[论文翻译]适度规律的体育锻炼有助于缓解血吸虫感染对大脑认知功能的系统性影响

由 发布于 血吸虫胶质细胞免疫细胞
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Moderate regular physical exercise can help in alleviating the systemic impact of schistosomiasis infection on brain cognitive function

适度规律的体育锻炼有助于缓解血吸虫感染对大脑认知功能的系统性影响

Inssaf Berkiks 1,2*, Nada Abdel Aziz 1,2,3, Blessing Moses 1,2, Tiroyaone Brombacher 1,2† and Frank Brombacher 1,2†

Inssaf Berkiks 1,2*、Nada Abdel Aziz 1,2,3、Blessing Moses 1,2、Tiroyaone Brombacher 1,2† 和 Frank Brombacher 1,2†

1Cytokines and Diseases Group, International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Division of Immunology, Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa, 2Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Diseases and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa, 3 Biotechnology Department, Faculty of Science, Cairo University, Cairo, Egypt

1细胞因子与疾病研究组，国际遗传工程与生物技术中心开普敦分部，健康科学学院传染病与分子医学研究所免疫学部，开普敦大学，南非开普敦，2非洲传染病研究威康中心，健康科学学院传染病与分子医学研究所(IDM)，开普敦大学，南非开普敦，3生物技术系，理学院，开罗大学，埃及开罗

One of the major consequences of schistosomiasis is its impact on brain function, and despite its severity, the underlying mechanism(s) remain inadequately understood, highlighting a knowledge gap in the disease. The symptoms can vary from headaches to profound cognitive impairment. Besides, the potential influence of physical exercise in mitigating cognitive deficits has received little attention. In our study, we utilized a murine model of Sch is to soma mansoni infection to investigate the cognitive impact of schistosomiasis. Our aims were multifaceted: to pinpoint the specific cognitive domains affected during the infection in adult mice, to unravel the complex interplay between glial and immune cells within the central nervous system (CNS), and crucially, to explore the potential therapeutic role of regular physical exercise in counteracting the deleterious effects of schistosomiasis on the CNS. Our findings unveiled that while acute infection did not disrupt simple and complex learning or spatial reference memory, it did induce significant deficits in recall memory—a critical aspect of cognitive function. Furthermore, our investigation unearthed profound alterations in the immune and glial cell populations within the CNS. Notably, we observed marked changes in $\mathsf{C D4^{+}}$ T cells and eosinophils in the meninges, as well as alterations in glial cell dynamics within the hippocampus and other brain regions. These alterations were characterized by heightened microglial activation, diminished astrocyte reactivity and a shift towards a pro inflammatory milieu within the CNS. We also provided insights into the transformative potential of regular moderate physical exercise in partially alleviating cognitive and neuro inflammatory consequences of schistosomiasis. Remarkably, exercise decreased glial cell production of TNFa, suggesting a shift towards a less proinflammatory environment. Collectively, our study provided compelling evidence of the intricate interplay between schistosomiasis infection and cognitive function, underscoring the critical need for further exploration in this area. Furthermore, our findings demonstrated the positive effects of physical activities on mitigating the cognitive burden of schistosomiasis, offering new hope for patients afflicted by this debilitating disease.

血吸虫病的主要后果之一是对大脑功能的影响，尽管病情严重，但其潜在机制仍未得到充分理解，这凸显了该疾病领域的知识空白。症状可能从头痛到严重认知障碍不等。此外，体育锻炼在缓解认知缺陷方面的潜在影响很少受到关注。在我们的研究中，我们利用曼氏血吸虫感染的小鼠模型来研究血吸虫病的认知影响。我们的目标是多方面的：确定成年小鼠感染期间受影响的具体认知领域，揭示中枢神经系统(CNS)中胶质细胞和免疫细胞之间复杂的相互作用，并关键地探索定期体育锻炼在抵消血吸虫病对中枢神经系统有害影响方面的潜在治疗作用。

我们的研究结果表明，虽然急性感染不会破坏简单和复杂的学习或空间参考记忆，但它确实会导致回忆记忆（认知功能的一个关键方面）的显著缺陷。此外，我们的研究发现了中枢神经系统中免疫细胞和胶质细胞群的深刻变化。值得注意的是，我们观察到脑膜中$\mathsf{C D4^{+}}$T细胞和嗜酸性粒细胞的显著变化，以及海马体和其他大脑区域中胶质细胞动力学的改变。这些变化的特点是：小胶质细胞激活增强，星形胶质细胞反应性降低，以及中枢神经系统向促炎环境的转变。

我们还深入探讨了定期适度体育锻炼在部分缓解血吸虫病认知和神经炎症后果方面的转化潜力。值得注意的是，运动降低了胶质细胞TNFa的产生，表明环境向促炎性较低的方向转变。总的来说，我们的研究提供了强有力的证据，证明了血吸虫感染与认知功能之间复杂的相互作用，并强调了在这一领域进一步探索的迫切需要。此外，我们的研究结果表明，体育活动在减轻血吸虫病认知负担方面具有积极作用，为受这种衰弱性疾病困扰的患者带来了新的希望。

KEYWORDS s histos omi as is, morris water maze, neuro infection, physical activities, Sch is to soma mansoni

关键词：组织学、莫里斯水迷宫、神经感染、体力活动、曼氏血吸虫

Introduction

引言

Schistosomiasis is one of the most debilitating neglected tropical diseases, afflicting approximately 240 million individuals and resulting in around 280,000 deaths annually (1–3). Furthermore, it imposes a staggering burden of up to 4 million disability-adjusted life years (DALYs) (3). Schistosomiasis typically progresses from an acute phase characterized by non-specific symptoms like fever and muscle aches to a chronic phase where the worms reside in the host’s blood vessels, leading to organ damage (3). The pathology of schistosomiasis stems from the systemic immune response triggered by the presence of eggs trapped within the tissues leading to granuloma formation (4). Although it is rare, schistosomiasis can be also directly involved in the brain and spinal cord, causing a condition known as neuro schistosomiasis. Despite being under-diagnosed, this form affects at least $2-5%$ of the 200 million individuals infected worldwide (5, 6) making it the second most common presentation of S. mansoni infection (7).

血吸虫病是最令人衰弱的被忽视热带病之一，困扰着约2.4亿人，每年导致约28万人死亡 (1-3)。此外，它造成了高达400万伤残调整寿命年 (DALYs) 的惊人负担 (3)。该疾病通常从以发热、肌肉酸痛等非特异性症状为特征的急性期，进展至成虫寄宿于宿主血管的慢性期，最终导致器官损伤 (3)。血吸虫病的病理机制源于虫卵滞留组织引发的全身免疫反应，进而形成肉芽肿 (4)。尽管较为罕见，血吸虫也可直接侵袭脑和脊髓，引发神经血吸虫病。虽然诊断不足，但全球2亿感染者中至少有$2-5%$会罹患这种类型 (5,6)，使其成为曼氏血吸虫感染的第二常见临床表现 (7)。

The systemic effects of S. mansoni infection on neurological function can develop at any stage of the disease (5, 6). The key pathologic feature is driven by sch is to some eggs in the central nervous system (CNS) resulting in gran u loma to us formation. Over time, these granulomas can cause focal neurological damage. Some neurological symptoms may also occur due to systemic inflammatory responses to the parasitic infection, which can be secondary to the release of cytokines and other inflammatory mediators (5, 6). There are two main forms of neuro schist oso mi as which are cerebral and spinal. Cerebral schistosomiasis is mediated by S. japonicum, and rarely S. mansoni, whereby the frontal and parietal lobes are often affected. Symptoms may include seizures, headaches, focal neurologic deficits, and altered mental status. On the other hand, spinal schistosomiasis is more commonly associated with S. mansoni infection whereby the thoracic spinal cord is predominantly affected. The clinical presentation can include para pares is, quad rip are s is, back pain, sensory deficits, and bladder and bowel dysfunction (5, 6). Of note, schistosomiasis also negatively impacts cognitive function. Research indicates that children who are moderately to heavily infected with helminths tend to score lower on cognitive function tests and achieve less in education compared to children who are either uninfected or lightly infected (8, 9). A recent attempt to understand the underlying mechanism demonstrated that

曼氏血吸虫感染对神经功能的系统性影响可在疾病任何阶段发生 (5, 6)。其关键病理特征是由中枢神经系统 (CNS) 中的虫卵引发肉芽肿形成。随时间推移，这些肉芽肿可导致局灶性神经损伤。某些神经系统症状也可能源于对寄生虫感染的系统性炎症反应，这可能是细胞因子和其他炎症介质释放的继发效应 (5, 6)。

神经血吸虫病主要有两种形式：脑型和脊髓型。脑型血吸虫病主要由日本血吸虫引起，少数由曼氏血吸虫导致，常累及额叶和顶叶，症状包括癫痫发作、头痛、局灶性神经功能缺损及精神状态改变。脊髓型血吸虫病则更常见于曼氏血吸虫感染，主要影响胸段脊髓，临床表现可包括轻瘫、四肢瘫痪、背痛、感觉障碍及膀胱肠道功能障碍 (5, 6)。

值得注意的是，血吸虫病还会对认知功能产生负面影响。研究表明，与未感染或轻度感染的儿童相比，中重度感染蠕虫的儿童在认知功能测试中得分较低，教育成就也相对有限 (8, 9)。最近一项针对潜在机制的研究表明...

S. mansoni infection led to impairment of spatial learning and memory capacity. The phenotype was associated with enhanced microglia and astrocytes number and reactivity (10). This was however characterized in mice at the post-natal stage, leaving the mechanisms underlying the impact of schistosomiasis on the adult brain unclear. It is worth mentioning that murine models to study neuro schistosomiasis provides valuable insights due to their genetic tract ability, allowing for the exploration of pathogen-host interactions and therapeutic strategies. However, one of the main limitations is the significant biological differences between murine and human hosts that may not fully recapitulate the complexity of human neurological involvement or the diverse clinical presentations seen in neuro schistosomiasis. Consequently, while murine studies can highlight potential mechanisms and treatments, they may always need translational validation directly on human patients.

曼氏血吸虫感染导致空间学习和记忆能力受损。该表型与小胶质细胞和星形胶质细胞数量及反应性增加相关 [10]。然而这一特征仅在出生后阶段的小鼠中被观察到，使得血吸虫病对成人大脑影响的机制仍不明确。值得指出的是，由于遗传可操作性，研究神经系统血吸虫病的鼠类模型为探索病原体-宿主相互作用及治疗策略提供了重要见解。但主要局限在于鼠类与人类宿主存在显著生物学差异，可能无法完全复现人类神经系统受累的复杂性或神经系统血吸虫病的多样化临床表现。因此，尽管鼠类研究能揭示潜在机制和治疗方法，但始终需要直接在人类患者身上进行转化验证。

Pra zi quant el is the mainstay drug for treating schistosomiasis. However, in cases of neuro schistosomiasis, its efficacy in reducing neurological symptoms is unclear, as the drug may not always effectively cross the blood-brain barrier (8). Corticosteroids are typically administered alongside pra zi quant el to reduce inflammation caused by the dying parasites (5, 6). However, corticosteroids carry significant side effects that can potentially harm the host, making the exploration of safe natural alternatives imperative. One such non-invasive approach known for its ability to down regulate immune response and confer protection against chronic inflammation is physical activities (PA) (11). Reports demonstrated that regular moderate exercise have protective and anti-inflammatory impacts, thereby enhancing CNS functionality (12). This is achieved through various pathways i) transient increase in IL-6 that drives the production of anti-inflammatory mediators IL-10 and Interleukin-1 receptor antagonist (IL-1RA), ii) stimulating the adrenal gland cortex and medulla to produce adrenaline and g luc oc or tico id, respectively, iii) reducing the expression of toll-like receptor on monocytes, and/or iv) diminishing the circulating number of pre-inflammatory monocytes. These pathways collectively contribute to the reduction of pro-inflammatory mediators and bolster the ability of immune cells within the CNS to adopt an anti-inflammatory phenotype (11, 12). Generation of the anti-inflammatory phenotype fosters neuro plasticity, neuro genesis, neuro protection, and support hippocampus-mediated learning and memory (13, 14). The question of whether PA can effectively mitigate the proinflammatory impact of schistosomiasis on the brain remains open and warrants further investigation.

吡喹酮 (praziquantel) 是治疗血吸虫病的主要药物。然而，在神经型血吸虫病病例中，其对缓解神经系统症状的疗效尚不明确，因为该药物可能无法有效穿透血脑屏障 (8)。临床上通常将皮质类固醇与吡喹酮联用以减轻寄生虫死亡引发的炎症反应 (5, 6)。但皮质类固醇会带来严重副作用，可能对宿主造成伤害，因此探索安全的天然替代方案势在必行。

体育活动 (PA) 作为一种非侵入性干预手段，已知具有下调免疫反应和抵御慢性炎症的作用 (11)。研究表明，规律的中等强度运动能产生保护性和抗炎效应，从而提升中枢神经系统 (CNS) 功能 (12)。其作用机制包括：i) 短暂升高 IL-6 以促进抗炎介质 IL-10 和白细胞介素-1受体拮抗剂 (IL-1RA) 的生成；ii) 刺激肾上腺皮质和髓质分别分泌肾上腺素和糖皮质激素；iii) 降低单核细胞表面 Toll 样受体的表达；和/或 iv) 减少循环系统中促炎性单核细胞数量。这些通路共同作用可减少促炎介质，并增强 CNS 内免疫细胞向抗炎表型转化的能力 (11, 12)。抗炎表型的形成能促进神经可塑性、神经发生、神经保护，并支持海马体介导的学习记忆功能 (13, 14)。

目前尚不清楚体育活动能否有效缓解血吸虫病对大脑的促炎影响，这一问题值得深入研究。

In the present study, we aimed to 1) dissect the impact of S. mansoni infection on the specific learning and memory domains in adult mice, 2) characterize the cellular changes in CNS in response to schistosomiasis infection, and to 3) finally to evaluate the impact of PA in alleviating schistosomiasis-induced changes in the CNS.

本研究旨在：1) 剖析曼氏血吸虫(S. mansoni)感染对成年小鼠特定学习记忆功能域的影响；2) 阐明血吸虫病感染引发的中枢神经系统细胞变化特征；3) 最终评估吡哆胺(PA)对缓解血吸虫病所致中枢神经系统改变的作用。

Materials and methods

材料与方法

Animals

动物

Wildtype mice on BALB/c background were used, ad all mice were maintained in specific-pathogen-free barrier conditions in individually ventilated cages at the University of Cape Town biosafety level 2 animal facility. Experimental mice were sex and age-matched and used at 8 weeks of age. All the experimental work was done in strict accordance with the recommendations of the South African national guidelines and of the University of Cape Town practice for laboratory animal procedures as in ethics protocols, 020-007, approved by the Animal Research Ethics Committee of the Faculty of Health Science, University of Cape Town. All efforts were made to minimize animal suffering. Upon reaching the study experimental endpoint and/or the protocoldefined humane endpoint, animals were euthanized under this study by exposure to an excess of Halothane $(4%$ in air) for 5 minutes. Death was confirmed either by neck dislocation or ex san gui nation by cardiac puncture. Death was not a predetermined endpoint in any of the arms of this study.

采用BALB/c背景的野生型小鼠，所有小鼠在开普敦大学生物安全2级动物设施中，于特定无病原体屏障条件下的独立通风笼中饲养。实验小鼠性别与年龄匹配，使用周龄为8周。所有实验工作均严格按照南非国家指南和开普敦大学实验室动物操作规范（伦理协议020-007，经开普敦大学健康科学学院动物研究伦理委员会批准）的建议进行。尽一切努力减少动物痛苦。当达到研究实验终点和/或方案定义的人道终点时，动物在本研究中通过暴露于过量氟烷（空气中4%）5分钟实施安乐死。通过颈部脱位或心脏穿刺放血确认死亡。死亡并非本研究任何组别的预设终点。

Morris Water Maze

莫里斯水迷宫 (Morris Water Maze)

The Morris Water Maze (MWM) is a widely-used behavioral testing procedure designed to study spatial learning and memory in rodents, particularly rats and mice (15). The MWM task involved a training phase where mice performed four swim trials 1 min each per day (with 5 minutes interval between trials) for 4 consecutive days to locate a plexiglass circular platform ( $10\mathrm{cm}$ in diameter), which was placed approximately $0.5\mathrm{cm}$ below the water level in an open circular $123\mathrm{cm}$ diameter MWM. The water temperature was controlled using an automated water heater and made to e qui lib rate with the room temperature maintained at $20{-}24^{\circ}\mathrm{C}$ . During the training phase of the task, each mouse was allowed a maximum of 60 seconds to locate and climb onto the platform. Once the mouse had located the platform, it was given approximately 10 seconds to remain on the platform. Mice that failed to locate the platform within 60 seconds were gently guided to the platform and allowed to acclimatize for 10 seconds before returning to the home cage. During this phase, the test was measuring learning memory and spatial cues. On the 5th day, a probe trial was performed with the platform removed in order to test reference memory (observations were based on the number of and latency to platform crossings). Each mouse was given a maximum of 60 seconds in the MWM to find and cross the platform location. On days 6 and 7, the platform is placed in the quadrant opposite the original training quadrant, and the mouse was retrained for four trials each day (i.e. 60sec swim with approximately 5mins interval $\textrm{x4}$ trials). On day 8 mice were introduced to the pool with a visible platform in a third quadrant, placed approximately $0.5~\mathrm{cm}$ above water level to test learning memory. All data was recorded using the EthoVision XT 8 automated tracking system (Noldus Information Technology, VA).

莫里斯水迷宫 (MWM) 是一种广泛使用的行为测试程序，用于研究啮齿动物（特别是大鼠和小鼠）的空间学习和记忆能力 [15]。MWM 任务包括训练阶段：小鼠连续4天每天进行4次游泳试验（每次1分钟，试验间隔5分钟），以定位一个直径 $10\mathrm{cm}$ 的有机玻璃圆形平台，该平台置于直径 $123\mathrm{cm}$ 的圆形MWM中，平台边缘距水面约 $0.5\mathrm{cm}$。水温通过自动加热器控制，并与室温（维持在 $20{-}24^{\circ}\mathrm{C}$）保持平衡。在训练阶段，每只小鼠最多有60秒时间定位并爬上平台。成功定位平台后，小鼠可在平台上停留约10秒。未能在60秒内找到平台的小鼠会被轻柔引导至平台，并适应10秒后返回饲养笼。此阶段测试的是学习记忆和空间线索能力。第5天进行探测试验（移除平台以测试参考记忆，观测指标为平台穿越次数和潜伏期），每只小鼠在MWM中最多有60秒时间寻找并穿越原平台位置。第6-7天将平台置于与原训练象限相对的象限中，每天重新训练4次试验（即每次游泳60秒，间隔约5分钟，共 $\textrm{x4}$ 次试验）。第8天在水面以上约 $0.5~\mathrm{cm}$ 处设置可见平台（位于第三象限）以测试学习记忆。所有数据均通过EthoVision XT 8自动追踪系统（Noldus Information Technology, VA）记录。

S. mansoni infection

S. mansoni 感染

Prior to percutaneous infection with S. mansoni cercariae, animals between 7 to 8 weeks age were anesthetized by intra peritoneal injection of a cocktail of Ketamine $(100~\mathrm{mg/kg})$ and Xylazine $\mathrm{10~mg/kg)}$ and monitored for 5 mins to confirm deep anesthesia. Anesthesia was confirmed by the absence of pedal reflex (toe pinch) and eyeblink reflex amid a regular respiratory rate. The anesthesia duration was of a maximum of 30 minutes. During the anesthesia phase, animals were exposed to an infra-red lamp to help them maintain their core body temperature. This procedure was performed and dully cared for by trained and authorized researchers. Then, mice were percutaneous ly infected via the abdomen, using stainless-steel rings, with 0 or 35 viable cercariae of a Puerto Rican strain of S. mansoni obtained from infected Bio mph al aria glabrata snails (NMRI strain, NR-21962, provided by Biomedical Research Institute, Rockville, MD) for control or infected group, respectively. Post-infection, animals were monitored until regaining of consciousness and moistened food was added to the cage bedding.

在经皮感染曼氏血吸虫尾蚴前，对7至8周龄的动物通过腹腔注射氯胺酮（100 mg/kg）和甲苯噻嗪（10 mg/kg）混合麻醉剂进行麻醉，并监测5分钟以确认深度麻醉。麻醉状态通过足部反射（趾捏）消失、规律呼吸频率下的眨眼反射消失来确认。麻醉持续时间最长30分钟。麻醉期间，动物暴露于红外灯下以维持核心体温。该操作由经过培训且获得授权的研究人员规范执行并全程监护。随后，分别使用不锈钢环经腹部对对照组和感染组小鼠进行经皮感染，接种剂量为0或35条具有活性的波多黎各株曼氏血吸虫尾蚴（该虫株采自受感染的光滑双脐螺（NMRI品系，NR-21962，由马里兰州罗克维尔生物医学研究所提供）。感染后持续监测动物直至恢复意识，并在垫料中添加湿润饲料。

Experiments design

实验设计

The MWM put mice to a swimming task for 8 consecutive days, hence right after day 8 of MWM we attribute potential biological effects to the immediate impact of physical activity. To evaluate the long-term effects of the initial physical activities on the animals, we repeated the MWM test at 11 weeks post-infection, during the chronic stage, to further examine any lasting impacts. Thus, our experimental design included physical activities as a factor, and infection status as another independent factor, generating 4 experimental groups: noninfected non-trained (NINT), non-infected trained (NIT), infected non-trained (INT), and infected trained (IT).

MWM让小鼠连续8天进行游泳任务，因此在MWM第8天结束后，我们将潜在的生物效应归因于体力活动的直接影响。为了评估初始体力活动对动物的长期影响，我们在感染后11周（慢性阶段）重复了MWM测试，以进一步考察任何持久性影响。因此，我们的实验设计将体力活动作为一个因素，感染状态作为另一个独立因素，共生成4个实验组：未感染未训练(NINT)、未感染训练(NIT)、感染未训练(INT)和感染训练(IT)。

Eggs detection

鸡蛋检测

Mice were euthanized at acute stage (8 weeks post-infection). Eggs were purified from KOH digested liver, ileum, and spinal cord and counted at $40\mathrm{x}$ magnifications as previously described (16–18). The method for determining collagen production through hydroxy pro line content was conducted as outlined in reference (18). Briefly, liver samples of specific weight were hydrolyzed in $6\mathrm{M}$ hydrochloric acid at $110^{\circ}\mathrm{C}$ overnight, followed by filtration using Whatman filter papers. The resulting filtrate was then neutralized using $1%$ phenol phthalein and titrated with $10\mathrm{M}$ sodium hydroxide. A portion of this filtrate was combined with iso prop an ol and introduced to a solution of chloramine-T and citrate buffer $\mathrm{\check{pH}}$ 6.0). To this mixture, Ehrlich’s reagent (comprising $25\mathrm{g}$ of pdimethyl-amino-benz aldehyde and $37.5~\mathrm{ml}$ of $60%$ perchloric acid) was added. The absorbance was measured at $570~\mathrm{nm}$ employing a VersaMax microplate spectrophotometer from Molecular Devices. The concentration of hydroxy pro line was determined using a standard of 4-hydroxy-L-proline (Calbiochem, San Diego, CA, US) with the results being reported in micrograms of hydroxy pro line per weight of liver tissue, which contained $10\Lambda4$ eggs.

小鼠在急性期（感染后8周）被安乐死。虫卵通过KOH消化的肝脏、回肠和脊髓纯化，并按先前描述的方法在40倍放大倍数下计数[16-18]。通过羟脯氨酸含量测定胶原蛋白生成的方法参照文献[18]进行。简言之，特定重量的肝脏样本在6M盐酸中110℃过夜水解，随后用Whatman滤纸过滤。所得滤液用1%酚酞中和，并用10M氢氧化钠滴定。取部分滤液与异丙醇混合，加入氯胺-T和柠檬酸盐缓冲液(pH 6.0)溶液中。向该混合液中加入Ehrlich试剂（含25g对二甲氨基苯甲醛和37.5ml 60%高氯酸）。使用Molecular Devices公司的VersaMax酶标仪在570nm波长处测定吸光度。羟脯氨酸浓度以4-羟基-L-脯氨酸（美国加州圣地亚哥Calbiochem公司）为标准品进行测定，结果以每克含10^4个虫卵的肝组织中羟脯氨酸微克数表示。

Physical exercise using Morris Water Maze

使用莫里斯水迷宫进行体育锻炼

Cognitive function was assessed at the acute stage, 8 weeks postinfection, using the Morris Water Maze (MWM) task for 8 consecutive days to verify the immediate impact of exercise. To evaluate the long-term effects of the initial exercise on the animals, we repeated the Morris Water Maze (MWM) test at 11 weeks postinfection, during the chronic stage, to further examine any lasting impacts. using Morris Water Maze (MWM) platform for 8 consecutive days. MWM is a widely-used behavioral testing procedure designed to study spatial learning and memory in rodents, particularly rats and mice (15). The MWM task involved a training phase During the training phase, where mice were given performed four swim trials per day (with 5 minutes interval between trials) for 4 consecutive days to locate a plexiglass circular platform ( $10\mathrm{cm}$ in diameter), which was placed approximately $0.5~\mathrm{cm}$ below the water level in an open circular $123\mathrm{cm}$ diameter MWM. The water temperature normally was controlled using an automated water heater and made to e qui lib rate with the room temperature maintained at $20{-}24^{\circ}\mathrm{C}$ During the training phase of the task, each mouse was allowed a maximum of 60 seconds to locate and climb onto the platform. Once the mouse has had located the platform, it was given approximately 10 seconds to remain on the platform. Mice that failed to locate the platform within 60 seconds were gently guided to the platform and allowed to acclimatize for 10 seconds before returning to the home cage. During this phase, the test help in measuring learning memory and spatial cues On the 5th day, a probe trial was performed with the platform removed in order to test reference memory (observations were based on helped in measuring learning memory and spatial cues. On the 5th day, a probe trial was performed with the platform removed in order to test reference memory (observations were based on the number of and latency to platform crossings). Each mouse was given a maximum of 60 seconds in the MWM to find and cross the platform location. On days 6 and 7, the platform is placed in the quadrant opposite the original training quadrant, and the mouse was retrained for four trials each day (i.e 60sec swim with approximately 5mins interval $\textbf{x4}$ trials). On day 8 mice were introduced to the pool with a visible platform in a third quadrant, placed approximately $0.5~\mathrm{cm}$ above water level to test learning memory. All data will be recorded using the EthoVision XT 8 automated tracking system (Noldus Information Technology, VA).

在急性期（感染后8周）采用莫里斯水迷宫（Morris Water Maze, MWM）任务连续测试8天以评估运动干预的即时影响。为探究初期运动对动物的长期效应，我们在慢性期（感染后11周）再次进行MWM测试，持续8天以观察持续作用。MWM是广泛应用于啮齿类动物（特别是大鼠和小鼠）空间学习与记忆研究的标准化行为测试范式[15]。

实验流程包括：

  1. 训练阶段（第1-4天）：每天进行4次游泳训练（每次间隔5分钟），小鼠需在直径123厘米的圆形MWM水池中定位直径10厘米的亚克力平台（平台隐藏于水面下0.5厘米）。水温通过自动加热装置维持在20-24°C与环境温度平衡。每只小鼠有60秒最大寻台时间，成功登台后允许停留10秒。未能在时限内定位平台的小鼠将被轻柔引导至平台并适应10秒后放回饲养笼。该阶段主要测量空间学习能力与线索记忆。
  2. 探查测试（第5天）：撤除平台进行60秒参考记忆测试，记录小鼠穿越原平台位置的次数和潜伏期。
  3. 反向训练（第6-7天）：将平台移至原训练象限的对侧象限，每天进行4次游泳训练（每次60秒，间隔约5分钟）。
  4. 可视平台测试（第8天）：在第三象限设置高出水面0.5厘米的可见平台，检验学习记忆能力。

所有数据通过EthoVision XT 8自动追踪系统（Noldus Information Technology, VA）采集。

Intestinal contract ili ty

肠道收缩性

A measure of schistosomiasis infection was conducted 8 weeks post-infection. After eu than i zing the mice, Approximately $1~\mathrm{cm}$ of jejunum segments were removed from the small intestine of all groups. The smooth muscle contractile responses were measured using a water-jacketed organ bath (Panlab, Spain), connected to transducers and the PowerLabTM system (AD Instruments, Australia). This setup feeds and translates the signals to a computer for measuring tissue isometric tensions. The tissues were weighed on an analytical scale before being stimulated with varying concentrations of ACh (−9 to $^{-3}$ LOG [M]) to determine the isometric contractile responses.

感染后8周对血吸虫病感染程度进行了测量。处死小鼠后，从各组小鼠小肠中取出约$1~\mathrm{cm}$的空肠段。使用水套式器官浴槽(Panlab, 西班牙)连接传感器和PowerLabTM系统(AD Instruments, 澳大利亚)测量平滑肌收缩反应。该装置将信号传输并转换至计算机，用于测量组织等长张力。组织在用不同浓度ACh(−9至$^{-3}$ LOG [M])刺激以确定等长收缩反应前，先在天平上称重。

Cells isolation

细胞分离

One day after MWM, animals were euthanized and perfused thoroughly with ice-cold PBS $\mathrm{(pH 7.4)}$ for 5 minutes. Following perfusion, heads were removed, and skulls were cleared of all tissue. Surgical scissors were utilized to sequentially remove the tops of the skulls in a clockwise manner. Subsequently, the skulls were promptly placed in ice-cold RPMI media. Meninges were meticulously extracted from the interior surfaces of the skulls and the brain surfaces using forceps (19). The hippocampus and prefrontal cortex were then separated from the brain parenchyma using surgical forceps. Part of hippocampus and prefrontal cortex was used for histology while the rest was used for single cell suspension. Single-cell suspensions from the meninges, hippocampus, and prefrontal cortex were prepared through enzymatic digestion in RPMI containing 220 $\mathrm{U/mg}$ Collagen as e IV (Gibco, Waltham, Massachusetts), $13~\mathrm{U/mg}$ DNase I (Sigma, St. Louis, Missouri), and $5%$ iFCS (in activated fetal calf serum) (Gibco) in RPMI supplemented with 2mM MgCl2, 2mM CaCl2, $20%$ FBS, and $2\textrm{m M}$ HEPES. The digested tissue was mechanically disrupted, filtered through a $100\mathrm{m}$ mesh, and then enriched for leukocytes by cent ri fuga tion $\mathbf{\zeta}{600}\mathbf{\zeta}{8},\mathbf{\zeta}_{10}$ minutes, no brakes) through $40%$ Percoll (Merck) (20).

MWM实验后一天，对动物实施安乐死并用冰PBS (pH 7.4) 灌注5分钟。灌注后摘除头部并清除颅骨所有组织。用手术剪沿顺时针方向逐步去除颅顶，随后立即将颅骨置于冰RPMI培养基中。使用镊子从颅骨内表面和脑表面精细剥离脑膜 (19)。随后用手术镊从脑实质分离海马体和前额叶皮层，部分组织用于组织学检测，其余用于单细胞悬液制备。

通过酶消化法在含以下成分的RPMI培养基中制备脑膜、海马体和前额叶皮层单细胞悬液：220 U/mg胶原酶IV (Gibco)、13 U/mg DNase I (Sigma)、5% iFCS (灭活胎牛血清)，补充2mM MgCl2、2mM CaCl2、20% FBS和2mM HEPES。消化后的组织经机械破碎后通过100μm滤网过滤，继而采用40% Percoll (Merck) 通过离心 (600g，8-10分钟，无刹车) 进行白细胞富集 (20)。

Flow cytometry

流式细胞术

Antibodies used for flow cytometry analysis were as follows: CD3e (500A2), CD4 (RM4-5), CD8a (53-6.7), CD19 (1D3), CD44 (IM7), CD62L (MEL-14), IFN-g (XMG1.2), IL-4 (11B11), IL-13 (eBio13A), TNFa, CXCR5, CD11b, SiglecF, NK1.1, CD45, F4/80, MHC II, PD1, and GFAP purchased from BD Bioscience s (Franklin Lakes, New Jersey) and e Bioscience (San Diego, California). For staining of cell surface markers, cells $(1\mathrm{x}10^{6})$ were labeled and washed in PBS containing $1%$ BSA (Roche, Switzerland) and $0.1%$ $\mathrm{NAN}{3}$ (FACS buffer). For detection of intracellular cytokines, cells were seeded at a density of $2\mathrm{x}10^{6}$ cells/well in a complete RPMI culture medium and stimulated with $50~\mathrm{ng/ml}$ phorbol myristate acetate (PMA), $250~\mathrm{ng/ml}$ ionomycin and $200~\mathrm{M}$ monensin (all from Sigma) for $8{\cdot}12\mathrm{hr}$ at $37^{\circ}\mathrm{C}$ in a humidified atmosphere containing $5%$ $\mathrm{CO}_{2}$ . After the incubation period, cells were harvested, washed, fixed in $2%$ (w/v) para formaldehyde, per me abi liz ed with $0.5%$ saponin buffer, and then stained for cytokine production as previously described (17, 18). The acquisition was performed using BD L SR For tessa (BD Bioscience s), and data were analyzed using FlowJo software (Treestar, Ashland, Oregon). Uniform Manifold Approximation and Projection (UMAP) was used for data visualization. It is a nonlinear dimensionality-reduction technique (21) available as a FlowJo plugin.

流式细胞分析所用抗体如下：CD3e (500A2)、CD4 (RM4-5)、CD8a (53-6.7)、CD19 (1D3)、CD44 (IM7)、CD62L (MEL-14)、IFN-γ (XMG1.2)、IL-4 (11B11)、IL-13 (eBio13A)、TNF-α、CXCR5、CD11b、SiglecF、NK1.1、CD45、F4/80、MHC II、PD1和GFAP，购自BD Biosciences（美国新泽西州富兰克林湖）和eBioscience（美国加州圣地亚哥）。

细胞表面标志物染色时，将 $(1\mathrm{x}10^{6})$ 个细胞在含 $1%$ BSA（瑞士罗氏）和 $0.1%$ $\mathrm{NAN}{3}$ 的PBS（FACS缓冲液）中标记并洗涤。检测细胞内细胞因子时，将细胞以 $2\mathrm{x}10^{6}$ 个/孔的密度接种于完全RPMI培养基中，用 $50~\mathrm{ng/ml}$ 佛波醇肉豆蔻酸酯（PMA）、$250~\mathrm{ng/ml}$ 离子霉素和 $200~\mathrm{M}$ 莫能菌素（均购自Sigma）在 $37^{\circ}\mathrm{C}$、含 $5%$ $\mathrm{CO}_{2}$ 的湿润气氛中刺激 $8{\cdot}12\mathrm{~小时}$。孵育后，收集细胞并洗涤，用 $2%$ （w/v）多聚甲醛固定，$0.5%$ 皂苷缓冲液透化，随后按文献方法（17, 18）进行细胞因子染色。

数据采集使用BD LSR Fortessa（BD Biosciences），分析采用FlowJo软件（美国俄勒冈州阿什兰Treestar）。数据可视化采用均匀流形逼近与投影（UMAP），这是一种非线性降维技术（21），可通过FlowJo插件实现。

Quantitative real-time RT-PCR

实时定量RT-PCR

RNA from parenchyma single cell suspension was reverse transcribed by Transcript or First Strand cDNA Synthesis Kit (Roche) according to manufacturer’s instructions. Real-time reverse transcribed PCR (QRT-PCR) was performed with Light Cycle r 480 SYBR Green I Master mix in Light Cycle r 480 II (Roche) and gene-specific primers (IDT, CA, USA). Fold change in gene expression was calculated by the DDCt method and normalized to Hprt1 which was used as internal control as described (22).

采用 Transcriptor First Strand cDNA Synthesis Kit (Roche) 试剂盒，按照制造商说明对薄壁组织单细胞悬浮液中的 RNA 进行反转录。使用 LightCycler 480 SYBR Green I Master mix 在 LightCycler 480 II (Roche) 系统上进行实时荧光定量 PCR (QRT-PCR)，并采用 IDT (CA, USA) 公司提供的基因特异性引物。通过 DDCt 法计算基因表达变化倍数，并以 Hprt1 作为内参基因进行标准化处理 (22)。

The primers used are as follow (Table 1):

所用引物如下 (表 1):

TABLE 1 The primers used in qPCR.

表 1: qPCR所用引物。

编号 基因 方向 序列
1 HPRT 正向 5'-GTT GGA TAT GCC CTT GAC-3'
反向 5'-AGGACT AGA ACA CCT GCT-3'
2 Muscarinic M5 正向 5'-CTC TGC TGG CAG TAC TTG GTC- 3'
反向 5'-GTG AGC CGGTTT TCT CTT CTT - 3'
3 Muscarinic M2 正向 5'-TGA AAA CAC GGT TTC CAC TTC-3'
反向 5'-GAT GGA GGAGGC TTC TTT TTG- 3'
4 Muscarinic M1 正向 5'-GGACAACAACAC CAGAGG AGA-3'
反向 5'-CGA GGT CAC TTT AGGGTA GGG-3'
5 IL-4 正向 5'-TCG GCA TTT TGA ACG AGG TC-3'
反向 5'-GAAAAGCCC GAA AGA GTC TC-3′
6 IL-6 正向 5'-CGT GGA AAT GAG AAA AGA GTT GTG-3'

(Continued)

（接上文）

TABLE 1 Continued

表 1 续表

No Gene Direction Sequences
Reverse 5'-ATCTCT CTGAAG GACTCT GGC T-3'
IL-13 Forward 5'-CTCCCT CTGACC CTT AAG GAG-3'
Reverse 5'-GAAGGGGCC GTG GCGAAACAG-3'
8 CCL2 Forward 5'-CTCTCT CTT CCT CCA CCA CCA T-3'
Reverse 5'-TGGGGC GTT AAC TGCATCTG-3'
9 TNFα Forward 5'-TCT CATCAGTTCTAT GGCCC-3'
Reverse 5'-GGGAGT AGACAA GGTACAAC-3'

Tissue homogenate for cytokine analysis

用于细胞因子分析的组织匀浆

Brain parenchyma (hippocampus and prefrontal cortex) was collected and homogenized in RIPA buffer. Cytokines (TNFa, IL-6, and MCP all from BD Pharmingen) were measured in the protein extracts by sandwich ELISA as described previously (17, 18). Cytokine values were normalized according to the protein content measured by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, catalogue no. 23225).

收集脑实质（海马体和前额叶皮层）并在RIPA缓冲液中匀浆。采用夹心ELISA法检测蛋白提取物中的细胞因子（TNFa、IL-6和MCP，均购自BD Pharmingen），方法如前所述[17, 18]。细胞因子数值根据Pierce BCA蛋白检测试剂盒（Thermo Fisher Scientific，货号23225）测定的蛋白含量进行标准化。

Immuno fluorescence

免疫荧光

First, mice were anesthetized using isoflurane and perfused with ice-cold PBS and $4%$ para formaldehyde (PFA) through the heart. Next, free-floating coronal sections from the brain or spinal cord were sectioned at $30~\mathrm{m}$ using a Leica cryostat. The sections were then immersed in a blocking solution consisting of PBS with $2%$ Normal Goat Serum, $1%$ BSA, $1%$ Triton , $0.05%$ Tween-20, and $0.05%$ sodium azide for 1 hour at room temperature. The sections were subsequently incubated overnight at $4^{\circ}\mathrm{C}$ with primary antibodies, including rabbit anti-Ach (Abcam), rabbit anti-Iba1 (Abcam) at a concentration of 1:500 and 1:400, and rabbit antiIba1(Abcam), at a concentration of 1:500 and 1:400, respectively. After washing the sections with PBS, they were incubated with flu or oph ore-conjugated secondary antibodies at a dilution of 1:400 for 2 hours. Finally, the sections were stained with DAPI at a concentration of $10~{}{\mathrm{g/ml}}$ for 10 minutes at room temperature, mounted on glass slides using moviol and antifade mounting medium from Thermo Fisher Scientific.

首先，用异氟烷麻醉小鼠，并通过心脏灌注冰冷的PBS和4%多聚甲醛(PFA)。随后使用Leica冰冻切片机将大脑或脊髓的游离冠状切片切成30微米厚度。切片在室温下浸入封闭液(含2%正常山羊血清、1% BSA、1% Triton X-100、0.05% Tween-20和0.05%叠氮化钠的PBS溶液)中封闭1小时。接着将切片在4℃下与一抗(包括1:500稀释的兔抗Ach抗体[Abcam]、1:400稀释的兔抗Iba1抗体[Abcam])孵育过夜。PBS洗涤后，切片与1:400稀释的荧光标记二抗孵育2小时。最后用10微克/毫升DAPI室温染色10分钟，采用Thermo Fisher Scientific的moviol抗淬灭封片剂将切片封固于载玻片上。

Immuno his to chemistry

免疫组织化学

Brain or spinal cord tissue was sectioned at a thickness of $9\mathrm{m}$ using OCT compound and a cryostat. The sections were mounted on glass slides and dried for 2 hours. Following this, the slides were stained in $0.1%$ cresyl violet solution for 5 minutes, then rinsed with running distilled water. The sections were further dehydrated using graded alcohols, cleared in xylene, and finally mounted with antifade medium (Mowiol).

使用 OCT 复合物和冷冻切片机将脑或脊髓组织切片，厚度为 $9\mathrm{m}$。切片置于载玻片上干燥 2 小时，随后在 $0.1%$ 甲苯胺蓝溶液中染色 5 分钟，并用流动蒸馏水冲洗。切片经梯度酒精脱水、二甲苯透明，最后用抗褪色封片剂 (Mowiol) 封固。

Statistics

统计

Statistical analysis was conducted using GraphPad Prism 6 software and SPSS 20. Data were calculated as mean $\pm\mathrm{SEM}$ . Statistical significance was determined using the unpaired Student’s t-test and One-Way ANOVA with Bonferroni’s post-test after testing for normality and homogeneity in one category independent data. Two-way ANOVA was used to compare two categorical independent variables (infection and PA), and also the interaction between them, defining differences to uninfected mice as significant $(^{},P\leq0.05;^{}$ , $P\le0.01$ ; $^{***}$ , $P\le0.001$ ). Two-way ANOVA was also employed to examine the interaction effects of infection and training, enabling an understanding of both the separate and combined effects of these factors. This comprehensive approach provided a nuanced understanding of the collective impact of infection and training on experimental outcomes.

使用GraphPad Prism 6软件和SPSS 20进行统计分析。数据以均值$\pm\mathrm{SEM}$表示。在对单类别独立数据进行正态性和方差齐性检验后，采用非配对Student’s t检验和单因素方差分析（Bonferroni事后检验）确定统计学显著性。双因素方差分析用于比较两个分类自变量（感染和PA）及其交互作用，并将与未感染小鼠的差异定义为显著$(^{},P\leq0.05;^{}$, $P\le0.01$; $^{***}$, $P\le0.001$)。双因素方差分析还用于检验感染与训练的交互效应，从而理解这些因素的独立及联合作用。这种综合方法细致解析了感染和训练对实验结果的共同影响。

Results

结果

Acute schistosomiasis causes recall memory impairment

急性血吸虫病导致回忆记忆受损

In order to understand how systemic Schistosomiasis infection may affect the brain function, especially the behavior, we conducted MWM test at the acute stage (8 weeks post-infection to assess cognitive abilities.

为了解系统性血吸虫感染如何影响大脑功能，尤其是行为表现，我们在感染急性期（感染后8周）进行了Morris水迷宫（MWM）测试以评估认知能力。

The MWM analysis revealed some key findings. Firstly, S. mansoni infection did not significantly alter locomotor activity, as shown by the consistent distance moved (Figure 1A). Similarly, velocity measurements (Figure 1B) indicated no signs of anxietylike behavior. Furthermore, there were no significant differences in immobility time (Figure 1C), center-moving duration (Figure 1D), or immobility time ratio (Figure 1E), suggesting the absence of any phenotypes-like behavior in infected mice.

MWM分析揭示了一些关键发现。首先，曼氏血吸虫感染并未显著改变运动活动能力，移动距离保持稳定（图1A）。同样，速度测量结果（图1B）显示无焦虑样行为迹象。此外，静止时间（图1C）、中心移动时长（图1D）及静止时间占比（图1E）均无显著差异，表明感染小鼠未出现任何表型样行为。

Both infected and non-infected mice demonstrated comparable spatial learning abilities, with a similar reduction in the time taken to reach the platform across learning sessions (Figure 1F). To further assess spatial reference memory, a probe test was performed 24 hours after the last learning trial. No significant differences were observed between the groups in time spent in the platform’s quadrant (Figure 1Gi), latency to cross the platform area (Figure 1Gii), or the number of platform crossings (Figure 1Giii), indicating that spatial memory remained intact in infected mice after 8 weeks post infection.

感染组与未感染组小鼠均表现出相当的空间学习能力，其到达平台所需时间在各学习阶段呈现相似的缩短趋势 (图 1F)。为进一步评估空间参考记忆，末次学习测试24小时后进行探针实验。两组在平台象限停留时间 (图 1Gi)、穿越平台区域延迟时间 (图 1Gii) 及平台穿越次数 (图 1Giii) 方面均无显著差异，表明感染后8周小鼠的空间记忆功能保持完整。

However, during the reversal trial, when the platform was relocated to a different quadrant, infected mice displayed a significant increase in latency to find the platform on day 8 compared to controls (Figure 1H). Additionally, infected mice spent more time in the arena (Figure 1I), although the distance they travelled remained similar to that of non-infected mice (Figure 1J). These results suggest that while complex learning abilities were not impaired, schistosomiasis infection negatively impacted recall memory.

然而，在反向试验中，当平台被重新放置到不同象限时，与对照组相比，感染小鼠在第8天找到平台的潜伏期显著增加 (图 1H)。此外，感染小鼠在实验区域停留的时间更长 (图 1I)，尽管它们的移动距离与非感染小鼠相似 (图 1J)。这些结果表明，虽然复杂学习能力未受损害，但血吸虫感染对回忆记忆产生了负面影响。

To our knowledge, this is the first study to dissect cognitive behavior in this context and shed light on the impact of schistosomiasis on recall system rather than the entire cognitive outcomes.

据我们所知，这是首个在此背景下剖析认知行为的研究，揭示了血吸虫病对记忆系统而非整体认知结果的影响。

Impact of physical activities on peripheral and CNS responses following schistosomiasis infection

血吸虫感染后体力活动对外周和中枢神经系统反应的影响

Research shows that schistosomiasis infection harms intestinal function by causing inflammation and fibrosis, which disrupt normal muscle contractions needed for gut movement. When parasite eggs lodge in intestinal tissues, they trigger immune responses that lead to chronic inflammation, tissue damage, and changes in muscle activity, impairing digestion and nutrient absorption. In our study, we specifically looked at how physical activity (PA) impacts intestinal contract ili ty after infection. Surprisingly, despite infection, the hyper contract ili ty of the intestine was similar between noninfected, non-trained (NINT) and infected, non-trained (INT) groups (Supplementary Figure S1A). These results suggest that at this stage of infection, the presence of parasite eggs may not significantly alter muscle contract ili ty. However, as noted in other studies, exercise slightly increased hyper contract ili ty in both noninfected trained (NIT) and infected trained (IT) groups (Supplementary Figure S1A). While increased intestinal contract ili ty can enhance gut motility and digestion, the implications of this change in the context of infection warrant further investigation.

研究表明，血吸虫感染通过引发炎症和纤维化损害肠道功能，破坏肠道蠕动所需的正常肌肉收缩。当寄生虫卵滞留于肠道组织时，会引发免疫反应，导致慢性炎症、组织损伤和肌肉活动改变，从而损害消化和营养吸收功能。本研究中，我们重点探究了体力活动（PA）对感染后肠道收缩能力的影响。出乎意料的是，尽管存在感染，未感染未训练组（NINT）与感染未训练组（INT）的肠道过度收缩程度相近（补充图 S1A）。这些结果表明，在当前感染阶段，寄生虫卵的存在可能不会显著改变肌肉收缩能力。但如其他研究所述，运动使未感染训练组（NIT）和感染训练组（IT）的肠道过度收缩程度均轻微增强（补充图 S1A）。虽然肠道收缩能力增强可促进肠道蠕动和消化功能，但这种变化在感染背景下的影响仍需进一步研究。

Considering the potential link between enhanced intestine contract ili ty and the expulsion of schistosomiasis eggs (23), we examined the effects of PA on egg burden and egg-driven immuno pathology. Unexpectedly, our findings indicated that PA led to an increase in egg burden in the liver (Supplementary Figure S1B) and small intestine (Supplementary Figure S1C) in the IT group compared to the INT group. Furthermore, his to pathological analysis revealed that training exacerbated liver granuloma size (Supplementary Figures S1F, G) and increased tissue fibrosis in the liver (Supplementary Figure S1H). These unexpected outcomes underscore the intricate relationship between PA and schistosomiasis progression, necessitating further exploration to unravel underlying mechanisms and potential implications for disease management.

考虑到增强的肠道收缩能力与血吸虫卵排出之间的潜在联系 [23]，我们研究了体育锻炼 (PA) 对虫卵负荷及虫卵驱动免疫病理的影响。出乎意料的是，研究结果表明，与INT组相比，IT组的体育锻炼导致肝脏（补充图 S1B）和小肠（补充图 S1C）中的虫卵负荷增加。此外，组织病理学分析显示，训练加剧了肝脏肉芽肿体积（补充图 S1F、G）并增加了肝脏组织纤维化（补充图 S1H）。这些意外结果凸显了体育锻炼与血吸虫病进展之间复杂的关系，需要进一步探索以阐明潜在机制及其对疾病管理的潜在影响。

In our investigation of egg infiltration in the central nervous system (CNS), specifically the spinal cord and brain parenchyma, his to logical assessments yielded noteworthy results. As we expected, there was a lack of egg infiltration in the spinal cord (Supplementary

在对中枢神经系统(CNS)(特别是脊髓和脑实质)中虫卵浸润的研究中，组织学评估得出了值得注意的结果。正如预期的那样，脊髓中未发现虫卵浸润(补充材料


Acute schistosomiasis infection can impair recall memory. (A) Distance moved by the mice in MWM, (B) the villosity, (C) Immubility time, (D) Centre moving duration, (E) Ratio of immobility time, (F) Spatial learning was assessed during the first four days whereby latency to platform was assessed. A probe test was then done on day 5, and (Gi) the time percentage spent in the right training quadrant, (Gii) the latency to cross the virtual platform, and (Gii) the number of times the mice crossed the platform were recorded. (H) Reversal trial was then performed from day 6 to day 8 and the latency to the new platform was measured. (I) Duration spent on the arena during each trial on day 8. (J) Distance travelled in each day. Results are pooled from two different experiments with 6-8 mice per group. Data are expressed as mean $\pm$ S.E.M. NS, $\mathsf{P}>0.05$ ; $^{\star}P<0.05,$ $^{\star\star}P<0.001,$ $^{\star\star\star}P<$ 0.0001 by two-tailed unpaired Student t test and repeated measures ANOVA. Figures S2A, B) and brain parenchyma, particularly in the prefrontal cortex (PFC) and hippocampus (HPC), for both the infected non-trained (INT) and infected trained (IT) groups (Figure 2A).

急性血吸虫感染可损害回忆记忆。(A) 小鼠在 Morris 水迷宫 (MWM) 中的移动距离，(B) 绒毛结构，(C) 静止时间，(D) 中心区域移动时长，(E) 静止时间占比，(F) 前四天评估空间学习能力（以抵达平台潜伏期为指标）。第5天进行探测试验，记录：(Gi) 正确训练象限停留时间百分比，(Gii) 穿越虚拟平台潜伏期，(Giii) 平台穿越次数。(H) 第6至8天进行逆转试验，测量抵达新平台的潜伏期。(I) 第8天每次试验在竞技场的停留时长。(J) 每日运动总距离。数据合并自两组独立实验（每组6-8只小鼠），以均值±标准误表示。NS：P>0.05；*P<0.05，**P<0.001，***P<0.0001（双尾非配对t检验与重复测量方差分析）。图S2A-B及脑实质（特别是前额叶皮层(PFC)和海马体(HPC)）在感染未训练组(INT)与感染训练组(IT)中的表现（图2A）。

Our study found that while schistosomiasis infection and PA did not affect intestinal muscle contract ili ty in non-trained infected groups. Exercise increased egg burden in the liver and intestines, worsened liver granuloma size, and increased fibrosis, highlighting a complex interaction between PA and infection progression.

我们的研究发现，虽然血吸虫感染和体力活动 (PA) 对未训练感染组的肠道肌肉收缩力没有影响，但运动会增加肝脏和肠道中的虫卵负荷，加剧肝脏肉芽肿大小，并加重纤维化，凸显了体力活动与感染进程之间复杂的相互作用。

Impact of schistosomiasis infection and physical activity on choline r gic system and cytokine expression in the nervous system

血吸虫病感染与体力活动对神经系统胆碱能系统及细胞因子表达的影响

We next analyzed the impact of schistosomiasis infection and its interaction with PA on the choline r gic system. In spinal cord, the number of cells producing acetylcholine (ACh) showed a significant increase in the INT group compared to the NINT group (Supplementary Figures S2Ci-C). This indicated that schistosomiasis infection affected ACh production in the spinal cord. However, PA in the IT group restored ACh production to the baseline level, suggesting that exercise could mitigate the impact of infection on ACh production (Supplementary Figures S2C-Ci). On other hand, the expression of muscarinic receptors (M1, M2, and M5) (Figures 2Fi-Fiii) was examined in the HPC and PFC (Supplementary Figures S2Diii, Dvi). In the hippocampus, M1, M2, and M5 expression levels were remarkably increased in the INT group compared to the NINT group. However, M5 was the only receptor to decrease significantly in the IT group with PA, and statistical analysis showed a significant interaction effect. This suggests that while M1 and M2 are generally up regulated with infection, M5 expression may be uniquely modulated by the combination of infection and PA. For PFC, no change was noted in either $C c l2,I l I3,I l4,M I,M2,$ or M5 expression between the different groups Supplementary Figures S2Di, Dvi.

我们接下来分析了血吸虫感染及其与体力活动(PA)相互作用对胆碱能系统的影响。在脊髓中，INT组产生乙酰胆碱(ACh)的细胞数量较NINT组显著增加(补充图S2Ci-C)，表明血吸虫感染影响了脊髓中的ACh生成。然而IT组通过PA干预使ACh生成恢复至基线水平，提示运动可减轻感染对ACh生成的影响(补充图S2C-Ci)。另一方面，我们检测了海马(HPC)和前额叶皮层(PFC)中毒蕈碱受体(M1、M2和M5)的表达情况(图2Fi-Fiii)(补充图S2Diii, Dvi)。海马区中，INT组的M1、M2和M5表达水平较NINT组显著升高。但M5是唯一在PA干预的IT组中显著降低的受体，统计学分析显示显著的交互效应，这表明虽然M1和M2通常随感染上调，但M5表达可能被感染与PA的组合独特调控。对于前额叶皮层，各组间$Ccl2, Il13, Il4, M1, M2,$或M5表达均未观察到变化(补充图S2Di, Dvi)。

We further characterized cytokine expression in brain parenchyma. Significant effect was observed in the expression of $\mathrm{TNF}$ in the HPC, with PA modulating its impact on tnfa expression dependent on infection presence (Figure 2Di). PA restored tnfa expression to baseline levels compared to the INT group (Figure 2Di). Similar, but not significant, tnfa trend was noted between the different groups (Figure 2Dii).

我们进一步分析了脑实质中的细胞因子表达。在海马体(HPC)中观察到TNFα( $\mathrm{TNF}$ )表达存在显著效应，其中PA(假性过敏)对其tnfa表达的影响取决于感染状态(图2Di)。与INT组相比，PA使tnfa表达恢复至基线水平(图2Di)。各组间tnfa表达趋势相似但未达显著水平(图2Dii)。

For Il6, the INT group exhibited a significant elevation in Il6 expression compared to the NINT group in HPC (Figure 2Diii) and PFC (Figure 2Ei). However, PA did not significantly alter Il6 expression. Training of the infected mice helped in partially restoring Il6 expression (Figures 2Diii, Ei). Similar to Il6, the INT group showed a significant increase in $I l4$ expression compared to the NINT group (Figure 2Dvi), and PA did not have a significant impact on Il4 expression.

对于Il6，INT组在HPC(图2Diii)和PFC(图2Ei)中表现出比NINT组显著升高的Il6表达水平。然而PA并未显著改变Il6表达。感染小鼠的训练有助于部分恢复Il6表达(图2Diii，Ei)。与Il6类似，INT组相比NINT组显示出$I l4$表达的显著增加(图2Dvi)，且PA对Il4表达无显著影响。

Analyzing the expression of $C c l2$ (Figure 2Div) and Il13 (Figure 2Dv), no significant differences were observed between the INT, NINT, and NIT groups. However, it was worth noting that PA in the infected group significantly enhanced Il13 expression compared to the rest (Figure 2Dv). In summary, Muscarinic receptors, particularly the M2 subtype, can modulate inflammation by inhibiting pro-inflammatory cytokines like TNFa and IL-6. Conversely, elevated cytokines can alter muscarinic receptor expression and function, impacting neuro inflammation and cognitive processes. This reciprocal relationship suggests that schistosomiasis-induced cytokine changes may influence muscarinic receptor activity, affecting neuro inflammatory responses and brain function.

分析 $C c l2$ (图 2Div) 和 Il13 (图 2Dv) 的表达水平时，INT、NINT 和 NIT 组间未观察到显著差异。但值得注意的是，感染组中的 PA 显著增强了 Il13 的表达 (图 2Dv)。综上所述，毒蕈碱受体 (尤其是 M2 亚型) 可通过抑制 TNFa 和 IL-6 等促炎细胞因子来调节炎症反应。反之，细胞因子水平升高也会改变毒蕈碱受体的表达与功能，进而影响神经炎症和认知过程。这种双向作用关系表明，血吸虫病引发的细胞因子变化可能调控毒蕈碱受体活性，最终影响神经炎症反应与脑功能。

Physical activities helps reduce the impact of schistosomiasis on microglia and myeloid cell phenotypes in the brain

体育活动有助于减轻血吸虫病对大脑中小胶质细胞和髓系细胞表型的影响

In the prefrontal cortex, PA’s impact on cell numbers depended on the presence of infection (Figures 2B, 2Ci), with infection and PA independently influencing microglia accumulation (Figures 2B, 2Ci). Moreover, the INT group exhibited higher cell numbers compared to the non-infected trained (NIT) and non-infected non-trained (NINT) groups (Figures 2B, 2Ci).

在前额叶皮层中，PA对细胞数量的影响取决于感染的存在 (图 2B, 2Ci)，感染和PA独立影响小胶质细胞的积累 (图 2B, 2Ci)。此外，与非感染训练组 (NIT) 和非感染非训练组 (NINT) 相比，INT组表现出更高的细胞数量 (图 2B, 2Ci)。

The occupied area or cell distribution in the PFC revealed significant interaction effects, indicating the dependency of PAs impact on infection presence (Figure 2Cii). Additionally, main effects of PA and infection were observed, influencing the occupied area independently (Figure 2Cii). The INT group had a reduced occupied area compared to the NIT and IT groups (Figure 2Cii). Microglia in the HPC were not significantly affected by infection or PA (Figures 2Ciii, Civ), indicating the resilience of this cell population to the experimental conditions. Next, we delved into the impact of schistosomiasis infection in brain cells parenchyma using flow cytometry (Figures 3A, Bii). The findings revealed a significant increase in microglia frequency (Figure 3Aiii), TNFa production (Figures 3Ai, Aii), and activation status, marked by higher MHC class II (MHCII) expression (Figure 3Aiii) in HPC in response to schistosomiasis infection compared to the NINT group. In contrast, PA had no effect on microglia activation level in infected animals (Figure 3Aiv).Notably, a statistical analysis highlighted a significant interaction effect between the infection and training for TNFa production in the HPC (Figures 3Ai, Aii),. Of interest, and consistent with our previous observation (Figure 2Di), PA in IT group restored TNFa production to a level similar to NINT and NIT groups. Furthermore, PA did not impact microglia MHCII expression or the production of IL-4 or IL-13 in the HPC (Figures 3iii, Aiv). In the PFC comparable microglia frequency (Figures 3Bii, Biii), activation (Figure 3Biv), and TNFa production (Figures 3B, Bi) were observed in response to schistosomiasis infection and/or PA. Statistical analysis did not reveal significant effects on TNFa production in the PFC (Figure 3Bi).

前额皮层(PFC)的占据面积或细胞分布显示出显著的交互效应，表明体育活动(PA)的影响取决于感染状态(图2Cii)。此外，PA和感染的主效应均被观察到，可独立影响占据面积(图2Cii)。INT组的占据面积较NIT和IT组有所减少(图2Cii)。海马区(HPC)的小胶质细胞未受感染或PA的显著影响(图2Ciii、2Civ)，表明该细胞群体对实验条件具有抗性。随后我们通过流式细胞术深入研究了血吸虫感染对脑实质细胞的影响(图3A、3Bii)。结果显示：与NINT组相比，感染组HPC区小胶质细胞频率(图3Aiii)、TNFa分泌量(图3Ai、3Aii)以及以MHCII分子高表达为标志的激活状态(图3Aiii)均显著增加。值得注意的是，PA对感染动物的HPC区小胶质细胞激活水平无影响(图3Aiv)。统计分析特别指出，HPC区TNFa分泌存在感染与训练的显著交互效应(图3Ai、3Aii)。值得关注的是，与我们先前的发现一致(图2Di)，IT组的PA干预使TNFa分泌恢复至与NINT和NIT组相当的水平。此外，PA对HPC区小胶质细胞MHCII表达及IL-4、IL-13分泌均无影响(图3Aiii、3Aiv)。在PFC区域，血吸虫感染和/或PA引发了类似的小胶质细胞频率变化(图3Bii、3Biii)、激活状态(图3Biv)和TNFa分泌(图3B、3Bi)。统计分析未发现PFC区TNFa分泌存在显著效应(图3Bi)。

Considering the anticipated type 2 immune response induced by S. mansoni infection, we examined microglia’s production of IL-4 and IL13. Surprisingly, infected non-trained animals produced similar levels of IL-4 and IL-13 in the HPC compared with the non-infected group (Figure 3Aiii). In the PFC, while no significant change in IL-4 production was noted, the INT group exhibited a notable reduction in IL-13 production (Figure 3Biii). After PA, no noticeable differences in IL-4 or IL-13 production by microglia in the PFC were observed in the IT group compared to the NIT group (Figure 3Biv).

考虑到曼氏血吸虫感染预计会引发的2型免疫反应，我们检测了小胶质细胞产生的IL-4和IL13。出乎意料的是，在HPC区域，未训练感染组动物产生的IL-4和IL-13水平与未感染组相当(图 3Aiii)。在PFC区域，虽然IL-4产量未见显著变化，但INT组的IL-13产量明显降低(图 3Biii)。PA处理后，IT组与NIT组相比，PFC区小胶质细胞的IL-4或IL-13产量均未观察到显著差异(图 3Biv)。

For myeloid cell subsets, there were similar frequency of monocytes and eosinophils before (Supplementary Figure S3Ci)

对于髓系细胞亚群，单核细胞和嗜酸性粒细胞在治疗前频率相似（补充图 S3Ci）


FIGURE 2 Impact of schistosomiasis infection and/or physical activities on brain parenchyma. (A) Eggs infiltration in prefrontal cortex (upper row) and hippocampus (lower row) was assessed using H&E staining (original magnification 500um). (B) Immuno fluorescence staining of CA3 area in hippocampus to to show Tmem 119 positive cells area occupied 50um, (the quant if i cation of cell presence within a defined spatial with 50um dept, specifically calculated as the number of cells per cubic millimeter $(m m^{3})$ (Ci) Total cell number in $\mathsf{m m}^{2}$ and (Cii) area occupied in prefrontal cortex. (Ciii) Total cell number in $\mathsf{m m}^{2}$ and (Civ) area occupied in hippocampus. (Di) Tnfa mRNA expression relative to HPRT housekeeping gene determined using qRT-PCR from hippocampus. (Dii) TNFa concentration in hippocampus homogenate using ELISA. mRNA expression level of (Diii) Il6, (Div) Ccl2, (Dv) Il13, and (Dvi) $I/4$ relative to HPRT housekeeping gene determined using qRT-PCR in hippocampus. mRNA expression level of (Ei) Il6 relative to HPRT housekeeping gene determined using qRT-PCR in prefrontal cortex. Muscarinic receptors expression was assessed using qPCR, mRNA expression level of (Fi) M1, (Fii) $M2,$ and (Fiii) M5 relative to HPRT housekeeping gene determined using qRT-PCR in hippocampus. Results are pooled from two different experiments with 6-8 mice per group. Data are expressed as mean $\pm$ S.E.M. NS, $\mathsf{P}>0.05$ $^{\star}P<0.05$ $^{\star\star}P<$ 0.001, $\star\star\star\vert P<0.0001$ by two-way ANOVA followed by Bonferroni.

图 2: 血吸虫感染和/或体力活动对脑实质的影响。(A) 采用H&E染色评估前额叶皮层(上图)和海马体(下图)中的虫卵浸润情况(原始放大倍数500μm)。(B) 海马体CA3区免疫荧光染色显示Tmem119阳性细胞区域占50μm(在50μm深度的限定空间内量化细胞存在情况，具体计算为每立方毫米$(mm^{3})$的细胞数)。(Ci) 前额叶皮层中$\mathsf{mm}^{2}$内的总细胞数和(Cii)占据区域面积。(Ciii) 海马体中$\mathsf{mm}^{2}$内的总细胞数和(Civ)占据区域面积。(Di) 通过qRT-PCR测定海马体中Tnfa mRNA相对于HPRT管家基因的表达量。(Dii) ELISA检测海马体匀浆中TNFa浓度。(Diii) Il6、(Div) Ccl2、(Dv) Il13和(Dvi) $I/4$ mRNA相对于HPRT管家基因的表达量通过qRT-PCR在海马体中测定。(Ei) 前额叶皮层中Il6 mRNA相对于HPRT管家基因的表达量通过qRT-PCR测定。采用qPCR评估毒蕈碱受体表达，(Fi) M1、(Fii) $M2$和(Fiii) M5 mRNA相对于HPRT管家基因的表达量通过qRT-PCR在海马体中测定。结果汇总自两组独立实验，每组6-8只小鼠。数据表示为均值$\pm$标准误。NS表示$\mathsf{P}>0.05$，$^{\star}P<0.05$，$^{\star\star}P<0.001$，$\star\star\star\vert P<0.0001$(双向方差分析后Bonferroni校正)。

FIGURE 3

图 3:


The impact of schistosomiasis infection and/or physical activities on microglia and myeloid cells phenotype. Wildtype BALB/c mice were infected with 0 or 35 S. mansoni cercariae and then euthanized 8 weeks post-infection and different brain regions were collected for characterization using flow cytometry. (A) UMAP vi sul aiz ation of Microglia in hippocampus (HPC) using flow cytometry. (Ai) Frequency of microglia producing TNFa in hippocampus. (Aii) TNFa expression by microglia depicted in (Aii). (Aiii) microglia frequency, MHC class II expression, IL-4, and IL-13 production before and after PA in hippocampus (Aiv). Frequency of microglia producing TNFa in prefrontal cortex (B). TNFa expression by microglia depicted in (Bi, Bii) Representative flow cytometry of microglia and CD11b population. (Biii) microglia frequency, MHC class II expression, IL-4, and IL-3 production before and after PA in prefrontal cortex (Biii, Biv). Results are pooled from two different experiments with 6-8 mice per group. Data are expressed as mean $\pm\mathsf{S}.\mathsf{E}.\mathsf{M}$ . NS, $\mathsf{P}>0.05$ ; $^{\star}P<0.05.$ , $^{\star\star}P<0.001$ by two-way ANOVA followed by Bonferroni or non-parametric test after testing the homogeneity and normality.

血吸虫感染和/或体力活动对小胶质细胞及髓系细胞表型的影响。野生型BALB/c小鼠分别感染0或35条曼氏血吸虫尾蚴，感染8周后处死并采集不同脑区，通过流式细胞术进行表征。(A) 海马体(HPC)小胶质细胞的UMAP可视化分析。(Ai) 海马体产生TNFa的小胶质细胞频率。(Aii) (Aii)所示小胶质细胞的TNFa表达水平。(Aiii) 体力活动(PA)前后海马体小胶质细胞频率、MHC II类分子表达及IL-4、IL-13分泌情况(Aiv)。前额叶皮层产生TNFa的小胶质细胞频率(B)。(Bi, Bii)展示小胶质细胞及CD11b细胞群的典型流式图。(Biii) 前额叶皮层在体力活动前后的小胶质细胞频率、MHC II类分子表达及IL-4、IL-3分泌水平(Biii, Biv)。数据整合自两组独立实验，每组6-8只小鼠。结果以均值$\pm\mathsf{S}.\mathsf{E}.\mathsf{M}$表示。NS表示$\mathsf{P}>0.05$；$^{\star}P<0.05$，$^{\star\star}P<0.001$（经方差齐性与正态性检验后，采用双向ANOVA结合Bonferroni或非参数检验）。

and after PA (Supplementary Figure S3Cii) of S. mansoni infected animals compared with non-infected controls in HPC and PFC. We also noted comparable frequency of $\mathrm{CD11b^{+}}$ cells (Supplementary Figure S3A, B) and their production of IL-4 (Supplementary Figures S3Aiii, Biii) and IL-13 (Supplementary Figures S3Aiv, Biv) in HPC and PFC, respectively, in NINT, INT and IT groups. to summarize this part, our results suggest that while schistosomiasis infection did not significantly impact the CD11b cell phenotype, it did enhance microglia polarization towards an microglia producing TNFa phenotype, particularly in the hippocampus. Importantly, PA might play a role in mitigating microglia adoption of the microglia producing TNFa phenotype by restoring $\mathrm{TNF}$ production to baseline levels. These findings underscore the potential influence of PA in modulating microglia response in the context of schistosomiasis infection and emphasize the need for further investigation into the underlying mechanisms and implications for disease progression.

与未感染对照组相比，曼氏血吸虫感染动物的海马区(HPC)和前额叶皮层(PFC)在PA干预前后(补充图S3Cii)均显示出变化。我们同时观察到，在NINT、INT和IT组中，HPC与PFC区域的$\mathrm{CD11b^{+}}$细胞频率(补充图S3A、B)及其IL-4(补充图S3Aiii、Biii)和IL-13(补充图S3Aiv、Biv)分泌水平均保持相当。综上所述，本部分结果表明：虽然血吸虫感染未显著影响CD11b细胞表型，但会促进小胶质细胞向分泌TNFa的表型极化，这种现象在海马区尤为明显。值得注意的是，PA可能通过将$\mathrm{TNF}$分泌恢复至基线水平，从而抑制小胶质细胞向分泌TNFa表型的转化。这些发现揭示了PA在调节血吸虫感染中小胶质细胞应答方面的潜在作用，并强调需要进一步探究其内在机制及对疾病进展的影响。

Impact of schistosomiasis and training on astrocytes phenotype

血吸虫病及训练对星形胶质细胞表型的影响

Expanding our examination to include other glial cells, specifically astrocytes $\mathrm{GFAP+}$ cells, we probed into their activity, antigen presentation capacity, and cytokine production in response to schistosomiasis infection.

将研究范围扩展到其他胶质细胞，特别是星形胶质细胞 $\mathrm{GFAP+}$ 细胞，我们探究了它们在血吸虫感染中的活性、抗原呈递能力和细胞因子产生情况。

GFAP-positive astrocytes are generally associated with functions like forming glial scars, modulating inflammation, and protecting surrounding neural tissue.

GFAP阳性星形胶质细胞通常与形成胶质瘢痕、调节炎症和保护周围神经组织等功能相关。

To understand the changed happening in GFAP $+.$ Astrocytes, we measured GFAP expression (Figure 4A). Surprisingly, our results revealed a reduction in GFAP expression due to S. mansoni infection in the infected non-trained (INT) compared to noninfected non-trained (NINT) group (Figure 4Bi). Additionally, PA did not alter astrocyte reactivity, with the IT group showing similar GFAP expression compared to the NIT group (Figure 4Bii).

为了解GFAP$+.$星形胶质细胞的变化，我们检测了GFAP表达水平（图4A）。令人惊讶的是，与未感染未训练组（NINT）相比，感染未训练组（INT）中曼氏血吸虫感染导致GFAP表达降低（图4Bi）。此外，体育锻炼（PA）并未改变星形胶质细胞反应性，感染训练组（IT）与未感染训练组（NIT）的GFAP表达水平相近（图4Bii）。

Furthermore, our results indicated that schistosomiasis infection and/or PA led to a significant reduction in astrocyte antigen presentation capacity, marked by lower MHCII expression (Figures 4Bi, Bii). The statistical analysis revealed a significant interaction effect, emphasizing the interdependence of infection and PA on astrocyte antigen presentation capacity (Figures 4Bi, Bii. However, no significant main effects of PA or infection alone on MHCII expression were observed. These changes may signify a loss of $\mathrm{GFAP+}$ astrocytes that maybe responsible for modulating the inflammation or protecting the neural tissue induced by infection. Importantly, there were no significant alterations in the production of type 2 cytokines, namely IL-4 and IL-13, between the INT and NINT groups (Figure 4Bi) or between the NIT and IT groups (Figure 4Bii). Of particular note, PA exerted a positive influence on astrocyte production of TNFa compared to the NINT control group. Similarly, schistosomiasis infection induced a significant increase in $\mathrm{TNF}$ production by astrocytes, which returned to baseline levels after training. These compelling findings highlight that, akin to microglia, PA effectively restored TNFa production by astrocytes to baseline levels post-S. mansoni infection. We can conclude that, our investigation into GFAP $^+$ astrocyte responses underlined the intricate dynamics influenced by schistosomiasis infection and PA. The restoration of $\mathrm{TNF}$ production emphasizes the potential of exercise to modulate astrocyte $\mathrm{GFAP+}$ , offering valuable insights into the broader implications for disease progression and therapeutic strategies. In here, It’s important to note that GFAP does not capture the full diversity of astrocyte subtypes and states, such as those involved in metabolic support, neurotransmitter regulation, or synaptic modulation. Thus additional study should be conducted.

此外，我们的研究结果表明，血吸虫感染和/或PA（physical activity，体力活动）显著降低了星形胶质细胞的抗原呈递能力，表现为MHCII表达水平下降（图4Bi、Bii）。统计分析显示存在显著的交互效应，强调了感染与PA对星形胶质细胞抗原呈递能力的共同影响（图4Bi、Bii）。然而，单独PA或感染对MHCII表达均未产生显著主效应。这些变化可能意味着具有调节炎症或保护神经组织功能的$\mathrm{GFAP+}$星形胶质细胞数量减少。值得注意的是，INT组与NINT组之间（图4Bi）、NIT组与IT组之间（图4Bii）的2型细胞因子（IL-4和IL-13）产量均未出现显著差异。特别需要指出的是，与NINT对照组相比，PA对星形胶质细胞产生TNFα具有正向调节作用。同样，血吸虫感染会显著提升星形胶质细胞的$\mathrm{TNF}$产量，而训练后该指标恢复至基线水平。这些重要发现表明，与小胶质细胞类似，PA能有效使曼氏血吸虫感染后的星形胶质细胞TNFα产量回归基线。我们可以得出结论：针对$\mathrm{GFAP^+}$星形胶质细胞反应的研究揭示了血吸虫感染与PA共同作用的复杂动态机制。$\mathrm{TNF}$产量的恢复证明了运动调节$\mathrm{GFAP+}$星形胶质细胞的潜力，为疾病进展及治疗策略提供了更广阔的视角。需要说明的是，GFAP并不能反映星形胶质细胞亚型与功能状态（如代谢支持、神经递质调节或突触调控等）的完整多样性，因此仍需开展进一步研究。


FIGURE 4 Impact of schistosomiasis infection and/or physical activities on astrocyte phenotype. (A) Representative flow cytometry of $G F A P^{+}$ astrocytes in hippocampus. (Bi) Frequency of GFAP astrocytes, MHC class II expression, and astrocytes producing IL-4 and IL-13 before and (Bii) after training. (Biii) IL-4 (left) and IL-13 (right) expression by astrocytes depicted in (Bi, Bii). (C) Frequency of astrocytes producing TNFa assessed using flow cytometry. Results are pooled from two different experiments with 6-8 mice per group. Data are expressed as mean $\pm$ S.E.M. NS, $\mathsf{P}>0.05$ $^{\star}P<$ 0.05, $^{\star\star}P<0.001$ , $\star\star\star\vert P<0.0001$ by two-way ANOVA followed by Bonferroni.

图 4: 血吸虫感染和/或体力活动对星形胶质细胞表型的影响。(A) 海马区 $G F A P^{+}$ 星形胶质细胞的代表性流式细胞术分析。(Bi) 训练前 GFAP 星形胶质细胞频率、MHC II 类分子表达及产生 IL-4 和 IL-13 的星形胶质细胞比例，(Bii) 训练后相应指标。(Biii) (Bi, Bii) 中星形胶质细胞表达的 IL-4（左）和 IL-13（右）。(C) 流式细胞术检测产生 TNFa 的星形胶质细胞频率。数据汇总自两组独立实验，每组 6-8 只小鼠。结果以均值 $\pm$ 标准误表示。NS: $\mathsf{P}>0.05$，$^{\star}P<0.05$，$^{\star\star}P<0.001$，$\star\star\star\vert P<0.0001$（双因素方差分析后 Bonferroni 校正）。

Schistosomiasis infection alters meningeal lymphocyte composition

血吸虫感染改变脑膜淋巴细胞组成

In our pursuit to characterize the influence of schistosomiasis infection on meningeal lymphocyte composition, we employed flow cytometry to phenotype lymphoid and myeloid cell subsets in the meninges of both non-infected and schistosomiasis-infected mice, both before and after PA. Examining T cell subsets (Figure 5A), we noted that while a similar frequency of naïve $\mathrm{CD4^{+}}$ T cells in the meninges upon S. mansoni infection (Figure 5Bi), the infection led to a significant increase in the frequency of effector T cells (TEM, Figure 5Bii). This effect was influenced by PA,. Additionally, infection alone had a significant effect on both TEM and central memory T cell (TCM) subsets (Figures 5Bii, Biii), highlighting an increase in these T cell subsets in the meninges. However, PA alone did not significantly affect the frequency of TEM or TCM $\mathrm{CD4^{+}}$ T cell subsets.

为了探究血吸虫感染对脑膜淋巴细胞组成的影响，我们采用流式细胞术对感染前后小鼠脑膜中的淋巴和髓系细胞亚群进行表型分析。在T细胞亚群分析中（图5A），虽然曼氏血吸虫感染后脑膜中初始型$\mathrm{CD4^{+}}$T细胞比例保持稳定（图5Bi），但感染导致效应T细胞（TEM，图5Bii）频率显著增加，且该效应受PA影响。此外，单独感染对TEM和中央记忆T细胞（TCM）亚群均有显著影响（图5Bii、Biii），表现为脑膜中这两类T细胞亚群的增加。但单独PA处理对TEM或TCM型$\mathrm{CD4^{+}}$T细胞亚群频率无显著影响。

Further characterization of the $\mathrm{CD4^{+}}$ T cell population in the meninges revealed higher PD1 expression, indicating T cell exhaustion (Figure 5Biv). While PA alone did not significantly impact PD1 expression, it effectively countered the schistosomiasis-induced alteration of $\mathrm{CD4+}$ T cell populations. PA reduced the frequency of TEM $\mathrm{CD4^{+}}$ T populations without affecting other sub populations or PD1 expression compared to the infected (INT) group (Figures 5Bi-Biv).

对脑膜中 $\mathrm{CD4^{+}}$ T细胞群的进一步表征显示PD1表达升高，表明T细胞耗竭（图 5Biv）。虽然单独使用PA对PD1表达无显著影响，但它有效抵消了血吸虫病诱导的 $\mathrm{CD4+}$ T细胞群改变。与感染(INT)组相比，PA降低了TEM $\mathrm{CD4^{+}}$ T细胞群的频率，且未影响其他亚群或PD1表达（图 5Bi-Biv）。

Analyzing the $\mathrm{CD8^{+}}$ T cell population, schistosomiasis infection led to a significant increase in the naïve $\mathrm{CD8^{+}}$ T cell population (Figure 5Ci). The impact of training on the frequency of naïve $\mathrm{CD}8^{+}\mathrm{T}$ cells was influenced by schistosomiasis infection, as indicated by a significant interaction. Notably, PA did not alter the frequency of $\mathrm{CD8^{+}}$ T cell subsets (naïve, TEM, and TCM) compared to the training group (Figures 5Ci-Ciii). Moreover, PA helped in partially restoring the basal frequency of naïve CD8 (Figure 5Ci) and TEM-CD8 (Figure 5Cii) subsets, demonstrating its beneficial impact on these subsets in the meninges after infections. Additionally, PA reduced $\mathrm{CD8^{+}}$ T cell exhaustion, indicated by decreased PD1 expression (Figure 5Civ),.

分析 $\mathrm{CD8^{+}}$ T细胞群时发现，血吸虫感染导致初始 $\mathrm{CD8^{+}}$ T细胞群显著增加（图 5Ci）。训练对初始 $\mathrm{CD}8^{+}\mathrm{T}$ 细胞频率的影响与血吸虫感染存在显著交互作用。值得注意的是，与训练组相比，PA（体育锻炼）未改变 $\mathrm{CD8^{+}}$ T细胞亚群（初始、TEM和TCM）的频率（图 5Ci-Ciii）。此外，PA有助于部分恢复初始CD8（图 5Ci）和TEM-CD8（图 5Cii）亚群的基础频率，表明其对感染后脑膜中这些亚群的有益影响。同时，PA通过降低PD1表达（图 5Civ）减轻了 $\mathrm{CD8^{+}}$ T细胞耗竭。

Further analyses unveiled a significant interaction between PA and infection in the eosinophil population, emphasizing the positive influence of training in mitigating the increase in eosinophils induced by schistosomiasis infection (Figure 5D). Additionally, wide-screen analyses demonstrated an increase in various markers (CD62L, CXCR5, SiglecF, CD44, CD45) in response to S. mansoni infection, which was reduced through PA in the IT group compared to the INT group (Supplementary Figure S4). Furthermore, no significant changes were observed in the $\mathrm{CD19^{+}}$ B cell population (Figure 5E) or $\mathrm{NK1.1^{+}}$ cell population (Figure 5F) in the meninges of INT compared to NINT group.

进一步分析揭示了体力活动(PA)与感染在嗜酸性粒细胞群中的显著交互作用，强调了训练对减轻血吸虫感染引起的嗜酸性粒细胞增多的积极影响(图5D)。此外，广谱分析显示，曼氏血吸虫感染导致多种标志物(CD62L、CXCR5、SiglecF、CD44、CD45)升高，而IT组通过体力活动干预，这些标志物水平较INT组有所降低(补充图S4)。另外，与NINT组相比，INT组脑膜中的$\mathrm{CD19^{+}}$B细胞群(图5E)或$\mathrm{NK1.1^{+}}$细胞群(图5F)均未观察到显著变化。

In the brain parenchyma, particularly the HPC, schistosomiasis infection did not induce major changes in lymphoid cell populations. However, PA had a notable impact, enhancing the $\mathrm{CD4^{+}}$ T cell (Figure 5G) and $\operatorname{CD19^{+}~B}$ cell populations (Figure 5I). Interestingly, the increase in the $\mathrm{CD4^{+}}$ T cell population was not driven by the central memory T cell (TCM) population, which was down regulated before and after schistosomiasis infection (Figure 5H) but possibly through another T cell subset like TEM. Moreover, an increase in the hippocampus CD $19^{+}$ cell population was observed in the S. mansoni infection group after PA (IT) compared to the INT group (Figure 5I), this increase is not related to the infection but is related to the benefit impact of PA on hippocampus.

在脑实质，尤其是海马体(HPC)中，血吸虫感染并未引起淋巴细胞的显著变化。然而，体育锻炼(PA)产生了显著影响：提升了$\mathrm{CD4^{+}}$T细胞(图5G)和$\operatorname{CD19^{+}~B}$细胞群(图5I)。值得注意的是，$\mathrm{CD4^{+}}$T细胞的增加并非由中枢记忆T细胞(TCM)驱动——该细胞群在血吸虫感染前后均呈现下调趋势(图5H)，而可能是通过TEM等其他T细胞亚群实现的。此外，与INT组相比，曼氏血吸虫感染组(IT)在锻炼后海马区CD$19^{+}$细胞群出现增长(图5I)，这种增长与感染无关，而是源于体育锻炼对海马体的积极影响。

In conclusion, our data illustrate the nuanced effects of schistosomiasis infection and PA on lymphoid and myeloid populations in the meninges and brain parenchyma. PA emerges as a potential mitigator of infection-induced changes in these populations, providing valuable insights into the intricate interplay between PA and immune responses in the context of schistosomiasis.

综上所述，我们的数据揭示了血吸虫感染和体育锻炼(PA)对脑膜及脑实质中淋巴系与髓系细胞群的微妙影响。研究表明，体育锻炼可能缓解感染引发的这些细胞群变化，为理解血吸虫病背景下体育锻炼与免疫反应间的复杂相互作用提供了重要见解。

Discussion

讨论

Schistosomiasis infection correlated with diminished cognitive performance, particularly evident in school-aged children afflicted with heavy or moderate infections, leading to educational, learning, and memory deficits (24, 25). Both the specific cognitive domain affected and the mechanism underlying its pathogen es is is in completely understood. The current therapeutic approaches carry a lot of side effects, thereby finding a natural alternative is imperative. Whether PA may help in alleviating these consequences is an open question that yet to be explored. In our study, we investigated the effects of schistosomiasis infection on cognitive function in adult mice, delved into the cellular-level alterations in the CNS during the acute stage of infection, and evaluated the potential benefits of PA in mitigating schistosomiasis-induced systemic inflammation on CNS. First, our findings revealed that during the acute infection in adult mice, schistosomiasis infection did not significantly impair simple and complex learning or spatial reference memory, however; it did adversely affect recall memory. At the cellular level, we observed significant alterations in T cell populations and eosinophils within the meninges, accompanied by dys regulated cytokine production in the hippocampus. Additionally, an increase in microglial frequency and activation status, along with heightened TNFa production in response to the infection. Moreover, the infection promoted the expression of muscarinic receptors in the hippocampus, while reducing the number and antigen presentation capacity of astrocytes. Interestingly, PA in infected animals partially mitigated schistosomiasis-induced changes in glial and immune cell phenotypes within the brain parenchyma and meninges, respectively. Most notably, exercise reduced the production of TNFa by glial cells, indicating a shift towards a less proinflammatory environment. Collectively, these unprecedented findings shed light on the immune and glial cell alterations within the CNS induced by schistosomiasis and underscore the potential of PA in ameliorating schistosomiasis-induced CNS alterations.

血吸虫感染与认知能力下降相关，尤其在中重度感染的学龄儿童中表现明显，导致教育、学习和记忆缺陷 (24, 25)。其具体影响的认知领域及致病机制尚未完全阐明。现有疗法副作用较多，因此寻找天然替代方案至关重要。PA（物理活动）是否能缓解这些后果仍有待探索。本研究调查了血吸虫感染对成年小鼠认知功能的影响，探究了感染急性期中枢神经系统（CNS）的细胞水平变化，并评估了PA在减轻血吸虫感染引发的全身炎症对CNS影响的潜在益处。

首先，我们发现成年小鼠在急性感染期间，血吸虫感染未显著损害简单或复杂学习能力及空间参考记忆，但确实对回忆记忆产生负面影响。在细胞层面，观察到脑膜中T细胞群和嗜酸性粒细胞显著改变，同时海马体细胞因子分泌失调。此外，小胶质细胞频率和激活状态增加，感染还促使TNFα产量上升。感染还促进了海马体毒蕈碱受体表达，同时降低了星形胶质细胞的数量和抗原呈递能力。

值得注意的是，感染动物的PA部分缓解了血吸虫引发的脑实质和脑膜中胶质细胞及免疫细胞表型变化。最显著的是，运动降低了胶质细胞TNFα的产量，表明促炎环境有所改善。这些前所未有的发现揭示了血吸虫感染引起的中枢神经系统免疫及胶质细胞变化，并凸显了PA在改善血吸虫诱导的CNS改变方面的潜力。


FIGURE 5 Impact of schistosomiasis infection and/or physical activities on lymphoid and myeloid cells in meninges and brain parenchyma. (A) Representative flow cytometry of $\mathsf{C D4}^{+}$ and $\mathsf{C D8^{+}}$ T cell populations (upper row) and CD62L+ CD44+ central memory and CD62L $\mathsf{C D44}^{+}$ effector T cell subsets (lower row) in the meninges. (Bi) Frequency of naïve, (Bii) effector memory (TEM), and (Biii) central memory (TCM) subsets and (Biv) the expression of PD1 by $\mathsf{C D4^{+}}$ T cells. (Ci) Frequency of naïve, (Cii) effector memory (TEM), and (Ciii) central memory (TCM) subsets and (Civ) the expression of PD1 by $\mathsf{C D8^{+}}$ T cells. Frequency of (D) eosinophils, (E) $\mathsf{C D19^{+}}$ B cells, and $\mathbb{N}\mathbb{K}\mathbb{1}.1^{+}$ cells in the meninges. (G) Frequency of total $\mathsf{C D4}^{+}$ T cells and (H) TCM subset in the HPC. (I) Frequency of CD19+ B cells in the HPC. Results are pooled from two different experiments with 6-8 mice per group. Data are expressed as mean $\pm$ S.E.M. NS, $\mathsf{P}>0.05$ ; $^{\star}P<0.05.$ $^{\star\star}P<0.001$ , $\star\star\star\vert P<0.0001$ $\star\star\star\vert P<0.000$ . by ANOVA followed by Bonferoni, or un parametric test.

图 5: 血吸虫感染和/或体力活动对脑膜及脑实质中淋巴系和髓系细胞的影响。(A) 脑膜中 $\mathsf{CD4}^{+}$ 和 $\mathsf{CD8^{+}}$ T细胞群(上行)及CD62L+ CD44+中枢记忆与CD62L $\mathsf{CD44}^{+}$ 效应T细胞亚群(下行)的代表性流式细胞术分析。(Bi) 初始、(Bii) 效应记忆(TEM)、(Biii) 中枢记忆(TCM)亚群频率及(Biv) $\mathsf{CD4^{+}}$ T细胞PD1表达。(Ci) 初始、(Cii) 效应记忆(TEM)、(Ciii) 中枢记忆(TCM)亚群频率及(Civ) $\mathsf{CD8^{+}}$ T细胞PD1表达。(D) 脑膜中嗜酸性粒细胞、(E) $\mathsf{CD19^{+}}$ B细胞及 $\mathbb{NK1.1}^{+}$ 细胞频率。(G) 海马区(HPC)总 $\mathsf{CD4}^{+}$ T细胞及(H) TCM亚群频率。(I) HPC区CD19+ B细胞频率。结果汇总自两组独立实验(每组6-8只小鼠)。数据以均值 $\pm$ 标准误表示。NS: $\mathsf{P}>0.05$；$^{\star}P<0.05$；$^{\star\star}P<0.001$；$\star\star\star\vert P<0.0001$；$\star\star\star\vert P<0.000$ (经ANOVA及Bonferroni检验或非参数检验)。

Several studies have indicated that schistosomiasis infection causes significant impairment of brain cognitive functions (3, 25). Whereas schistosomiasis impact on cognitive functions were assessed at the preclinical and clinical stages (5, 10), information is very scarce about the specific cognitive domain(s) impacted in adult age. Recent paper on the impact of schistosomiasis in post-natal mice showed that infected animals suffered from impairment in spatial learning and memory formation during acute schistosomiasis (10). By contrast, our findings indicated unaffected simple and complex learning as well as spatial reference memory in adult mice. The infection, however; resulted in impairment in the recall cognitive behavior. Two potential explanations may elucidate these disparities: firstly, schistosomiasis may exert varying impacts on cognitive domains depending on the host’s maturation stage; and secondly, a higher infection inoculum used in the previous study could lead to more severe impacts of schistosomiasis infection on CNS function. Future experiments involving different infection doses administered to mice at various maturation stages will be instrumental in developing a more comprehensive understanding in that regard. Regarding affective behavior, emotions-associated behaviour, schistosomiasis infection did neither alter locomotor activity nor anxiety levels, as infected mice exhibited similar distances moved and velocities compared to uninfected controls. These findings are consistent with a previous report showing that infection of postnatal mice did neither alter locomotion nor anxiety level (10), and in contrast with others that demonstrated impact of helminth infection on the locomotion, suggesting specific species-neuro behavior impact (26, 27). Together, these findings suggested that while schistosomiasis infection had a specific effect on spatial reference memory, it had minimal influence on motor performance and affective behavior in adult mice.

多项研究表明，血吸虫感染会导致大脑认知功能显著受损[3,25]。虽然已有研究在临床前和临床阶段评估了血吸虫病对认知功能的影响[5,10]，但关于其对成年期特定认知领域影响的信息非常匮乏。近期关于血吸虫病对产后小鼠影响的论文显示，感染动物在急性血吸虫病期间会出现空间学习和记忆形成障碍[10]。相比之下，我们的研究结果表明成年小鼠的简单/复杂学习能力和空间参考记忆均未受影响，但感染确实导致了回忆认知行为的损伤。两种潜在解释可能阐明这些差异：其一，血吸虫病可能根据宿主成熟阶段对认知领域产生不同影响；其二，先前研究使用的更高感染剂量可能导致血吸虫感染对中枢神经系统功能造成更严重影响。未来针对不同成熟阶段小鼠施用不同感染剂量的实验，将有助于形成更全面的认知。在情感行为方面，血吸虫感染既未改变运动活性也未影响焦虑水平，因为感染小鼠与未感染对照组表现出相似的运动距离和速度。这些发现与先前显示产后小鼠感染既未改变运动能力也未影响焦虑水平的报告一致[10]，而与证明蠕虫感染影响运动能力的研究形成对比，提示存在特定物种-神经行为影响[26,27]。综上表明，虽然血吸虫感染对空间参考记忆有特定影响，但对成年小鼠运动表现和情感行为的影响微乎其微。

Due to the spine and the large size of S. mansoni eggs, they are almost unable to traverse the blood brain barrier and they usually infiltrate the lower domain of the spinal cord (5, 28, 29). In agreement, our his to logical examination of brain sections indicated absence of eggs translocation into the brain. We also noted absence of egg infestation in the spinal cord likely due to the acute stage of the disease, which is one of the limitation in this study. Therefore, the noted schistosomiasis impact on the CNS was most likely through the systemic inflammation. This systemic inflammation led to significant alteration in T cell profile in meninges. In fact, there was a remarkable increase in effector and central memory $\mathrm{CD4^{+}}$ T cell population that was associated with increased cell exhaustion as indicated by higher level of PD-1 expression. We also noted an increase in naïve $\mathrm{CD8^{+}}$ T cell population. These alterations in T cell population in the brain could be the causal of significant impairment of hippo camp al-dependent spatial learning and memory acquisition (30, 31). Hence, the changes in T cell population could be driving the schistosomiasis-induced impairment of the recall cognitive functions.

由于曼氏血吸虫卵的棘刺和较大体积，它们几乎无法穿过血脑屏障，通常只浸润脊髓下部区域 (5, 28, 29)。与此一致的是，我们对脑组织切片的病理学检查显示虫卵并未转移至脑部。同时观察到脊髓中也未出现虫卵浸润，这可能是疾病处于急性期的缘故，也是本研究的局限性之一。因此，血吸虫病对中枢神经系统的影响很可能是通过系统性炎症实现的。这种系统性炎症导致脑膜中T细胞谱系发生显著改变：效应记忆T细胞和中央记忆$\mathrm{CD4^{+}}$T细胞群显著增加，且伴随PD-1表达水平升高显示的细胞耗竭现象；同时 naïve $\mathrm{CD8^{+}}$T细胞群也有所增加。这些脑内T细胞群的改变可能是导致海马依赖的空间学习和记忆获取能力显著受损的原因 (30, 31)。由此可见，T细胞群的变化可能是血吸虫病诱发回忆认知功能障碍的驱动因素。

Schistosomiasis infection led also to a significant increase in the frequency of eosinophil population in the meninges, the phenomenon previously demonstrated in T. regenti infection (26). Further investigation is warranted to determine whether the elevated infiltration of eosinophils was driven by increased production of IL-5 by T cells (32).

血吸虫感染还导致脑膜中嗜酸性粒细胞群频率显著增加，这一现象在T. regenti感染中已有报道[26]。需进一步研究以确定嗜酸性粒细胞浸润增加是否由T细胞产生的IL-5增多驱动[32]。

By affecting the cell dynamics in the meninges, neuro inflammation in the brain parenchyma was highly anticipated. In agreement, there was an increase in microglia frequency and activation in hippocampus, while maintaining similar frequency and reactivity in prefrontal cortex, suggesting a profound impact of schistosomiasis infection on HPC compared to prefrontal cortex at adult stage. Significant increase in microglia was also recorded in the CNS of schist oso mati dae infected mice (26). Investigating microglia polarization indicated a significant increase in $\mathrm{TNF}$ production in the infected group compared to noninfected non-trained control. While TNFa production can promote inflammation and edema in addition to its toxic effects on neuronal structure and myelin, it could also promote neural cell survival and proliferation (13). The tendency of one effect over another is context dependent. In the present study, the predominant production of TNFa over type 2 cytokines (IL-4 and IL-13) could suggest the domination of pro-inflammatory effects of TNFa during neuro schistosomiasis after 8 weeks post-infections. Microglia production of TNFa may initiated neuro inflammation and aggravate neural tissue injury. It could further impair the neuro genesis and syn ap to genesis in hippocampus (33) which may explain the dys regulation in recall memory (34, 35). The pre domination of pro-inflammatory environment in hippocampus during neuro schistosomiasis could be the cause of the increased cell infiltration and reduction in the area occupied [the quant if i cation of cell presence within a defined spatial area, specifically calculated as the number of cells per cubic millimeter $(\mathrm{mm}^{3})$ ] in the prefrontal cortex but not the hippocampus.

通过影响脑膜中的细胞动态，脑实质中的神经炎症备受关注。与此一致的是，海马区小胶质细胞频率和活化程度增加，而前额叶皮层保持相似的频率和反应性，表明血吸虫感染对成年阶段海马区（HPC）的影响远大于前额叶皮层。感染日本血吸虫的小鼠中枢神经系统中也记录到小胶质细胞的显著增加[26]。对小胶质细胞极化的研究表明，与未感染未训练的对照组相比，感染组中$\mathrm{TNF}$的产生显著增加。虽然TNFα的产生除了对神经元结构和髓鞘的毒性作用外，还可能促进炎症和水肿，但它也可能促进神经细胞的存活和增殖[13]。其中一种效应占主导地位取决于具体情境。在本研究中，TNFα的产生量超过2型细胞因子（IL-4和IL-13），可能表明感染后8周的神经血吸虫病期间TNFα的促炎效应占主导。小胶质细胞产生的TNFα可能引发神经炎症并加剧神经组织损伤，还可能进一步损害海马区的神经发生和突触发生[33]，这可以解释回忆记忆的失调[34, 35]。神经血吸虫病期间海马区促炎环境的主导可能是前额叶皮层（而非海马区）细胞浸润增加和占据面积减少[定义为特定空间区域内细胞数量的量化，具体计算为每立方毫米$(\mathrm{mm}^{3})$的细胞数]的原因。

Cytokine profile in brain parenchyma was also significantly altered. We noted a consistent increase in IL-6 in hippocampus and prefrontal cortex, and IL-4 and TNFa in HPC. The higher level of IL-6 and IL-4 is consistent with the previous study demonstrated an increase in the same cytokines in cerebrospinal fluid (CSF) in spinal cord schistosomiasis (SCS) patients (36). Schistosomiasis infection at ovi position stage, $>6$ weeks post-infection, is characterized by the domination of type 2 immunity (4). Hence, the production of IL-4 and IL-6 could be helping in skewing the immune response to type 2 immunity in the CNS to down modulate pro-inflammatory immune response and diminish reactive oxygen and nitrogen species production that may damage the tissue. In fact, they could possibly mitigate the increased expression level of TNFa in hippocampus produced by microglia and astrocytes in response to the infection. The increase in TNFa is in disagreement with previous attempts by other investigator showing lower TNFa in CSF of SCS patients (36) and similar TNFa in prefrontal cortex in schistosomiasis-infected mice at postnatal stage (10). Of note, IL-6 may also induce nervous tissue lesion. Future studies will be needed to dissect the independent functional contributions of these cytokine to schistosomiasis effects on CNS.

脑实质中的细胞因子谱也发生了显著改变。我们注意到海马体和前额叶皮层中IL-6持续升高，而海马体中IL-4和TNFa水平上升。IL-6与IL-4的升高趋势与先前研究一致，该研究显示脊髓血吸虫病(SCS)患者脑脊液(CSF)中相同细胞因子水平升高[36]。血吸虫感染在产卵阶段（感染后>6周）以2型免疫反应为主导特征[4]。因此，IL-4和IL-6的产生可能有助于中枢神经系统向2型免疫反应偏移，从而下调促炎性免疫反应，减少可能损伤组织的活性氧和氮物种生成。事实上，它们可能减轻小胶质细胞和星形胶质细胞响应感染时在海马体产生的TNFa表达水平升高。TNFa的升高与其他研究者的发现存在矛盾——既往研究显示SCS患者脑脊液中TNFa较低[36]，而产后阶段感染血吸虫的小鼠前额叶皮层中TNFa水平相当[10]。值得注意的是，IL-6也可能诱导神经组织损伤。未来研究需要解析这些细胞因子对血吸虫病影响中枢神经系统的独立功能贡献。

Schistosomiasis infection further induced alteration in the choline r gic system. This could be due to the reliance of schistosomiasis parasites on the neurotransmitters produced by the host for the activation of their nervous system (37). Schistosomiasis nervous system is principal in the parasites survival, motility, nutrient uptake, and reproduction. ACh is one of the most important neurotransmitters needed for muscles contraction and nutrient absorption (38–40). Enhanced ACh expression by the cells in the spinal cord could then be driven by the parasites in favor of their survival. The increased production of ACh was paralleled by enhanced expression of the muscarinic receptors in the hippocampus namely, M1, M2 and M5. Recently, it was shown that systemic schistosomiasis infection results in enhanced phosphor yl ation of Tau, micro tubule associated protein (10). Increased uptake of Tau by neurons through M1 and M3 receptors enhanced microglia activation while maintaining similar GFAP expression level (41). Our results partially goes in line with these findings as we noted increase in microglia population and similar GFAP expression. However, the frequency of $\mathrm{GFAP^{+}}$ astrocytes was significantly diminished in infected animals and were characterized by their potency of producing higher level of TNFa. The changes in muscarinic receptors expression could be playing a role in the noted immune dys regulation (42).

血吸虫感染进一步引起胆碱能系统的改变。这可能是由于血吸虫依赖宿主产生的神经递质来激活其神经系统 (37)。血吸虫神经系统对寄生虫的生存、运动、营养摄取和繁殖至关重要。乙酰胆碱 (ACh) 是肌肉收缩和营养吸收所需的最重要神经递质之一 (38-40)。脊髓细胞中ACh表达增强可能是寄生虫为促进自身存活所驱动的。ACh产量增加的同时，海马区毒蕈碱受体（M1、M2和M5）的表达也相应增强。近期研究表明，系统性血吸虫感染会增强Tau蛋白（微管相关蛋白）的磷酸化 (10)。神经元通过M1和M3受体对Tau蛋白的摄取增加，在保持相似GFAP表达水平的同时增强了小胶质细胞的活化 (41)。我们的结果部分支持这些发现，因为观察到小胶质细胞数量增加和GFAP表达水平相近。但感染动物中$\mathrm{GFAP^{+}}$星形胶质细胞频率显著降低，其特征是产生更高水平的TNFa。毒蕈碱受体表达的变化可能在观察到的免疫失调中发挥作用 (42)。

The utilization of physical activity (PA) as an adjunctive therapy in the treatment of neuro schistosomiasis represents an innovative approach to modulate host immune responses and potentially ameliorate disease outcomes. Pra zi quant el (PZQ) remains the primary anti helm in tic drug for treating schistosomiasis, and its efficacy is wellestablished; it can reduce neuro schistosomiasis symptoms as schoolaged children treated with PZQ often show enhanced cognitive performance, including improvements in attention, memory, and problem-solving skills. However, while PZQ effectively kills the parasites, it does not directly address the inflammatory symptoms and complications associated with neuro schistosomiasis. Consequently, adjunctive therapies like corticosteroids are frequently needed to manage symptoms, which comes with their own set of potential side effects (5, 6). Previous studies have found a protective effect of PA against inflammation-associated diseases (11, 12).

将体力活动(PA)作为神经血吸虫病的辅助治疗手段，是一种通过调节宿主免疫反应来潜在改善疾病预后的创新方法。吡喹酮(PZQ)目前仍是治疗血吸虫病的主要抗蠕虫药物，其疗效已得到充分证实：该药物能减轻神经血吸虫病症状，经PZQ治疗的学龄儿童常表现出认知能力提升，包括注意力、记忆力和问题解决能力的改善。然而，PZQ虽能有效杀灭寄生虫，却无法直接缓解神经血吸虫病相关的炎症症状和并发症。因此，临床上常需联用皮质类固醇等辅助疗法来控制症状，但这些药物本身也存在潜在副作用(5, 6)。既往研究表明，体力活动对炎症相关疾病具有保护作用(11, 12)。

Therefore, in our study, we explored the intricate effects of an 8- day PA regimen on glial and immune cells within the CNS of schistosomiasis-infected subjects. Our data revealed that PA not only mitigated neuro inflammation but also fostered a milieu conducive to neuro protection. The dampening of TNFa production and the concurrent increase in IL-13 within the hippocampus suggested a potential shift toward an anti-inflammatory state. This was particularly pertinent given the neurotoxic potential of chronic TNFa elevation and the neuro protective role IL-13 can play. Exerciseinduced modulation of glial cell activity, characterized by the normalization of pro-inflammatory cytokine expression, indicated that PA exerted a regulatory influence over CNS innate immunity, which could have broad implications for limiting the neurological impact of schistosomiasis.

因此，在我们的研究中，我们探讨了8天体育锻炼(PA)方案对血吸虫感染受试者中枢神经系统(CNS)中胶质细胞和免疫细胞的复杂影响。我们的数据显示，PA不仅能减轻神经炎症，还能营造有利于神经保护的环境。海马体中TNFa产生的抑制与IL-13的同步增加，表明可能向抗炎状态转变。这一点尤为重要，因为慢性TNFa升高具有神经毒性潜力，而IL-13可发挥神经保护作用。运动诱导的胶质细胞活性调节（以促炎细胞因子表达正常化为特征）表明，PA对中枢神经系统先天免疫具有调节作用，这可能对限制血吸虫感染的神经学影响具有广泛意义。

Furthermore, PA had a restorative effect on neurotransmitter system markers, such as ACh in the spinal cord and M5 muscarinic receptors in the hippocampus, which may indicate a broader impact on CNS function and disease resilience. The PA regimen positively influenced lymphocyte populations, reducing the frequency of CD4 $^+$ and $\mathrm{CD8+}$ effector memory T cells and eosinophils within the meninges. These could further alleviate meningeal inflammation and reduce the risk of long-term structural CNS damage. These neuro-immunological adjustments underscored the capacity of PA to act beyond a simple physical enhancer and position it as a powerful modulator of central immune and neural processes.

此外，PA（物理活动）对神经递质系统标志物具有恢复作用，例如脊髓中的ACh（乙酰胆碱）和海马中的M5毒蕈碱受体，这可能表明其对中枢神经系统（CNS）功能和疾病抵抗力的广泛影响。PA方案对淋巴细胞群产生积极影响，降低了脑膜内CD4$^+$和$\mathrm{CD8+}$效应记忆T细胞以及嗜酸性粒细胞的频率。这些变化可能进一步缓解脑膜炎症，并降低长期CNS结构损伤的风险。这些神经免疫学调整突显了PA不仅作为简单的身体增强剂，还能作为中枢免疫和神经过程的有力调节器。

However, despite these beneficial CNS effects, PA unexpectedly influenced other disease dynamics. We previously demonstrated that gut smooth muscles hyper contract ili ty is crucial for egg expulsion and any defects in this process led to higher susceptibility to the infection and premature death (23). Consequently, we expected that enhancing gut smooth muscle hyper contract ili ty by PA would help in reducing egg burden but in contrary we noted significant increase in the egg burden after training. This unexpected finding suggested that PA could somehow have interfered with the egg expulsion process, possibly by redirecting the body’s activity in another direction. We also noted worsened the egg-induced granuloma and fibrosis. One possible explanation could be that increase in IL-6 and C-reactive protein usually generated during PA could have led to the exacerbation of the disease immuno pathology (11). Given these findings, it is crucial to understand the balance between beneficial and potentially detrimental effects of PA. Our results thus necessitate a careful consideration of the role of exercise in the management of schistosomiasis, highlighting the importance of future research to delineate the optimal types and intensities of PA that could improve disease outcomes without worsening the peripheral pathology, particularly concerning liver health and fibrosis, which are not ameliorated by PA according to previous studies.

然而，尽管存在这些有益的中枢神经系统效应，体力活动（PA）却意外影响了其他疾病动态。我们先前证明肠道平滑肌过度收缩性对虫卵排出至关重要，该过程的任何缺陷都会导致更高的感染易感性和过早死亡 [23]。因此，我们预期通过PA增强肠道平滑肌过度收缩性将有助于减轻虫卵负荷，但相反地，我们观察到训练后虫卵负荷显著增加。这一意外发现表明，PA可能通过将身体活动导向其他方向，从而干扰了虫卵排出过程。我们还注意到虫卵诱导的肉芽肿和纤维化恶化。一种可能的解释是，PA期间通常产生的IL-6和C反应蛋白增加可能导致疾病免疫病理学的恶化 [11]。鉴于这些发现，理解PA的有益效应与潜在有害效应之间的平衡至关重要。因此，我们的研究结果需要谨慎考虑运动在血吸虫病管理中的作用，并强调未来研究的重要性，以明确能够改善疾病结局而不加重外周病理（尤其是肝脏健康和纤维化）的最佳PA类型和强度——根据先前研究，PA并未改善这些方面。

Overall, our results demonstrated the impact of schistosomiasis infection on the cognitive function and cellular composition and phenotype in the CNS and how regular moderate PA could help in mitigating the impact of schistosomiasis on the CNS. Our results indicated impairment specifically in the recall memory during acute stage of the infection in adult mice. We further demonstrated the changes in glial and immune cells in CNS. Our results illustrated changes in T cells and eosinophils in the meninges which were accompanied by higher the propensity of glial cell to produce proinflammatory cytokines, particularly TNFa during schistosomiasis infection. We then highlighted how PA alleviated the schistosomiasis-induced impacts on the CNS. Together, our data support that moderate regular PA could be a natural non-invasive strategy that could help in mitigating the impact of S. mansoni infection on CNS.

总体而言，我们的研究结果证明了血吸虫感染对中枢神经系统认知功能、细胞组成及表型的影响，以及定期适度体力活动（PA）如何帮助减轻血吸虫对中枢神经系统的影响。结果表明，成年小鼠在感染急性期特别表现出回忆记忆能力的损伤。我们进一步展示了中枢神经系统中胶质细胞和免疫细胞的变化。研究数据揭示了脑膜中T细胞和嗜酸性粒细胞的变化，这些变化伴随着胶质细胞在血吸虫感染期间产生促炎细胞因子（尤其是TNFa）的倾向增强。随后我们重点阐述了体力活动如何缓解血吸虫感染对中枢神经系统的影响。综合数据表明，定期适度体力活动可能成为一种天然无创策略，有助于减轻曼氏血吸虫感染对中枢神经系统的影响。

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