poecilia latipinna TNFR13C [XP_014910891]; Haplochromis burtoni TNFR13C [XP_005947083]; Neolamprologus brichardi TNFR13C [XP_006798027]; Esox lucius TNFR13B [XP_010863669]; Danio rerio TNFR13B [XP_009304656] and TNFR17 [XP_009304977]; Cyprinus carpio TNFR17 [KTF73008].
Fig 2.
Clustal W alignment of BAFF-R (TNFR13C) from trout (KX894509), salmon (XP_014059270), P. latipinna (XP_014910891), human (NP_443177) and mouse (NP_082351).
The location of the cysteine rich domains (CRD), transmembrane domain (TM) and TRAF binding domain (TRAF domain) are shown above the alignment. Cysteine residues conserved in all species forming disulfide bonds in CRD regions are shown in red and the predicted disulfide bond is indicated with brackets. Residues implicated in receptor binding to BAFF and APRIL are shown in green and those that are known to limit APRIL binding in mammals are indicated in blue.
Fig 3.
Clustal W alignment of BCMA (TNFR17) from trout (KX894511), salmon (XP_014034440), zebrafish (XP_009304977), human (NP_001183) and mouse (NP_035738).
The location of the cysteine rich domains (CRD), transmembrane domain (TM) and TRAF binding domain (TRAF domain) are shown above the alignment. Residues predicted as essential for TRAF binding are boxed (TRAF binding sequence), cysteine residues forming disulfide bonds in CRD regions are shown in red and the predicted disulfide bonds are indicated with brackets. Residues implicated in BAFF and/or APRIL binding are shown in green.
Fig 4.
Clustal W alignment of TACI (TNFR13B) from trout (KX894510), salmon (XP_014034414), Esox lucius (XP_010863669), human (NP_036584) and mouse (NP_067324).
The location of the cysteine rich domains (CRD), transmembrane domain (TM) and TRAF binding domain (TRAF domain) are shown above the alignment. Residues predicted as essential for TRAF binding are boxed (TRAF binding sequence), cysteine residues conserved in all species forming disulfide bonds on each CRD region are shown in red and the predicted disulfide bonds are indicated with brackets. Residues implicated in BAFF and/or APRIL binding are shown in green.
Fig 5.
Constitutive levels of transcription of BAFF-R, BCMA and TACI in different tissues.
The amount of BAFF-R (A), BCMA (B) and TACI (C) mRNA in PBLs and tissues (spleen, head kidney (HK), skin, hindgut, gills, brain, liver and muscle) from 3 naïve perfused fish was estimated by real time PCR in duplicate samples. Data are shown as the gene expression relative to the expression of endogenous control EF-1α (mean + SD).
Fig 6.
Levels of transcription of BAFF-R, BCMA and TACI in early rainbow trout stages.
Transcriptional levels of BAFF-R (A), BCMA (B) and TACI (C) during trout early development at different stages. Data are shown as the gene expression relative to the expression of an endogenous control (EF-1α) (mean + SD, n = 5). DD: degree days; HAT: hatching; PFF: pre-first feeding; FF: first feeding; Fry: 3 weeks post-first feeding. *** Levels of expression significantly different to those observed in samples taken at 640 DD (p < 0.005).
Fig 7.
Transcription levels of BAFF-R, BCMA and TACI in B cells.
Constitutive levels of transcription of BAFF-R (A), BCMA (B) and TACI (C) in FACS isolated IgM+ B cells from different trout tissues were evaluated by real time PCR in duplicate. Results are shown as the gene expression relative to the expression of an endogenous control (EF-1α) (mean + SD, n = 5).
Fig 8.
Regulation of BAFF and APRIL subfamily ligands and receptors during PKD infection.
Posterior kidney tissue samples were obtained from rainbow trout naturally infected with PKD. Samples were classified according to their kidney swelling grade, as detailed in the Methods. All control fish had a kidney swelling grade of 0 (Control) and samples were named Grade 1 (G1), Grade 1–2 (G1-2), Grade 2 (G2) and Grade 3 (G3). Transcriptional levels of BAFF (A), APRIL (B) and BALM (C) ligands, as well as the BAFF-R (A), BCMA (B) and TACI (C) receptors were evaluated by real time PCR. Results are shown as the gene expression relative to the expression of an endogenous control (EF-1α) (mean ± SD) (Control, n = 10; G1, n = 4, G1-2, n = 7; G2, n = 11, G3, n = 9). Statistical differences between control and infected groups were analyzed with a 2-tailed Student´s t test where * P<0.05, ** P<0.01 and *** P < 0.005. A linear regression is also included (dotted line) to show the correlation between the expression of specific genes and the course of the infection. The Pearson product-moment correlation coefficients (r) are also given relative to kidney swelling grade (indicated in the plots).
Table 2.
Correlation of the expression of Ig and BAFF and APRIL subfamily ligands and receptors during the course of PKD infection.
Fig 9.
Effect of BAFF, APRIL and BALM on head kidney IgM+ B cell survival and immunoglobulin transcription.
(A) Head kidney leukocytes were incubated with BAFF (3 μg/ml), APRIL (3 μg/ml), BALM (3 μg/ml), LPS (50 μg/ml) or left unstimulated (control) for 3 days at 20°C. After this time, cells were reacted with an anti-IgM mAb and analyzed by flow cytometry. The total number of IgM+ B cells per 105 cells was measured and plotted for each individual fish under control or stimulation conditions. Each animal is represented by a broken line and the mean for each experiment is represented by a solid line (n = 9). (B, C) Head kidney leukocytes were incubated with BAFF (3 μg/ml), APRIL (3 μg/ml), BALM (3 μg/ml) or left unstimulated (control) for 24 h at 20°C. Thereafter RNA was extracted from total leukocytes and the transcription levels of total IgM (B) and total IgT (C) relative to the endogenous control gene EF-1α calculated for each sample and shown as mean + SD (n = 12). Statistical differences were evaluated by a two-tailed Student´s t test, where * p < 0.05, ** p < 0.01 and *** p < 0.005.