Mitochondrial genome variation affects humoral and cell-mediated innate immune responses and infection outcomes

The role of mitochondria in both adaptive and innate immune responses is increasingly recognized, but the role of natural mitochondrial DNA (mtDNA) variation as an immunomodulatory factor has received less attention. One reason for this is the difficulty of separating the effect of mtDNA from that of the nuclear genome. By utilizing the fruit fly Drosophila melanogaster, a powerful model system, we created cytoplasmic hybrids, aka. cybrid lines, where unique mtDNAs (mitotypes) were introgressed into a controlled isogenic nuclear background. We harnessed a panel of cybrid lines to study the effect of mtDNA variation on humoral and cell-mediated innate immune responses. Mitotypes exhibited heterogeneity in infection outcomes upon bacterial, viral and parasitoid infections. One mitotype of note, mtKSA2 appeared to be more immunocompetent when compared to other mitotypes. We performed transcriptomic profiling of uninfected flies and flies infected with bacteria or a virus and compared them to a different mitotype in the same nuclear background to find the mechanistic basis of the immunocompetence. We found that mtKSA2 caused an upregulation of TCA and OXPHOS related genes and a downregulation of a multiple genes encoding for antimicrobial peptides in uninfected flies. Upon infection, mtKSA2 flies produced unique transcriptomes that were separated by infection type and duration. When we examined immune cells (hemocytes) in mtKSA2 larvae, we noted an increase in hemocyte numbers. These hemocytes were activated in the absence of infection, increased their production of ROS, and showed evidence of increased encapsulation efficiency upon parasitoid wasp infection. Overall, our results show that mtDNA variation acts as an immunomodulatory factor in both humoral and cell-mediated innate immunity and that specific mitotypes provide enhanced protection against infections. 1-4 days old, mated females and males were infected with Staphylococcus aureus (PIG1) or Providencia rettgeri (DSM4542) by pricking the flies with a tungsten needle dipped in bacterial solution (OD600=0.1). Samples were collected 8 and 20 hours after infecting the flies. Systemic infection with DCV was introduced to 2-5 days old, mated females and males by pricking as described with the bacterial infections. DCV inoculum of 10^8 viral particles was used. Samples were collected 3 days after the infection with DCV. As controls, uninfected flies of similar age were collected.

线粒体在适应性免疫与固有免疫应答中的作用已得到日益广泛的认可，但天然线粒体DNA（mtDNA）变异作为免疫调节因子的相关研究却未受到足够关注。究其根源，主要在于难以区分mtDNA与核基因组各自的调控效应。本研究借助经典模式生物黑腹果蝇（Drosophila melanogaster）构建了胞质杂种（cybrid）品系，将独特的mtDNA（mitotypes，线粒体型）导入经严格控制的同基因核背景中。我们利用一组该类胞质杂种品系，探究mtDNA变异对体液免疫与细胞介导固有免疫的调控作用。不同线粒体型在细菌、病毒及寄生蜂感染后的感染结局中呈现显著异质性。其中值得关注的线粒体型mtKSA2，其免疫活性相较于其他线粒体型更为突出。我们对未感染果蝇以及感染细菌或病毒的果蝇开展转录组分析，并将其与同一核背景下的另一线粒体型果蝇进行比对，以解析该免疫活性的分子机制。研究发现，在未感染状态下，mtKSA2可上调三羧酸循环（TCA）与氧化磷酸化（OXPHOS）相关基因的表达，并下调多个编码抗菌肽的基因。感染后，mtKSA2果蝇的转录组特征呈现独特性，且可依据感染类型与感染时长进行区分。我们对mtKSA2幼虫的免疫细胞（hemocytes，血淋巴细胞）进行检测时，发现其血细胞数量显著增加。这些血细胞在未感染条件下即已被激活，活性氧（ROS）的产生量有所提升，且在寄生蜂感染后表现出更高的包囊效率。综上，本研究结果表明，mtDNA变异可作为体液免疫与细胞介导固有免疫的调节因子，且特定线粒体型可增强宿主对感染的防御能力。实验选取1-4日龄已交配的雌雄果蝇，使用蘸有细菌溶液（OD600=0.1）的钨针针刺接种，以感染金黄色葡萄球菌（Staphylococcus aureus，PIG1）或雷氏普罗威登斯菌（Providencia rettgeri，DSM4542），并分别于感染后8小时与20小时采集样本。参照上述细菌感染的针刺接种方式，对2-5日龄已交配的雌雄果蝇进行果蝇C病毒（DCV）全身感染，病毒接种量为10^8个病毒颗粒，并于感染后3天采集样本。对照组选取相同日龄的未感染果蝇。