Pregnancy Downregulates Plasmablast Metabolic Gene Expression Following Influenza without Altering Long-Term Antibody Function. Pregnancy Downregulates Plasmablast Metabolic Gene Expression Following Influenza without Altering Long-Term Antibody Function

The normal response to influenza A infection is to remain asymptomatic; indeed, a study of volunteers who tested positive for antibodies against 2009 H1N1 revealed that a majority did not experience symptoms. However, studies of influenza pandemics since the 20th century, and to a lesser degree seasonal flu, suggest that pregnancy increases influenza-associated morbidity and mortality. The confounding effect of pregnancy has been well established to adversely alter the clinical course of influenza, with short term complications to mother and baby apparent, and long-term consequences to the fetus hypothesized. However, how the condition of pregnancy impacts long-term maternal anti-influenza immunity remains contentious; while vaccination studies suggest humoral responses sufficient for protection, published research on adaptive immune responses against influenza formed during pregnancy is limited. This study expands upon published work from our lab and others by comprehensively examining compartmental and systemic changes following pandemic A/California/07/2009 influenza infection. Previously, we linked seasonal influenza infection to clinical observations of adverse outcomes in pregnancy, enhanced lung and placental histopathology, and reduced control of viral replication in lungs of infected pregnant mothers. Here, we observe that the results of a lower infectious dose of pandemic influenza are similar to that of seasonal influenza. Importantly, we expand upon work which suggests infection during pregnancy may also affect humoral immunity. Our observations of reduced early antibody hemagglutinin inhibition and virus neutralization which resolved within 8 weeks of delivery demonstrate that influenza infection impacts antibody maturation mechanisms without alterations to B cell frequency or antibody secretion. This hypothesis is further supported by plasmablast transcriptional data, which demonstrates downregulated B cell metabolism and post-translational modification systems only among pregnant animals. These findings corroborate a link between adverse pregnancy outcomes and severe pathology observed during influenza infection, and propose resolving humoral deficiencies following influenza infection confounded by pregnancy. Additional studies are required to specify the involvement of plasmablast metabolism with early humoral immunity abnormalities to best guide vaccination strategies and improve our understanding of the immunological consequences of pregnancy. Overall design: On day 12 of pregnancy (determined by 20% weight gain and/or presence of a copulation plug), pregnant and non-pregnant BALB/c mice were lightly anesthetized with isoflurane and infected intranasally with 30 µl 0.5xLD50 of mouse adapted A/California/07/2009, approximately 30 plaque forming units (p.f.u.). 10,000 plasmablasts (CD138+B220int) were sorted from the spleens of pregnant and non-pregnant influenza virus infected mice at 10 days post infection. Single-end RNA sequencing was performed on samples in duplicate to increase sequencing depth (101 base pair reads). Sequencing was checked for quality using FastQC (Barbaham Institute, United Kingdom). Processing of single end reads was performed within the R programming language. The Rsubread package align function (Liao et al., 2019) was used to align single end reads to the Mus musculus genome (GRCm38.93). BAM files of duplicate samples were merged prior to the counting of reads for each gene using the featureCounts function of the Rsubread package. Differential gene expression analysis was performed in R using the DESeq2 package (Love et al., 2014). Gene set enrichment analysis (Subramanian et al., 2005) was performed as previously described in pre-ranked mode with the clusterProfiler package (Yu et al., 2012). RNA sequencing was performed by the Yerkes National Primate Research Center, Non-human Primate Genomics Core.

甲型流感病毒（influenza A virus）感染的常见转归为无症状状态；事实上，一项针对2009年H1N1流感抗体阳性志愿者的研究显示，多数受试者未出现相关临床症状。然而，针对20世纪以来流感大流行以及程度较轻的季节性流感的相关研究表明，妊娠会升高流感相关的发病率与死亡率。妊娠带来的混杂效应已被证实会对流感的临床进程造成不良影响，可导致母婴出现短期并发症，且有假说提出其会对胎儿造成长期不良后果。不过，妊娠状态如何影响母体长期抗流感免疫能力这一问题仍存在争议；尽管疫苗研究提示可产生足以提供保护的体液免疫应答，但已发表的关于妊娠期间诱导形成的抗流感适应性免疫应答的研究仍较为有限。

本研究在本实验室及其他团队已发表工作的基础上，对大流行毒株A/California/07/2009感染后的免疫分区与全身免疫变化进行了全面分析。此前本团队已将季节性流感感染与妊娠不良临床结局、肺及胎盘组织病理损伤加重、感染妊娠小鼠肺内病毒复制调控能力减弱等现象建立关联。本研究观察到，低感染剂量的大流行流感病毒感染的结局与季节性流感相似。值得注意的是，本研究进一步拓展了关于妊娠期间感染可能影响体液免疫的相关研究：我们观察到，分娩后8周内即可恢复的早期抗体血凝抑制活性与病毒中和能力出现下降，这表明流感感染会影响抗体成熟机制，但并未改变B细胞频率或抗体分泌水平。浆母细胞（plasmablast）转录组数据进一步支持了这一假说——仅在妊娠小鼠体内，B细胞代谢与翻译后修饰系统呈现下调状态。

本研究结果证实了妊娠不良结局与流感感染期间严重病理损伤之间的关联，并提出妊娠相关的流感感染后体液免疫缺陷仍有待解决。未来仍需开展更多研究，明确浆母细胞代谢与早期体液免疫异常之间的关联，以优化疫苗接种策略，并加深我们对妊娠相关免疫后果的理解。

实验整体设计：
于妊娠第12天（通过体重增加20%和/或存在交配栓确认妊娠状态），将妊娠与非妊娠BALB/c小鼠用异氟烷（isoflurane）轻度麻醉，经鼻感染30 μl 0.5倍半数致死量（0.5×LD50）的小鼠适应性A/California/07/2009毒株，感染剂量约为30噬斑形成单位（plaque forming units, p.f.u.）。于感染后10天，从妊娠与非妊娠流感病毒感染小鼠的脾脏中分选10000个浆母细胞（CD138+B220int）。对样本进行双份单端RNA测序（RNA sequencing）以提升测序深度，读长为101碱基对。使用FastQC（英国巴布拉汉研究所）对测序数据进行质量质控。单端读段的数据分析在R编程语言环境中完成：采用Rsubread包的align函数（Liao等，2019）将读段比对至小鼠基因组（GRCm38.93）；将重复样本的BAM文件合并后，使用Rsubread包的featureCounts函数统计每个基因的读段计数。采用DESeq2包（Love等，2014）在R中完成差异基因表达分析。基因集富集分析（Subramanian等，2005）采用此前报道的预排序模式，通过clusterProfiler包（Yu等，2012）完成。RNA测序工作由耶基斯国家灵长类研究中心非人灵长类基因组核心实验室完成。