Challenging bovine foot skin fibroblasts with digital dermatitis treponemes: effects on global gene expression

Purpose, aims and objectives: Bovine digital dermatitis (BDD) is a painful and ulcerative, inflammatory, infectious disease of digital skin in dairy and beef cattle and is one of the most economically important causes of severe cattle lameness worldwide. A polytreponemal aetiology has been consistently implicated in BDD pathogenesis with three key phylogroups implicated globally: Treponema medium, Treponema phagedenis and Treponema pedis. This study aimed to investigate the hypothesis that the three Treponema phylogroups implement distict mechanisms of pathogenesis within cells of the bovine foot skin tissue during infection. Using a next generation RNA sequencing (RNA-Seq) approach, the dysregulation of global mRNA expression was investigated in primary bovine foot skin fibroblasts following challenge with representative strains of the three predominant BDD Treponema phylogroups (T19, T320A and T3552B respectively). Transcriptome profiles were compared to that of a commensal treponeme, Treponema ruminis strain Ru1, in attempts to identify pathogenic signatures. Results and conclusions: Here, for the first time, we implicate fibroblasts as a likely source of interleukin-8 dysregulation in BDD lesions. Despite treponemes reportedly having atypical LPS, a strong pro-inflammatory response was elicited by the BDD treponemes in a similar manner to the Gram-negative LPS control, which appeared to be mediated through interleukin-17 signalling. Along with interleukin-17, several interesting gene targets were identified which may help to inform and drive future vaccine development and therapeutic approaches; particularly those of RGS16, GRO1, MAFF and ZC3H12A, which were only stimulated by BDD treponemes. Interestingly, the three BDD treponemes were found to elicit very distinct mechanisms of pathogenesis in fibroblasts. T. phagedenis and T. pedis spirochaetes were found to upregulate several genes associated with apoptosis. Both T. medium and T. pedis spirochaetes, but notably not T. phagedenis, stimulated genes associated with actin rearrangement and a loss of cell adhesion, which may promote their persistence and deeper invasion within host tissues. The upregulation of antimicrobial peptide precursor, DEFB123, by T. phagedenis spirochaetes may present a microbial ecological advantage to all treponemes within BDD infected tissue, explaining their dominance within lesions. Interestingly, T. medium phylogroup was the least stimulatory and appeared to induce immunosuppression via the dysregulation of genes such as TSC22D3. Together with findings that the commensal, T. ruminis, significantly dysregulated over three times the number of host genes compared to each BDD treponeme; this suggests that, alike the syphilis pathogen (Treponema pallidum), BDD treponemes have evolved to become “stealth pathogens” and avoid triggering a substantial host immune/inflammatory response to promote persistence and facilitate invasion of host tissues. Overall, this study has highlighted the complexities of BDD pathogenesis; three key Treponema phylogroups appear to elicit very distinct mechanisms of pathogenesis in bovine foot skin cells. Several interesting gene targets have been identified from this study which may help to inform and drive future vaccine development and therapeutic approaches. RNA sequencing analysis was performed on RNA extracted from primary bovine foot skin fibroblast cell cultures following challenge for six hours with either control media (three experimental replicates), lipopolysaccharide from Salmonella enterica serotype Typhimurium (as an inflammatory stimulant control) (three experimental replicates), Treponema medium phylogroup strain T19 (three experimental replicates), Treponema phagedenis phylogroup strain T320A (three experimental replicates plus an extra technical replicate), Treponema pedis strain T3552B (three experimental replicates) or Treponema ruminis strain Ru1 (three experimental replicates plus an extra technical replicate). Following RNA extraction, these 20 samples underwent rRNA depletion and cDNA libraries were prepared using an Illumina NEBNext Ultra Directional RNA Library Prep Kit. Libraries undewent paired-end clustering and were subsequently sequenced on an Illumina HiSeq 4000 using version 1 chemistry, generating 2x 150 bp paired end reads. On average, we generated approximately 66 million sequenced reads per sample (range 44-102 million). FASTQ sequence files were trimmed to exclude adaptor sequences and poor quality reads using Cutadapt V1.2.1 and Sickle V1.200 respectively. Reference-based assembly was implemented using the Galaxy platform's main public server. Trimmed reads were mapped to the Bos taurus genome (UMD 3.1.1/bosTau8; Genbank assembly accession number: GCA_000003055.5) using TopHat2, with an overall average mapping rate of 86%. Mapped reads were assembled into transcripts using Cufflinks and a master transcriptome generated by Cuffmerge, using the Bos taurus genome as a reference. Differential gene expression profiles of fibroblasts (mean FPKM gene expression) were generated for each challenge group compared to the media control by implementing CuffDiff; using the master transcriptome assembly and mapped read (BAM) files for all corresponding replicates of the challenge and control group comparison. Differentially expressed genes with an adjusted p (q or FDR) value ≤ 0.05 were considered statistically significant, with a further log2 fold change cut-off (≥ 1 and ≤ -1) used to define biological significance; these criteria were used as a cut-off for SDE in all downstream analyses. Ingenuity pathway analysis was used to identify significantly enriched canonical pathways, diseases and biological functions. Technical replicates were obtained from cell monolayers seeded within different flasks from the same experimental culture.

研究背景与目的：牛数字性皮炎（Bovine digital dermatitis, BDD）是一种发生于奶牛和肉牛趾部皮肤的溃疡性、疼痛性炎症性传染病，是全球范围内导致肉牛严重跛行的最具经济重要性的病因之一。目前学界普遍认为，密螺旋体多病原病因与BDD的发病机制密切相关，全球范围内已明确三类关键系统发育群：中间密螺旋体（Treponema medium）、噬菌密螺旋体（Treponema phagedenis）和足密螺旋体（Treponema pedis）。本研究旨在验证这一假说：上述三类密螺旋体系统发育群在感染过程中，会通过截然不同的发病机制作用于牛足部皮肤组织细胞。本研究采用下一代RNA测序（next generation RNA sequencing, RNA-Seq）技术，探究了原代牛足部皮肤成纤维细胞在分别暴露于三类主要BDD相关密螺旋体系统发育群的代表性菌株（依次为T19、T320A和T3552B）后，全基因组mRNA表达的失调情况。同时将转录组谱与共生密螺旋体瘤胃密螺旋体（Treponema ruminis）菌株Ru1的转录组谱进行对比，以期鉴定出致病性特征标记。

研究结果与结论：本研究首次证实，成纤维细胞可能是BDD病灶中白细胞介素-8失调的来源。尽管此前研究表明密螺旋体的脂多糖（LPS）存在非典型性，但BDD相关密螺旋体仍可引发强烈的促炎反应，其机制与革兰氏阴性菌LPS对照相似，且该反应似乎通过白细胞介素-17信号通路介导。除白细胞介素-17外，本研究还鉴定出多个具有研究价值的基因靶点，可为未来疫苗开发与治疗策略提供参考，其中仅BDD相关密螺旋体可诱导激活的RGS16、GRO1、MAFF及ZC3H12A基因尤为值得关注。值得注意的是，三类BDD相关密螺旋体在成纤维细胞中引发的发病机制存在显著差异：噬菌密螺旋体和足密螺旋体可上调多个与细胞凋亡相关的基因；中间密螺旋体与足密螺旋体（而非噬菌密螺旋体）可激活与肌动蛋白重排及细胞黏附丧失相关的基因，这可能有助于其在宿主组织中定植并实现深层侵袭。此外，噬菌密螺旋体可上调抗菌肽前体DEFB123的表达，这或许能为BDD感染组织内的所有密螺旋体提供微生物生态优势，也解释了其在病灶中的优势地位。有趣的是，中间密螺旋体系统发育群的刺激活性最弱，其似乎通过调控TSC22D3等基因的表达来诱导免疫抑制。结合共生菌瘤胃密螺旋体相较于每株BDD相关密螺旋体，可显著调控三倍以上宿主基因的研究结果，这表明与梅毒病原体梅毒密螺旋体（Treponema pallidum）类似，BDD相关密螺旋体已进化为“隐匿病原体”，避免触发大规模宿主免疫/炎症反应，从而促进自身定植与宿主组织侵袭。总体而言，本研究揭示了BDD发病机制的复杂性：三类关键密螺旋体系统发育群在牛足部皮肤细胞中引发的发病机制存在显著差异。本研究鉴定出多个具有研究价值的基因靶点，可为未来疫苗开发与治疗策略提供参考。

RNA测序分析的实验流程如下：将原代牛足部皮肤成纤维细胞培养物分别以以下试剂处理6小时：对照培养基（3次生物学重复）、鼠伤寒沙门氏菌（Salmonella enterica serotype Typhimurium）脂多糖（作为促炎刺激对照，3次生物学重复）、中间密螺旋体菌株T19（3次生物学重复）、噬菌密螺旋体菌株T320A（3次生物学重复+1次技术重复）、足密螺旋体菌株T3552B（3次生物学重复）以及瘤胃密螺旋体菌株Ru1（3次生物学重复+1次技术重复）。RNA提取完成后，对这20个样本进行核糖体RNA（rRNA）去除，并使用Illumina NEBNext Ultra Directional RNA Library Prep Kit构建cDNA文库。文库完成双端聚类后，采用Illumina HiSeq 4000测序平台及v1测序化学试剂进行测序，生成2×150 bp的双端读长序列。每个样本平均生成约6600万条测序读长（范围为4400万至1.02亿条）。使用Cutadapt V1.2.1和Sickle V1.200分别对FASTQ格式的序列文件进行修剪，以去除接头序列与低质量读长。基于参考基因组的组装工作在Galaxy平台的公共主服务器上完成：将修剪后的读长序列比对至牛（Bos taurus）参考基因组（UMD 3.1.1/bosTau8；Genbank组装登录号：GCA_000003055.5），比对工具为TopHat2，整体平均比对率为86%。使用Cufflinks将比对后的读长组装为转录本，并以牛参考基因组为参照，通过Cuffmerge生成主转录组。采用Cuffdiff工具，以主转录组组装结果及对应处理组与对照组的所有重复样本的比对读长（BAM）文件为基础，生成各处理组相较于培养基对照组的成纤维细胞差异基因表达谱（以平均FPKM基因表达量计）。将校正后p值（q值或错误发现率FDR）≤0.05的差异表达基因视为具有统计学显著性，并进一步设置log2倍变化阈值（≥1且≤-1）以定义生物学显著性；该阈值标准用于所有后续分析中的显著差异表达基因（SDE）筛选。采用Ingenuity通路分析工具鉴定显著富集的经典通路、疾病及生物学功能。本研究的技术重复样本取自同一实验培养物中接种于不同培养瓶的细胞单层。