Pani Saswati, Ganguly Bristy, Mahapatra Smruti, Dash Smruti Prajnya, Das Rakesh, Saha Ashis, Samanta Mrinal
Immunology Laboratory, Fish Health Management Division (FHMD), Indian Council of Agricultural Research - Central Institute of Freshwater Aquaculture (ICAR-CIFA), Bhubaneswar, Odisha, India.
Reproductive Physiology and Endocrinology Laboratory, Fish Nutrition and Physiology Division (FNPD), Indian Council of Agricultural Research - Central Institute of Freshwater Aquaculture (ICAR-CIFA), Bhubaneswar, Odisha, India.
Front Immunol. 2025 Apr 25;16:1555048. doi: 10.3389/fimmu.2025.1555048. eCollection 2025.
Toll-like receptors (TLRs) play a vital role in the immune response by recognizing pathogen-associated molecular patterns (PAMPs) and triggering signaling pathways that activate innate immunity. In bony fish, TLR7 is essential for both antiviral and antibacterial defense; however, its interactions with a wide range of ligands and pathogens are still not well understood across various fish species. This study focuses on the identification and characterization of TLR7 in (LrTLR7) and aims to evaluate its response to pathogen challenges and stimulation by PAMPs.
To clone the TLR7 gene, RNA was extracted from kidney tissue using a standard protocol, followed by cDNA synthesis with commercial kits. The TLR7 gene was amplified by PCR, and the gel-purified product was cloned into the pGEM-T Easy vector. DNA sequencing and BLAST analysis confirmed the identity of the LrTLR7 gene. The ORF of LrTLR7 cDNA was predicted using ORF-finder, while structural motifs in the encoded protein were identified through SMART. Phylogenetic relationships were analyzed using MEGA7 to construct evolutionary trees. Gene expression profiles of LrTLR7 were evaluated by quantitative real-time PCR (qRT-PCR) across developmental stages, tissues/organs of rohu fingerlings, and during challenges with and infections, as well as LPS and Poly I:C stimulation. Mucosal RBCs and PBLs were isolated using density-gradient centrifugation with HiSep™ LSM 1077 (Himedia, India). Cultured gill (LRG) cells in Leibovitz's L-15 medium were infected with or at a multiplicity of infection (MOI) of 1, following established protocols.
LrTLR7 showed the closest phylogenetic affinity to TLR7 in . During embryonic development, LrTLR7 expression surged dramatically (111-fold, <0.05) in embryos at 120 h post-fertilization (hpf). In juveniles, the gene was ubiquitously expressed across tissues/organs, with peak expression in gills (2,000-fold). Following infection with or , LrTLR7 gene transcripts in the liver increased sharply at 6 hpi (93-fold and ~53,000-fold, respectively). In the infected fish, mucosal RBCs showed a ~500,000-fold upregulation (<0.05), while PBLs exhibited maximal responses at 24 hpi (5,000-fold for and ~10 million-fold for ). In the LRG cell line, LrTLR7 gene expression rose ~30-fold by 3 hpi. during infection. stimulation with LPS or poly I:C triggered a ~30,000-fold increase in hepatic LrTLR7 expression at 12 h post-stimulation, with kidney tissue showing secondary activation. Mucosal RBCs and PBLs displayed rapid (1-3 h) LrTLR7 upregulation following ligand exposure. Imiquimod and gardiquimod activated LrTLR7-signalling pathways in both and systems, elevating transcription of IRF7 and type I interferon genes.
Similar to higher vertebrates, LrTLR7 plays a crucial role in responding to pathogenic invasions and various PAMPs to induce innate immunity. Consequently, TLR7 in fish represents a significant target for immune activation using specific agonists or ligands, which could aid in the prevention of fish diseases.
Toll样受体（TLRs）通过识别病原体相关分子模式（PAMPs）并触发激活先天免疫的信号通路，在免疫反应中发挥至关重要的作用。在硬骨鱼中，TLR7对于抗病毒和抗菌防御均至关重要；然而，在不同鱼类物种中，其与多种配体和病原体的相互作用仍未得到充分了解。本研究聚焦于尼罗罗非鱼（LrTLR7）中TLR7的鉴定与表征，旨在评估其对病原体攻击和PAMPs刺激的反应。
为克隆TLR7基因，采用标准方案从尼罗罗非鱼肾脏组织中提取RNA，随后使用商业试剂盒进行cDNA合成。通过PCR扩增TLR7基因，将凝胶纯化产物克隆至pGEM-T Easy载体。DNA测序和BLAST分析确认了LrTLR7基因的身份。使用ORF-finder预测LrTLR7 cDNA的开放阅读框（ORF），同时通过SMART鉴定编码蛋白中的结构基序。使用MEGA7分析系统发育关系以构建进化树。通过定量实时PCR（qRT-PCR）评估LrTLR7在尼罗罗非鱼幼鱼发育阶段、组织/器官以及感染嗜水气单胞菌和无乳链球菌期间，以及LPS和Poly I:C刺激下的基因表达谱。使用HiSep™ LSM 1077（印度Himedia公司）通过密度梯度离心法分离黏膜红细胞（RBCs）和外周血淋巴细胞（PBLs）。按照既定方案，将莱博维茨L-15培养基中培养的尼罗罗非鱼鳃（LRG）细胞以感染复数（MOI）为1感染嗜水气单胞菌或无乳链球菌。
LrTLR7在系统发育上与虹鳟鱼中的TLR7亲缘关系最近。在胚胎发育过程中，受精后120小时（hpf）的胚胎中LrTLR7表达急剧上升（约111倍，<0.05）。在尼罗罗非鱼幼鱼中，该基因在各组织/器官中广泛表达，在鳃中表达量最高（约2000倍）。感染嗜水气单胞菌或无乳链球菌后，肝脏中LrTLR7基因转录本在感染后6小时急剧增加（分别约为93倍和53000倍）。在受感染的鱼中，黏膜RBCs上调约500000倍（<0.05），而PBLs在感染后2小时表现出最大反应（嗜水气单胞菌感染约为5000倍，无乳链球菌感染约为10000000倍）。在LRG细胞系中，感染嗜水气单胞菌后3小时LrTLR7基因表达上升约30倍。LPS或Poly I:C刺激后12小时，肝脏中LrTLR7表达增加约30000倍，肾脏组织出现二次激活。暴露于配体后，黏膜RBCs和PBLs中LrTLR7迅速上调（1 - 3小时）。咪喹莫特和加地喹莫特在体外和体内系统中均激活LrTLR7信号通路，提高IRF7和I型干扰素基因的转录水平。
与高等脊椎动物类似，LrTLR7在应对病原体入侵和各种PAMPs以诱导先天免疫方面发挥关键作用。因此，鱼类中的TLR7是使用特定激动剂或配体进行免疫激活的重要靶点，这有助于预防鱼类疾病。
