大弹涂鱼白细胞介素-34（IL-34）通过集落刺激因子-1受体1 (CSF-1R1) 调节单核/巨噬细胞功能的研究
Mudskipper interleukin-34 modulates the functions of monocytes/macrophages via the colony-stimulating factor-1 receptor 1
新型细胞因子白细胞介素-34（interleukin-34，IL-34）通过与集落刺激因子-1受体（the colony-stimulating factor-1 receptor，CSF-1R）结合，在先天免疫和炎症中发挥重要作用。目前，关于鱼类IL-34功能的研究还非常有限。本研究鉴定了大弹涂鱼（Boleophthalmus pectinirostris）的IL-34（BpIL-34），在序列和结构与其它已知的IL-34相似，与石斑鱼（Epinephelus coioides）的同源关系最近。mRNA水平的研究发现，BpIL-34能组成型表达于大弹涂鱼的各个组织，以大脑表达水平最高；迟缓爱德华氏菌（Edwardsiella tarda）感染能上调各组织的表达。本研究表达并纯化了BpIL-34的成熟肽（rBpIL-34），制备了抗体。肽N-糖苷酶F酶解发现单核/巨噬细胞的BpIL-34被N-糖基化。体外实验发现rBpIL-34能够增强单核/巨噬细胞的吞噬和杀菌活性，促进肿瘤坏死因子α（BpTNF-α）和BpIL-1β等促炎细胞因子的表达。RNA干扰实验结果表明，敲低大弹涂鱼CSF-1R1（BpCSF-1R1）后，rBpIL-34介导的单核/巨噬细胞功能的强化表现被显著抑制；BpCSF-1R2不具备此功能。综上，BpIL-34能够通过BpCSF-1R1调节大弹涂鱼单核/巨噬细胞的功能。Abstract:
Interleukin-34 (IL-34) is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colony-stimulating factor-1 receptor (CSF-1R). However, information on the function of IL-34 in fish remains limited. In the present study, we identified an IL-34 homolog from mudskippers (Boleophthalmus pectinirostris). In silico analysis showed that the mudskipper IL-34 (BpIL-34) was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper (Epinephelus coioides) homolog. BpIL-34 transcripts were constitutively expressed in various tissues, with the highest level of expression found in the brain. Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues. The recombinant mature BpIL-34 peptide (rBpIL-34) was purified and used to produce anti-rBpIL-34 IgG. Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages (MOs/MФs) was N-glycosylated. In vitro, rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs, as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factor α (BpTNF-α) and BpIL-1β in these cells. Furthermore, the knockdown of mudskipper CSF-1R1 (BpCSF-1R1), but not mudskipper BpCSF-1R2, significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФ function. In conclusion, our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.
Figure 1. Multiple alignment of amino acid sequences of BpIL-34 and related fish IL-34 sequences
Threshold for shading was >60%; similar residues are marked with a gray shadow, identical residues with a black shadow, and alignment gaps with “-”. BpIL-34: Mudskipper IL-34; DrIL-34: Zebrafish IL-34; OmIL-34: Rainbow trout IL-34; SaIL-34: Gilthead seabream IL-34; TrIL-34: Tiger puffer IL-34; SsIL-34: Atlantic salmon IL-34; IpIL-34: Channel catfish IL-34; CcIL-34: Common carp IL-34; OlIL-34: Japanese rice fish IL-34; EcIL-34: Orange-spotted grouper IL-34. Predicted cleavage site for signal peptide is marked as “↓”. Five conserved cysteine residues are marked as “*”. Two cysteine residues joined by solid line represent disulfide bond. Possible N-linked glycosylation sites of BpIL-34 are indicated by “▼”. RKx [R/K] K motif is double underlined. GenBank accession Nos. of sequences used are presented in Table 1.
Figure 2. Phylogenetic tree analysis of complete amino acid sequences of IL-34 and CSF-1 using neighbor-joining method
CSF-1 sequences as an outgroup. Percentage of bootstrap values is shown next to branches based on 1 000 bootstrap replications (shown only when >60%). Scale bar represents number of substitutions per base position. Site of mudskipper IL-34 is indicated by “▲”. GenBank accession Nos. of sequences used are listed in Table 1.
Figure 3. qRT-PCR analysis of BpIL-34 mRNA expression in tissues of healthy (A) and Edwardsiella tarda-infected mudskippers (B–E)
A: BpIL-34 mRNA expression relative to that of Bp18S rRNA was calculated using 2−ΔCT method. Values denoted by different letters are significantly different when compared by ANOVA (P<0.05); n=4. B–E: BpIL-34 mRNA expression relative to that of Bp18S rRNA was calculated using 2−ΔΔCT method. Tissues were collected at 4, 8, 12, and 24 h after bacterial infection. Data are expressed as means±SEM; n=4, *: P<0.05.
Figure 4. Prokaryotic expression and Western blot analysis of BpIL-34
A: SDS-PAGE analysis of prokaryotically expressed rBpIL-34. Lane M: Protein marker; 1, 2: Crude protein extracts from BL21 (DE3) transformed with pET-28a-BpIL-34 plasmid before and after IPTG induction, respectively; 3: Purified rBpIL-34. B: Western blot analysis of BpIL-34 using anti-rBpIL-34 IgG. NC: Negative control; 4: Purified rBpIL-34; 5: Proteins extracted from mudskipper MOs/MФs; 6: Proteins extracted from mudskipper MOs/MФs with PNGase F digestion.
Figure 5. Effects of rBpIL-34 on phagocytosis and bacterial killing of Edwardsiella tarda by mudskipper MO/MΦs
A: Effect of rBpIL-34 on phagocytosis of E. tarda by MOs/MΦs. Mudskipper MOs/MΦs were pre-treated with PBS or rBpIL-34 before adding FITC-E. tarda (MOI=10). After an additional 30 min incubation, phagocytosis of FITC-E. tarda was determined by flow cytometry. MFI is presented as fold-change over the value for PBS-treated group, which was assigned a unit of 100. B: Effect of rBpIL-34 on bacterial killing of E. tarda by MOs/MΦs. MOs/MΦs were infected with live E. tarda after treatment with PBS or rBpIL-34, and viability of E. tarda (MOI=10) was determined by qRT-PCR. Killing of E. tarda by mudskipper MOs/MФs was measured using CFU assay based on a standard curve. Data are expressed as means±SEM; n=4, *: P<0.05.
Figure 6. Effects of rBpIL-34 on mRNA expression of BpTNF-α (A), BpIL-1β (B), BpIL-6 (C), BpTGF-β (D), and BpIL-10 (E) in mudskipper MOs/MΦs
MOs/MΦs were treated with rBpIL-34 for 12 h. PBS-treated group was used as the control. mRNA expression levels of selected cytokines were normalized to those of Bp18S rRNA. Data are expressed as means±SEM; n=4, *: P<0.05.
Figure 7. RNAi-mediated gene knockdown of BpCSF-1R1 and BpCSF-1R2 in mudskipper MOs/MФs
A: qRT-PCR analysis of mRNA expression of BpCSF-1R1 and BpCSF-1R2 in primary MOs/MФs. B: qRT-PCR analysis of BpCSF-1R1 transcripts in BpCSF-1R1si- or BpCSF-1R2si-transfected MOs/MФs. C: qRT-PCR analysis of BpCSF-1R2 transcripts in BpCSF-1R1si- or BpCSF-1R2si-transfected MOs/MФs. MsiRNA-transfected MOs/MФs group was used as the control. mRNA levels of BpCSF-1R1 and BpCSF-1R2 were normalized to those of Bp18S rRNA. Data are expressed as means±SEM; n=4, *: P<0.05.
Figure 8. Effects of BpCSF-1R1 and BpCSF-1R2 knockdown on rBpIL-34-enhanced phagocytosis and bacterial killing of Edwardsiella tarda by mudskipper MOs/MΦs
A: Effect of BpCSF-1R1 or BpCSF-1R2 knockdown on rBpIL-34-enhanced phagocytosis of E. tarda by mudskipper MOs/MΦs. After transfection with BpCSF-1R1si, BpCSF-1R2si, or MsiRNA (control) for 48 h, mudskipper MOs/MФs were treated with rBpIL-34 for 12 h. Normal MOs/MΦs treated with rBpIL-34 (rBpIL-34 group) were used as controls. Thereafter, FITC-E. tarda were added at a MOI of 10 and incubated for an additional 30 min. Phagocytosis of FITC-E. tarda was determined by flow cytometry. Mean fluorescence intensity (MFI) is presented as a fold-change over value for rBpIL-34 group, which was assigned a value of 100. B: Effect of BpCSF-1R1 and BpCSF-1R2 knockdown on rBpIL-34-enhanced bacterial killing of E. tarda by mudskipper MOs/MΦs. After transfection with BpCSF-1R1si, BpCSF-1R2si, or MsiRNA for 48 h, mudskipper MOs/MФs were treated with rBpIL-34 for 12 h. Normal MOs/MΦs treated with rBpIL-34 (rBpIL-34 group) were used as controls. Live E. tarda were added at a MOI of 10 and incubated for an additional 30 min. Killing of E. tarda by mudskipper MOs/MФs was measured using a CFU assay based on standard curve. Data are expressed as means±SEM; n=4, *: P<0.05.
Figure 9. Effects of BpCSF-1R1 and BpCSF-1R2 knockdown on rBpIL-34-altered mRNA expression of selected cytokines in MOs/MФs
Mudskipper MOs/MФs were pre-treated with BpCSF-1R1si, BpCSF-1R2si, or MsiRNA (control) and further incubated with rBpIL-34 for 12 h. MsiRNA-treated group was used as the control. mRNA levels of BpTNF-α (A), BpIL-1β (B), BpIL-6 (C), BpTGF-β (D), and BpIL-10 (E) were normalized to those of Bp18S rRNA. Data are expressed as means±SEM; n=4, *: P<0.05.
Table 1. IL-34 and CSF-1 sequences used for multiple sequence alignment and phylogenetic tree analysis
GenBank accession No. Species Gene Latin name English name XM_020935293 Boleophthalmus pectinirostris Mudskipper IL-34 NM_001128701 Danio rerio Zebrafish IL-34 KM350155 Epinephelus coioides Orange-spotted grouper IL-34 NM_001257301 Oncorhynchus mykiss Rainbow trout IL-34 XM_030427145 Sparus aurata Gilthead seabream IL-34 NM_001305607 Takifugu rubripes Tiger puffer IL-34 XM_020104586 Paralichthys olivaceus Japanese flounder IL-34 XM_019360775 Oreochromis niloticus Nile tilapia IL-34 XM_010743578 Larimichthys crocea Large yellow croaker IL-34 XM_014124241 Salmo salar Atlantic salmon IL-34 XM_017465109 Ictalurus punctatus Channel catfish IL-34 XM_019087026 Cyprinus carpio Common carp IL-34 XM_011476357 Oryzias latipes Japanese rice fish IL-34 XM_027161223 Tachysurus fulvidraco Yellow catfish IL-34 XM_013138989 Esox lucius Northern pike IL-34 XM_026287434 Carassius auratus Goldfish IL-34 XM_007249177 Astyanax mexicanus Mexican tetra IL-34 MK297321 Ctenopharyngodon idella Grass carp IL-34 XM_020456685 Oncorhynchus kisutch Coho salmon IL-34 XM_024802984 Maylandia zebra Zebra mbuna IL-34 NM_152456 Homo sapiens Human IL-34 NM_001135100 Mus musculus Mouse IL-34 NM_001025766 Rattus norvegicus Rat IL-34 NM_001285975 Sus scrofa Pig IL-34 XM_022419217 Canis lupus familiaris Dog IL-34 XM_023637306 Equus caballus Horse IL-34 XM_018260639 Xenopus laevis African clawed frog IL-34 XM_003641892 Gallus gallus Chicken IL-34 XM_019535589 Crocodylus porosus Crocodile IL-34 NM_001100324 Bos taurus Cattle IL-34 NM_001114480 Danio rerio Zebrafish CSF-1 NM_001080076 Danio rerio Zebrafish CSF-1-2 NM_001124394 Oncorhynchus mykiss Rainbow trout CSF-1 NM_001160476 Oncorhynchus mykiss Rainbow trout CSF-1-2 KM350156 Epinephelus coioides Orange-spotted grouper CSF-1-2 NM_001280600 Xenopus laevis African clawed frog CSF-1 XM_017000369 Homo sapiens Human CSF-1 XM_024984551 Bos taurus Cattle CSF-1 XM_008761428 Rattus norvegicus Rat CSF-1 NM_001113530 Mus musculus Mouse CSF-1 NM_001193295 Gallus gallus Chicken CSF-1
Table 2. Oligonucleotide primers used in the qRT-PCR analysis of mudskipper genes
Gene GenBank accession No. Primer Nucleotide sequence (5′–3′) BpIL-34 XM_020935293 BpIL-34RF GCAGGAGCTTCCAGAGTCAG BpIL-34RR CCTCCAATGGGACCTGTCAC BpIL-1β KX492895 BpIL-1βF ACGAGTGGTGAATGTGGTCA BpIL-1βR GAACTGAGGTTGTGCTGCAA BpTNF-α KX492896 BpTNF-αF GGACAACAACGAGATCGTGA BpTNF-αR GTTCCACCGTGTGACTGATG BpIL-6 XM_020932674 BpIL-6F GCAGCACGTCAGAAGATGAGA BpIL-6R TCTCTGAGAAACTCGTGCAGC BpTGF-β XM_020928521 BpTGF-βF TCAAAGGACACTTGCACAGC BpTGF-βR CAGGGCCAAGATCTGTGAAT BpIL-10 XM_020936977 BpIL-10F GTGGAGGGGTTCCCTCTAAG BpIL-10R GTGCGGAGGTAAAAGCTCAG Bp18S rRNA KX492897 Bp18SF GGCCGTTCTTAGTTGGTGGA Bp18SR CCCGGACATCTAAGGGCATC BpCSF-1R1 XM_020921963 BpCSF-1R1F GGCCCACGTGTAAGGAGAAT BpCSF-1R1R TACTCCTCCCTCTGCACCTC BpCSF-1R2 XM_020941064 BpCSF-1R2F GTCTTGAGGTTGGACTCGGG BpCSF-1R2R CTCACACCTGTCGGTGAGTC
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