Transplantaion of Parabacteroides distasonis mitigated Aβ-associated changes in APP/PS1 mice via gut-brain interactions by attenuating neuroinflammation

Transplantaion of Parabacteroides distasonis mitigated Aβ-associated changes in APP/PS1 mice via gut-brain interactions by attenuating neuroinflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transplantaion of Parabacteroides distasonis mitigated Aβ-associated changes in APP/PS1 mice via gut-brain interactions by attenuating neuroinflammation jingwen Jiang, Yixi He, Hong Pan, Nanjie Xu, Yuyan Tan, dingya Sun, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4156881/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Alzheimer's Disease (AD) is a neuropathological condition marked by cognitive deterioration and chronic neuroinflammation. Previous investigations have unveiled a strong correlation between the gut microbiota and the progression of AD. In this study, our objective is to probe the effects of Parabacteroides distasonis ( P.distasonis ), previously found to be conspicuously diminished in AD patients, on the APP/PS1 mice model. Methods: To assess the impact of orally administered P.distasonis on gut microbiota and metabolites, we utilized 16s rDNA sequencing and GC-MS to analyze gut composition and short-chain fatty acids in APP/PS1 mice after one month of P.distasonis gavage. To investigate the effects of P.distasonis administration over a six-month period on APP/PS1 mice, we evaluated cognitive function using novel object recognition and Y-maze tests, assessed intestinal barrier integrity and AD-related pathological features with immunofluorescence, and analyzed immune cell subpopulations in intestine, blood, spleen, and brain tissues via flow cytometry. The Luminex assay was employed to detect inflammatory cytokine secretion in the same regions. Results: One-month oral administration of P.distasonis modulated the gut microbiota, elevated butyrate levels. Six-month oral administration of P.distasonis improved cognitive function in APP/PS1 mice, reducing Aβ deposition and inhibiting glial cell proliferation. It also amplified Treg cells within the gut, concomitant with the decreased Th1 proliferation and intestinal inflammation. Additionally, we observed the migration of peripheral CD4 + T cells to the brain through chemotaxis, accompanied by an increase in Treg cells and higher levels of anti-inflammatory factors such as IL-10 and TGF-β in the brain. Collectively, these multifaceted effects contributed to the alleviation of neuroinflammation. Conclusion: These findings underscore the potential of transplanting P.distasonis in alleviating AD-related pathology, suggesting a role for gut microbiota in neuroinflammation attenuation. Parabacteroides distasonis Alzheimer's Disease Treg cell Butyrate Chemotaxis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background Alzheimer's disease (AD) is a progressive neurodegenerative illness characterised by cognitive decline [ 1 , 2 ] . It is estimated that between 60 and 70 percent of all occurrences of dementia may be attributed to AD, making it the most prevalent form of dementia among the aged worldwide [ 3 ] . Recent cross-sectional data shows that 9.83 million people in China are living with AD [ 4 ] . The hallmark of AD biology is the accumulation of amyloid-beta (Aβ) peptide outside of cells and the aggregation of hyperphosphorylated tau within cells [ 5 , 6 ] . Therapeutic efforts are currently focused on targets within this framework that have the potential to substantially alter the clinical course of Alzheimer's disease patients. Several factors including age-related deterioration, degeneration of anatomical pathways, environmental influences, mitochondrial malfunction, immune system dysfunction, and genetic susceptibility have been identified as potential contributors to AD [ 7 ] . Apart from influencing the physiology and metabolism of the host [ 8 ] , the gut microbiota has been demonstrated to exert an effect on cognitive development and function [ 9 ] . Multiple studies have provided evidence indicating that the presence of intestinal flora may have an impact on the synthesis of various neurotransmitters and neuromodulators. This, in turn, can alter the communication between the gut and the brain, ultimately influencing brain function [ 10 , 11 ] . The bidirectional gut-brain axis facilitates the interaction between the gut microbiota and the central nervous system through various channels, including the neuro-immune system, the sympathetic and parasympathetic autonomic nervous system, and the neuroendocrine system [ 12 , 13 ] . The intestinal microbiota composition of APP/PS1 mice has been observed to undergo changes upon administration of antibiotics, resulting in a noteworthy rise in the abundance of genus Akkermansia and family Lachnospiraceae. The administration of this treatment is also associated with a decline in the levels of pro-inflammatory cytokines in circulation and a reduction in reactive gliosis surrounding Aβ plaques. Nevertheless, the safety and long-term advantages of this method are still a subject of debate [ 14 ] . The potential of investigating AD through the manipulation of the gut microbiota via targeted microbial transplantation is significant. Akbari et al. [ 15 ] executed a probiotic intervention study on 60 patients diagnosed with AD, administering Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, and Lactobacillus fermentum. The study findings indicated noteworthy enhancements in MMSE scores and a simultaneous reduction in peripheral inflammatory markers among the participants who received probiotic treatment. Additionally, research has demonstrated that probiotics can rectify microbiota dysbiosis and decrease Aβ accumulation within the brain [ 16 , 17 ] . Therefore, it is imperative to acknowledge the significance of the gut microbiota in the investigation of therapeutic approaches for AD. Parabacteroides, a class of gram-negative anaerobic bacteria that frequently inhabit the gut of various species [ 18 ] . Our preceding investigation has found a marked decline in the abundance of Parabacteroides in the fecal microbiota of AD patients compared to their healthy counterparts [ 19 ] . Previous study also demonstrated an inverse correlation between the existence of P.distasonis and colonic IL-1β levels as well as intestinal tumor burden in Apc1638N mice [ 20 ] . In addition, it is plausible that bacterial elements have the ability to traverse the blood-brain barrier and infiltrate the gut-associated lymphatic tissue (GALT), thereby eliciting responses from T-cells and other immune cells. Research has shown that specific chemicals produced by bacteria have the potential to stimulate the generation of effector-type T cells, thereby exerting an impact on neuroinflammation [ 21 ] . P.distasonis facilitated the development of IL-10 + FoxP3 + Tregs and anti-inflammatory CD4 + CD25 + T cells in germ-free mice [ 22 ] , thus, P.distasonis might hold promise for personalized therapy and nutritional supplements for inflammatory bowel diseases. In addition, extant data suggests that T lymphocytes have the ability to penetrate the brain and engage in inflammatory responses via chemotaxis [ 23 , 24 ] , a process that is intimately linked to the anomalous accumulation of Aβ in the brains of those afflicted with AD. To gain further insights into the impact of P.distasonis on AD and its underlying mechanism, we performed the study of administering the bacteria to APP/PS1 transgenic mice through gavage. 2. Methods 2.1 Ethics Statement All research conducted received approval from The Charles River Laboratories Institutional Animal Care and Use Committee (IACUC) under approval number P2021098. The study was reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) [ 25 ] . 2.2 Animals The APP/PS1 mice (B6.Cg-Tg(Thy1-APPSw,Thy1-PSEN1*L166P)21Jckr) were generously contributed by Mathias Jucker. In consideration of the pronounced gender effects noted in prior studies on gut microbiota, this investigation opted for a standardized approach by exclusively utilizing male mice [ 26 ] . Male mice of the APP/PS1 strain, which have a genetic background of C57Bl/6J, were crossbred with female mice of the C57Bl/6J wild-type strain. The male offsprings were subjected to genotyping through the utilisation of polymerase chain reaction. The control group consisted of age-matched male wild-type (WT) littermates lacking the APP/PS1 transgene. The rodents were accommodated in a climate-regulated setting with a temperature range of 20–22°C and a light/dark cycle of 12 hours each. 2.3 Microbial Strains The strain P.distasonis 8503 was procured from ATCC (Manassas, VA)and reconstituted in 1.9g Reinforced Clostridial Medium (Becton, Dickinson and Company, 218081) dispensed in 50ml aliquots in a glass bottle supplemented with 0.025g L-Cysteine hydrochloride monohydrate (Solarbio, C0011) and 5ul C 12 H 6 NNaO 4 (1mg/mL, Sigma-Aldrich, 199303) at 37℃ in an anaerobic chamber (MITSUBISHI, C-33) for 24 hr. The strain was identified by comparing the 16S rRNA gene sequences with the NCBI reference database ( https://www.ncbi.nlm.nih.gov/ , NCBI Reference Sequence: NC_009615.1). After centrifugation (8000× g rpm, 15 min at 4 ℃), culture pellets were washed with phosphate buffered saline (PBS; pH 7.2) (G4202, Servicebio, Wuhan, China) maintained in anaerobiosis. Cell pellets were suspended at 10 8 CFU/mL in anaerobic PBS containing 25% glycerol (56-81-5, Sangon Biotech, Shanghai, China).and suspensions were frozen in liquid nitrogen before storage at − 80 ℃. The cell suspension for oral administration in treatment group was prepared by suspending the cultured bacterial cells in oxygen-free PBS with a final cell density of 10 8 CFU per mL. A daily administration of suspension solution at a volume of 0.2 mL was implemented. The interventions were administered for a period of one month and six months, respectively. 2.4 Experimental design 3-month-old male APP/PS1 mice were randomly divided into two groups (n = 16 per group): (1) AD model (APP/PS1) and P.distasonis treatment group(APP/PS1 + P.distasonis ); (2) AD model (APP/PS1) and PBS treatment group (APP/PS1 + PBS). 3-month-old male WT mice treated with PBS were used as control group (WT + PBS, n = 16). All groups had access to food and water ad libitum. Figure 1 A displays the timeline of the experiment. 2.5 Fecal 16S rDNA gene sequencing The 4-month-old mice were anesthetized with approximately 0.6 mL of liquid isoflurane (Shanghai Yuyan Scientific Instrument Company, Shanghai, China, Cat# 100150) per liter of chamber volume. After anesthesia, fresh colon fecal samples (0.2–0.3 g each) were collected and stored. DNA extraction utilized the Qiagen QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany, 51504), with concentration and molecular weight assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis. The V3-V4 region of the bacterial 16S ribosomal RNA gene was amplified, sequenced on the Illumina MiSeq platform, and analyzed using QIIME (version 1.9.1) and UPARSE(version 7.1) for quality filtering, de-replication, and clustering. Chimeric sequences were removed by UCHIME. Taxonomic analysis employed the RDP classifier against the Silva (SSU123) 16S rDNA database, and gut microbiota characteristics were assessed using linear discriminant analysis effect size (LEfSe) for significant differences and effect size evaluation. 2.6 Liquid chromatography-mass spectrometry (LC-MS) for SCFA analysis. Colonic fecal samples were collected and immediately stored at -80°C, with each sample weighing between 0.5 to 1 gram. To measure short-chain fatty acids (SCFAs) in the fecal matter, we prepared standard solutions containing various concentrations of acetic, propionic, butyric, valeric, and hexanoic acid, along with 4-methylvaleric acid as an internal standard. After mixing each fecal sample with 0.5% phosphoric acid and ethyl acetate, we conducted gas chromatography-mass spectrometry (GC-MS) analysis using an Agilent DB-WAX capillary column. Quality control samples were included intermittently to ensure system consistency. MSD ChemStation software was employed for peak area and retention time extraction, and a standard curve was generated to quantify SCFA content in each sample. 2.7 Flow cytometric analysis The animals were anesthetized with isoflurane (Shanghai Yuyan Scientific Instrument Company, Cat# 100150) until respiratory arrest, typically within 2 minutes [ 27 ] . Following euthanasia, blood specimens were collected via cardiac puncture for obtaining peripheral blood mononuclear cells (PBMCs) through gradient centrifugation. Lamina propria lymphocytes (LPLs) were isolated using the Lamina Propria Dissociation Kit (Miltenyi Biotec, 130-097-410), while brain cells were obtained by digesting brain mononuclear cells using the Adult Brain Dissociation Kit (Miltenyi Biotec, 130-607-177). Debris Removal Solution (Miltenyi Biotec, 130-309-198) was used to clear cell debris from the brain tissue post-dissociation. Spleen cells were prepared by gentle mashing and then treated with 1×RBC Lysis Buffer (BD Pharmingen, 555899) to eliminate red blood cells. Flow cytometry analysis of surface antigens, including intracellular staining of Foxp3, IL-17, and IFN-γ producing T cells after stimulation, was performed as described [ 28 ] . For extracellular stains, surface staining was done in staining buffer (BD Pharmingen, 554656). Then, mononuclear cells were labeled with APC-Cy7-anti-mouse CD45 (BD Pharmingen, 561037); Percp-cy5.5-anti-mouse CD45 (BD Pharmingen, 561089); FITC-anti-mouse CD3(BD Pharmingen, 561827); pe-cy7-anti-mouse CD4(BD Pharmingen, 561099); PE-anti-mouse CD4(BD Pharmingen, 561829); APC- anti-mouse CD8(BD Pharmingen, 561093); BB700- anti-mouse CD8(BD Pharmingen, 563061); BV650-anti-mouse IFN-γ (BD Pharmingen, 563854); BV605-anti-mouse CD25 (BD Pharmingen, 563061); PE-anti-mouse FOXP3 (BD Pharmingen, 12-5773-80); BV421-anti-mouse IL17(BD Pharmingen, 566426); BV421-anti-mouse CCR7(BD Pharmingen, 562675); BV786-anti-mouse CCR6(BD Pharmingen, 740840). Stained cells were sorted by BD FACS Celestar flow cytometer (BD Biosciences) and the data were analyzed using FLOWJO software (v10, Tree star, Ashland, OR, USA). 2.8 Behavior testing 2.8.1 Novel object recognition test (NORT) The novel object recognition test (NORT), a common tool for assessing hippocampus-dependent recognition memory, leverages animals’ natural tendency to explore novel objects over familiar ones [ 29 ] . Conducted in a 40 cm x 40 cm open field arena, the test involves two phases. In the initial phase, animals encounter two identical objects, and after a 10-minute interval, one object is replaced in the subsequent phase(Fig. 3 A). The data, derived from video footage, is used to calculate the recognition index for novel object exploration, expressed as the time exploring the novel object divided by the sum of times exploring both objects, multiplied by 100%. Recognition index =(time spent with novel object-time spent with familiar object)/total time. 2.8.2 Y-maze spontaneous alternation test The Y-maze, a widely used tool for short-term memory assessment in mice, measures spontaneous alternation, reflecting spatial working memory. Utilizing a Y-shaped maze with three arms labeled A, B, and C, mice are allowed to explore freely for 8 minutes(refer to Fig. 3 B) [ 30 ] . Entries and triads are recorded to calculate the percentage of alternation. An entry is noted when all four limbs are within an arm. The alternation percentage, indicative of entering a different arm from the previous two entries, is calculated using the formula: Alternations / (Arm Entries − 2) * 100%. 2.9 Immunofluorescence staining At 9 months, the animals underwent behavioral testing and were euthanized afterwards and transcardially perfused with PBS followed by 4% buffered formalin solution. The prefrontal cortex, bilateral hippocampus and intestine of the mice were segregated in cold PBS and postfixed in 4% PFA overnight at 4°C. After 24 hours of fixation, the sample tissues were embedded in paraffin, cut into 3 µm sections. Intestinal tissues were subjected to rabbit ZO-1(1:100, Cell signaling, 13663S) and Claudin-1(1:400, Cell signaling, 13995S) staining. Immunofluorescence staining with rabbit anti-Aβ1–42 antibody (1:500, Cell signaling, 24090S), anti-GFAP antibody (1:200, Cell signaling, 12389S), anti-AIF-1/Iba1 Antibody(1:50, Cell signaling, 17198S) and anti-phospho-tau(Ser404) antibody(1:400, Cell signaling, 35834S) were performed on the brain sections. Following the incubation with the primary antibody, the slices underwent thorough rinsing with phosphate-buffered saline (PBS) and were subsequently subjected to treatment with secondary antibodies, namely Alexa 488- or Alexa 594-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, Cat# 111-585-003), at a dilution of 1:500 for a duration of 1 hour at a temperature of 37°C in the absence of light. Subsequently, the samples underwent three rounds of rinsing using PBS. The process of nuclear staining was carried out by utilising 4′,6-diamidino‐2‐phenylindole (DAPI, Beyotime, C1005) for a duration of 10 minutes, which was then followed by thorough washing in PBS. The slices were captured using fluorescence microscope (Zeiss-Axio Vert.A1, Germany) or digital slide scanner (Pannoramic DESK, 3DHISTEK, Hungary). 2.10 Western-blotting Intestine tissues were homogenized in Tissue lysate buffer (Absin, Shanghai, China, abs9225) supplemented with 1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA, Cat# P7626) and EASYpack Protease Inhibitor Cocktail (Roche, Basel, Switzerland, Cat# 5892970001) for 30 minutes at 4°C. The supernatant was obtained after centrifugation at 12,000×g for 20 minutes at 4°C. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay reagent (Beyotime, Cat# P0010). Subsequently, protein samples were denatured, separated by gel electrophoresis with 4–15% SDS-PAGE gels (BeyoGel Plus PAGE; Beyotime, Cat# P0520S), and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were probed with primary antibodies against Claudin-1 (1:1000, Proteintech Group, Cat# 13050-1-AP), ZO-1 (1:1000, Proteintech Group, Cat# 21773-1-AP) and β-actin (1:1000, Servicebio, Cat# GB111364) at 4°C overnight. Following this, horseradish peroxidase-conjugated secondary antibodies (Beyotime, Cat# A0208) were applied, and protein bands were visualized using enhanced chemiluminescence (Beyotime, Cat# P0018S) with the Tanon Gel Imaging System (Tanon, Shanghai, China, Cat# 1600). ImageJ software (version 1.53c) was employed for the analysis of the optical density ratio of proteins to β-actin. 2.11 Cytokine Measurement The prefrontal cortex, bilateral hippocampus and intestines of the mice were segregated in cold PBS and immediately stored at -80℃ for subsequent examination. The blood was centrifuged (12,000 g, 4°C, 20 minutes) and the plasma was frozen at 80°C prior to assay. The levels of cytokines, namely IL-1β, IL-6, IL-10, IL-17, and IFN-γ, were quantified in plasma, brain, and intestine tissue lysates. This was achieved by employing a Mouse Premixed Multi-Analyte kit (Magnetic Luminex assay; cat LXSAMSM; R&D Systems) in accordance with the manufacturer's instructions. xPONENT® 4.5 software was used to analyze the samples on the Luminex 200 cytometer and normalized to standard curve average values. The levels of IL-23 and TGF-β in plasma and tissue lysates were evaluated using specific ELISA kits, following the manufacturers’ instructions. The Meso Scale Discovery (MSD) method was utilised to quantify the concentration of Aβ 40 and Aβ 42 in lysates of brain samples in our investigation. The V-PLEX Aβ Peptide Panel 1 (4G8) (K15199E kits) (MSD, Rockville, Maryland, USA) were utilised to conduct the analysis. 2.12 Statistical analysis Analyses were carried out with IBM SPSS Statistics 20.0 and GraphPad Prism (v6.0). All data were expressed as mean ± SEM. All other data were analyzed by one-way ANOVA with Tukey-Kramer post hoc tests. Differences were deemed to be significant if p < 0.05. 3. Results Effects of Parabacteroides Distasonis Supplementation on Gut Microbiota Composition in APP/PS1 Mice In this study, we conducted an investigation into the impact of P.distasonis supplementation on the gut microbiota composition of APP/PS1 mice. Fecal samples were collected following one month of gavage administration, and 16S rDNA gene sequencing was performed. As anticipated, the supplementation of P.distasonis exhibited a noticeable impact on the gut microbiota composition. Following oral administration, the alpha diversity of the microbiota across the entire cohort remained unchanged. However, when stratified into two groups, the analysis revealed a notable rise in the Chao1 index among APP/PS1 mice receiving P.distasonis gavage, compared to the APP/PS1 + PBS group (p = 0.0207, Wilcoxon matched-pair rank test). Conversely, no significant differences were observed in the Shannon index between the APP/PS1 + PBS and APP/PS1 + P.distasonis groups (p > 0.05) (Fig. 1 B). Furthermore, the ANOSIM analysis provided additional evidence that beta diversity exhibited significant differences among the three groups (ANOSIM, R = 0.875 and P = 0.001). Subgroup analyses revealed a noticeable alteration in beta diversity between the APP/PS1 + PBS and APP/PS1 + P.distasonis groups (ANOSIM, R 2 = 4.0926, p = 0.007, Fig. 1 C). These results reinforce the notion that the supplementation of P.distasonis has a significant impact on the overall microbial composition and diversity in the gut of APP/PS1 mice. Additionally, LEfSe analysis was further applied to identify the bacterial taxa that exhibited significant differences between the APP/PS1 + PBS group and the APP/PS1 + P.distasonis group. The strict version of LEfSe was assigned to robustly identify abundant microbial taxa with a log LDA score above 2.0 that were statistically different(p < 0.05) between biological classes in this study. The major abundant bacterial taxa in the APP/PS1 + P.distasonis group were Bacteroidetes , Prevotellaceae , Desulfovibrionaceae , Desulfovibrio , as well as P.distasonis . In contrast, the major enriched bacterial taxa in the APP/PS1 + PBS group were the genus Firmicutes , Lachnospiraceae , and Costridia (Fig. 1 D&E). Effects of Parabacteroides Distasonis Supplementation on Short-Chain Fatty Acid Levels in the Gut of APP/PS1 Mice. The production of short-chain fatty acids (SCFAs) is a complex outcome arising from the dynamic interaction between diet and the gut microbiota within the gut environment [ 31 ] . To delve into the impact of gut microbiota alterations on SCFA levels, we employed the GC-MS technique to analyze SCFA concentrations in fecal metabolites. The outcomes indicated that while little disparity in gut microbiota metabolites existed between the control group and the APP/PS1 + PBS group, supplementation with P.distasonis significantly elevated butyrate levels (P < 0.05). Additionally, in comparison to the control group, the APP/PS1 + P.distasonis group exhibited a notable reduction in hexanoate levels (P < 0.05), as illustrated in Fig. 2 . Long term P.distasonis administration attenuated learning and memory deficits in APP/ PS1 mice To assess cognitive function and ascertain the long-term therapeutic effects of P.distasonis administration over a period of 6 months, we employed the Novel Object Recognition Test (NORT) and the Y-maze spontaneous alternation test. The NORT findings indicated a notable enhancement in object recognition capabilities in the P.distasonis -treated mice compared to the APP/PS1 + PBS group, as reflected in the higher discrimination index (P < 0.01) (Fig. 3 C). Furthermore, we utilized the Y-maze spontaneous alternation test to evaluate spatial working memory. The control group exhibited a significantly higher rate of accurate spontaneous alternation (64.22%±2.38%) than the APP/PS1 + PBS group (35.79%±3.85%, p < 0.001). However, the administration of P.distasonis elevated the alternation index of APP/PS1 mice to 54.24%±2.84% (p 0.05)( Fig. 3 D). These results collectively suggested that the prolonged administration of P.distasonis in the gut has the potential to enhance working memory in APP/PS1 mice. Long-term P.distasonis administration attenuates Aβ plaque burden and tau pathology in APP/PS1 mice To assess the impact of P.distasonis on Aβ plaque deposition in APP/PS1 mice, we utilized immunofluorescence staining to examine the burden of Aβ plaques in APP/PS1 mice following 6 months of gavage treatment. Remarkably, the cortex and hippocampus of P.distasonis -treated mice exhibited significantly reduced areas of Aβ-positive staining compared to the APP/PS1 + PBS group (Fig. 3 E&F). To further quantify changes in Aβ levels, MSD immunoassays were employed to measure the levels of Aβ 40 and Aβ 42 in brain tissue. The MSD analysis demonstrated a significant decrease in both Aβ 40 and Aβ 42 levels in brain homogenates from mice treated with P.distasonis in comparison to the PBS-treated APP/PS1 group (P < 0.0001) (Fig. 3 G). Specifically, phosphorylation of tau protein at Ser404 exhibited a significant increase in the cortex and hippocampus of the APP/PS1 + PBS group as depicted in Fig. 3 E&F, whereas P.distasonis treatment reversed this pathological alteration in APP/PS1 mice. Collectively, these findings suggest that supplementation with P.distasonis through gavage could alleviate characteristic pathological changes in the brains of APP/PS1 mice. Long-term administration of Parabacteroides distasonis reduces damage to the intestinal barrier and attenuates reactive glial activation and in APP/PS1 mice. To investigate the effect of P.distasonis on gut barrier permeability in APP/PS1 mice, we assessed gut barrier integrity via immunofluorescence after 6 months of continuous oral gavage in APP/PS1 mice. Tight junction proteins ZO-1 and Claudin-1 were used as indicators of gut barrier integrity, and their expression was examined through immunofluorescence staining. Results showed that the expression of ZO-1 and Claudin-1 per unit area in the intestines of APP/PS1-PBS mice was significantly lower than that of wild-type mice (P < 0.05 for both). Oral gavage with P.distasonis significantly increased the expression of ZO-1 and Claudin-1 in the intestines of APP/PS1 + P.distasonis mice (Fig. 4 A-D). These results suggested that P.distasonis can repair damaged mechanical barriers in the intestines and reduce abnormal intestinal permeability. We further investigated the neuroinflammatory response in the brains of mice by staining for activated microglia marked by Iba-1 positivity and reactive astrocytes labeled with GFAP. Strikingly, the APP/PS1 + P.distasonis group exhibited a significant reduction in Iba1-positive microglia and GFAP-positive astrocytes in the cortex and hippocampus regions compared to the APP/PS1 + PBS group (Fig. 4 E-H). This suggested that gut transplantation of P.distasonis effectively reduces the proliferation of glial cells and consequently alleviates immune-related pathologies in the brains of APP/PS1 mice. Administration of Parabacteroides distasonis modulates T-cell populations and inflammatory cytokine profiles in APP/PS1 mice Utilizing samples of APP/PS1 mice with one-month P.distasonis administration, we employed flow cytometry to investigate the subtypes of T-helper lymphocytes in the lamina propria of the small intestines, as well as the alterations in T cell populations on peripheral blood, spleen and brain, which play a critical role in maintaining immune equilibrium. Our findings exhibited a noteworthy rise in CD4 + CD25 + Foxp3 + (Treg) cells within the intestines of APP/PS1 mice treated with P.distasonis , in comparison to both the control group and APP/PS1 + PBS group (P < 0.01) (Fig. 5 A). Earlier studies have documented the anti-inflammatory role of P.distasonis in a mouse model of colitis by fostering the proliferation and differentiation of IL10 + CD4 + Foxp3 + Treg cells, thereby leading to a considerable anti-inflammatory effect [ 32 , 33 ] .Thus, our flow cytometry outcomes concerning T-cell subsets align with prior reports imply that P.distasonis might stimulate the proliferation and differentiation of Treg cells in the intestine. Conversely, the proportions of CD4 + IL-17 + (Th17) cells, whether in the confines of the small intestine or circulating in the blood, spleen, and brain, remained stoically unaltered between the APP/PS1 + PBS and APP/PS1 + P.distasonis groups (Fig. 1SA). Venturing further into our research, we extended our observations to APP/PS1 mice subjected to the prolonged embrace of a 6-month P.distasonis gavage. Within the small intestines of these mice, a sustained augmentation of Treg cell populations emerged, accompanied by a simultaneous attenuation of Th1 cell populations (P < 0.05 and P < 0.001, respectively) (Fig. 5 B). However, no significant differences in Th1, Th17, and Treg cell populations within the spleen were discerned. Notably, there was a significant elevation in the proportion of Treg cells in the peripheral blood of APP/PS1 + P. distasonis mice (P < 0.05). These findings underscore the profound impact of P.distasonis administration in fortifying the ranks of Treg cells within the small intestine, both in the short span of one month and the extended saga of 6 months, culminating in a significant surge of Treg cells in the peripheral blood after six months of APP/PS1 + P. distasonis intervention(Fig. 5 B&C). Subsequently, we conducted an analysis of CD4 + T cell subpopulations within the brains of mice. Flow cytometry unveiled a conspicuous upsurge in the proportion of Treg cells within the brains of mice treated with P.distasonis . This profound shift stood in stark contrast to the control group (p < 0.001) and the APP/PS1 + PBS group (p < 0.05)(Fig. 6 A). Does this administration of P.distasonis hold within its essence the gift of anti-inflammatory prowess? To unravel this enigma, we delved deeper into the intricacies of inflammatory cytokines inhabiting the same region after long-term gavage. The pro-inflammatory cytokine IL-1β exhibited notably higher levels in the intestines of APP/PS1 + PBS mice compare to WT + PBS group (P < 0.0001). Interestingly, in APP/PS1 mice administered 6-month P.distasonis , we observed a significant increase in the levels of anti-inflammatory cytokines IL-10 and TGF-β within the small intestine, standing in stark contrast to their counterparts in the APP/PS1 + PBS mice (P < 0.01 and P < 0.001, respectively). Additionally, the Th1-inducing cytokine IFN-γ stooped to significantly lower levels in APP/PS1 + P. distasonis mice compared to their APP/PS1 + PBS counterparts. Furthermore, our multiplex assays uncovered a significant upswing in the TGF-β levels (P < 0.05) and a noteworthy downturn in the pro-inflammatory cytokine IL-1β (P < 0.01) within the plasma of APP/PS1 + P.distasonis mice. Oral gavage of P.distasonis resulted in a significant increase in the secretion of anti-inflammatory cytokines, specifically IL-10 and TGF-β, while concurrently reducing the levels of the pro-inflammatory cytokine IL-1β, but had no significant effect on the Th1-inducing cytokine IFN-γ or the Th17-derived cytokine IL-17 in the brain(Fig. 6 B). Thus, our findings suggest that changes in inflammatory cytokine profiles corresponded to alterations in Treg cell populations. Long-term gavage with P. distasonis increases the migration of peripheral T cells towards the central nervous system in APP/PS1 mice In the intricate interplay of immunoregulation, T cells stationed in the bloodstream serve as vigilant sentinels, receiving signals from inflamed tissues and embarking on migratory journeys to enact their immune-modulating duties [ 34 ] . In order to enter the central nervous system, they first adhere to and halt at the blood-brain barrier or choroid plexus barrier. Subsequently, they traverse through the gaps between endothelial cells and gain entry into the tissue. This process is facilitated by chemokines and cell surface receptors, such as CCR7-CCL20 and LFA-1-ICAM-1 for T cells returning to lymph nodes, and CCR6-CCL20 for T cells crossing the choroid plexus into the central nervous system [ 23 ] . To explore the mechanisms behind the infiltration of peripheral CD4 + T cells into the brains of 9-month-old APP/PS1 mice following 6 months of P.distasonis oral gavage, our study examined the expression of chemokine receptors on CD4 + T cells in the brain and corresponding chemokine levels. The results revealed a significant increase in CD4 + T cells expressing the chemokine receptor CCR6 in the brains of APP/PS1 + P.distasonis mice compared to APP/PS1 + PBS mice (P < 0.05), as shown in Fig. 7 A. Additionally, the expression of CCL20, the ligand for CCR6, was significantly higher in the brains of APP/PS1 + P.distasonis mice compared to APP/PS1 + PBS mice (P < 0.05), as shown in Fig. 7 B. These findings indicate that peripheral CD4 + T cells have the capability to accumulate in the central nervous system via the CCR6/CCL20 chemotactic axis, while showing no such tendency via the CCR7/CCL21 chemotactic axis (Fig. 1SB). Discussion In our study, we explored the potential therapeutic effects of P.distasonis transplantation in the context of AD due to its previously demonstrated efficacy in mitigating various inflammatory conditions such as arthritis, obesity, metabolic dysfunctions, and modulating inflammatory markers [ 35 , 36 ] . Our previous investigations [ 19 ] has demonstrated a significant decrease in the prevalence of Parabacteroides in both fecal and blood samples from individuals diagnosed with AD compared to the control group (NC). This observation underscores the need to explore the efficacy and underlying mechanism of P.distasonis application in AD. In the present study, we investigated the effects of gut microbiota transplantation involving P.distasonis on APP/PS1 mice. Our findings from 16S rDNA sequencing highlight the substantial influence of transplanting P. distasonis on the composition and diversity of gut microbiota in APP/PS1 mice. Furthermore, this supplementation resulted in a marked alteration in the gut microbiota composition. In the APP/PS1 + P.distasonis group, Bacteroidetes , Prevotellaceae , Desulfovibrionaceae , and Desulfovibrio emerged as the dominant and prevalent bacterial taxa. In contrast, the genus Firmicutes , Lachnospiraceae , and Costridia were among the top enriched bacterial taxa in the APP/PS1 + PBS group (Fig. 1 D&E). Similarly, a study involving the administration of aloe polysaccharides (APs), primarily composed of mannose polysaccharides, led to a notable increase in the abundance of Bacteroides and Parabacteria , which are responsible for the production of short-chain fatty acids (SCFAs) in mouse feces. Conversely, APs reduced the prevalence of Firmicutes and Clostridium , establishing a positive correlation between the microbiota and SCFAs, with Parabacteroides playing a pivotal role in SCFA production [ 37 ] . Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced through the fermentation of indigestible carbohydrates by gut microorganisms. They play a vital role in promoting gut health by providing energy to intestinal cells, regulating microbiota, enhancing gut function, reducing inflammation and influencing gene expression. Changes in SCFA levels are associated with conditions like obesity, diabetes, and colitis, making them valuable markers for diagnosis and assessment [ 38 ] . In our study, the addition of P.distasonis to the gut of APP/PS1 mice led to a notable increase in butyrate levels. P. distasonis MRx0005 is known for its ability to produce short-chain fatty acids, with a particular emphasis on butyrate, which has demonstrated the capacity to alleviate neuroinflammation in in vitro studies [ 39 ] . Notably, our results also highlighted the prominent role of Prevotellaceae in the APP/PS1 + P.distasonis group. Prevotellaceae is closely associated with SCFA synthesis and has been demonstrated to rectify disturbances in gut barrier function and promote butyrate synthesis in colonic macrophages through the PPARα-CYP4X1 axis [ 40 ] . Butyrate serves as a primary energy source for the intestinal mucosa and plays a crucial regulatory role in maintaining gut microbial balance, gut barrier function, and the modulation of various genes involved in lipid metabolism, immunity, inflammation, differentiation, apoptosis, phagocytosis, and clearance of dead cells [ 41 ] . Several studies have also indicated that butyrate exhibits the potential to mitigate inflammatory responses and promotes the differentiation of Treg cells [ 42 ] . Our findings bring to light an intriguing connection. The elevated proportion of Treg cells observed in the small intestine of APP/PS1 mice fed with P.distasonis may be attributed to the augmented levels of butyrate synthesized by the gut microbiota. By elevating intestinal butyrate levels, the induction and differentiation of Treg cells in APP/PS1 mice are likely to be facilitated. Contrarily, butyrate exhibited a favorable impact, mitigating cognitive impairment, neuronal changes, and BDNF reduction in both in vivo and in vitro models [ 43 ] . It could potentially contribute as an additional factor to the observed cognitive enhancement following the administration of P. distasonis . Additionally, our study demonstrated that prolonged gavage of P.distasonis led to an increased proportion of Treg cells in the intestine, concurrently curbing the proliferative tendencies of Th1 cells. This culminated in a noteworthy reduction in disturbances to the intestinal barrier, a mitigation of intestinal inflammation, heightened levels of anti-inflammatory cytokines IL-10 and TGF-β within intestinal tissues, and a decrease in IFN-γ concentrations. Moreover, a reduction in the levels of the peripheral pro-inflammatory factor IL-1β within the circulatory system was observed, contributing to an overall alleviation of neuroinflammation and enhancement of cognitive function in APP/PS1 mice. This study highlights the pivotal role of Treg cells in modulating inflammatory responses. Our findings align with previous research showing the detrimental effects of pro-inflammatory cytokines such as IL-1β and tumor necrosis factor (TNF-α) play a critical role in inflammatory bowel disease by contributing to the breakdown of intestinal epithelial barrier function and the expression of tight junction proteins. It is imperative to underscore that the disturbance of gut microbiota equilibrium can compromise intestinal permeability, consequently setting off neuroinflammatory cascades. Prior to immune alterations in the central nervous system, a clear connection exists between imbalances in gut microbiota and the propagation of inflammatory molecules from the peripheral to the central nervous system. These molecules can compromise the integrity of both the intestinal blood barrier and the blood-brain barrier, facilitating the entry of gut-derived substances into the brain, ultimately triggering neuroinflammation. This process serves as a conduit for gut-originating molecules, toxins, and pathogens to infiltrate the brain parenchyma, provoking neuroinflammatory responses [ 44 – 46 ] . Our research also uncovered potential links between immune cells residing in the CNS and those in the gut. The activation of the immune system in both the gut and the brain is closely associated with reactions to neuroinflammation, responses to brain injuries, and alterations in neurogenesis and neural plasticity. [ 47 ] . In our one-month-duration experiment using gavage feeding, we observed the favorable influence of P.distasonis on gut microbiota and immune responses. In particular, the modulation of T cell subtypes, including Th1 cells, Th17 cells, and Treg cells, is of paramount importance. Th1 cells drive inflammation primarily through the production of IFN-γ, while Th17 cells promote cell damage and inflammation via IL-17 production. In contrast, Treg cells execute immunosuppressive functions by releasing anti-inflammatory cytokines, including IL-10 and TGF-β [ 48 ] . After the extended gavage administration of P.distasonis , a notable increase in the Treg cell proportion within the brain was observed, in conjunction with a reduction in Aβ deposition and the inhibition of glial cell proliferation. This augmentation significantly facilitated the manifestation of its anti-inflammatory attributes through the expression of cytokines, most notably the prominent IL-10 and TGF-β, thereby effectively mitigating instances of neuroinflammation. Research has previously highlighted the role of immune cells, including Treg cells, in the context of conditions like multiple sclerosis, where the gut microbiota stimulates the proliferation and differentiation of active Th1 cells, Th17 cells, and Treg cells within the gut, which subsequently migrate to the CNS and either promote or suppress the onset of inflammation [ 49 ] . It is essential to understand that immune cells can enter the brain parenchyma through meningeal lymphatic vessels, even in healthy brains [ 50 ] . The migration of T cells into inflamed tissues is guided by chemoattractants anchored on endothelial cells and the glycocalyx surface. This intricate process consists of multiple navigation signals, including target antigens, chemotactic factors, integrin ligands, and mechanistic markers of the inflammatory microenvironment [ 51 ] . Our findings support the presence of Treg cells in the brain during the chronic inflammatory phase of ischemic stroke mouse models, contributing to neural functional recovery. These brain-resident Treg cells utilize amplification and infiltration mechanisms guided by signaling molecules (IL-2, IL-33, 5-HT7) and chemokines (CCL1, CCL20). They effectively inhibit neurotoxicity and suppress astrocyte proliferation [ 52 ] .Previous research has suggested that Treg cells release cytokines such as IL-10 and IL-33, which contribute to the promotion of microglial polarization towards the M2 phenotype. M2 microglia, in turn, secrete IL-10 and TGF-β, further facilitating the polarization of Treg cells. This beneficial cycle suppresses the occurrence of neuroinflammatory responses, thereby providing a certain degree of protection [ 53 ] . The observed increase in brain Treg cells prompts a question: Do they locally multiply? This touches upon the complex immune mechanisms in the central nervous system. In the context of neuroinflammation, the brain hosts resident immune cells, including microglia and astrocytes. They aren't passive; they actively engage in immune responses. What's fascinating is how their interactions with peripheral immune cells, like T cells, impact the intricate web of brain inflammation. While the administration of P. distasonis appeared to markedly mitigate both Aβ and tau pathologies in APP/PS1 mice, its supplementation did not elicit commensurate effects on inflammation and other associated indices. It is conceivable that gastric administration of P. distasonis may engender supplementary neuroprotective mechanisms, thereby enhancing cognitive function. Further elucidation of these mechanisms merits scholarly investigation. In summary, the introduction of P.distasonis into the gut environment appears to play a significant role in ameliorating pathological alterations in a mouse model of AD. This beneficial effect is likely achieved through the modulation of gut microbiota composition, elevation of butyrate levels, stimulation of gut Treg cell proliferation, reduction of intestinal barrier impairment, and augmentation of central CD4 + T cell chemotaxis, particularly the increased presence of immunosuppressive Treg cells within the brain. These actions collectively contribute to the attenuation of neuroinflammation and enhancement of cognitive function in AD mice. Our research suggests that the supplementation of P.distasonis could potentially offer an alternative therapeutic approach for both the prevention and treatment of AD. Declarations Ethics approval and consent to participate All mouse experiments were approved by The Charles River Laboratories Institutional Animal Care and Use Committee (IACUC) under approval number P2021098. The study was reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) [25] . Consent for publication Not applicable. Fundings This work was supported by the National Natural Science Foundation of China, (82171401, 81971187 (to SC), (32100798(to YH)), grants from Shanghai Municipal Science and Technology Major Project, No. 2018SHZDZX05 (to SC) and Shanghai Municipal Education Commission, No. 2017-01-07-00-01-E00046 (to SC) and the China Postdoctoral Science Foundation(LCBSHZX017 (to YH)). Availability of data and materials The datasets analyzed during the current study are not publicly available due to ongoing research and confidentiality concerns. However, summarized data supporting the findings are presented in the article. Further inquiries can be directed to the corresponding author. Competing Intererts The authors declare that they have no financial or other conflicts of interest related to this research. Author contributions JJ was responsible for the design and conception of the research, the collection of data, the analysis and interpretation of the data, as well as the writing of the paper. YH get involved in data collection, analysis, and interpretation . HP&YT: the process of interpreting the data. NX: conceived and designed the experiments. SC&DS: the design and conception of the experimental research, the interpretation of the data and the editing of the paper Acknowledgements We would like to express our gratitude to all those who contributed to this research. References Grontvedt, G. R., Schroder, T. N., Sando, S. B. et al. Alzheimer's disease. Curr Biol[J]. 28, R645-R649. Armstrong, R. A. What causes alzheimer's disease? Folia Neuropathol[J]. 51, 169–188. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4156881","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284351910,"identity":"490ba000-7e0f-48b5-b0b4-cda0e6b95044","order_by":0,"name":"jingwen Jiang","email":"","orcid":"","institution":"1.\tDepartment and Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"jingwen","middleName":"","lastName":"Jiang","suffix":""},{"id":284351911,"identity":"c31eb324-fb68-49f0-af1d-338bb8e53016","order_by":1,"name":"Yixi He","email":"","orcid":"","institution":"1.\tDepartment and Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yixi","middleName":"","lastName":"He","suffix":""},{"id":284351912,"identity":"a2153fc7-24c8-427e-838d-7031ae1aa1f4","order_by":2,"name":"Hong Pan","email":"","orcid":"","institution":"1.\tDepartment and Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Pan","suffix":""},{"id":284351913,"identity":"28a470e1-1877-468e-a9f1-1fb56447f9e1","order_by":3,"name":"Nanjie Xu","email":"","orcid":"","institution":"5.\tDepartment of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai","correspondingAuthor":false,"prefix":"","firstName":"Nanjie","middleName":"","lastName":"Xu","suffix":""},{"id":284351914,"identity":"3ac77549-5e96-4557-b7e4-23059060c58e","order_by":4,"name":"Yuyan Tan","email":"","orcid":"","institution":"1.\tDepartment and Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuyan","middleName":"","lastName":"Tan","suffix":""},{"id":284351915,"identity":"e4f0c925-7970-4400-81a2-c0c80e3a7cf6","order_by":5,"name":"dingya Sun","email":"","orcid":"","institution":"4.\tDepartment of Neurobiology, Key Laboratory of Molecular Neurobiology of the Ministry of Education, Naval Medical University, Shanghai","correspondingAuthor":false,"prefix":"","firstName":"dingya","middleName":"","lastName":"Sun","suffix":""},{"id":284351916,"identity":"7f05aa68-2793-4593-b92a-95ca71bcbed9","order_by":6,"name":"Shengdi Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYDACCRDBZsPAOINELWmkazkMZRAD+Gc3H3v4o+x8PvPsBsYPH3MY5M0JWnLnWLoxz7nblo1zDjBLztzGYLizgYAWA4kcM2nGttsGjDMS2Jh5tzEkGBwgqCX/m+TPtnMkaclhk+BtO0CCFokbaWbSPOeSgVoSm4F+kTDcQEgL/4zkZ5I/yuwMDGckH/zwcZuNPEFb4MCwgbGBgYTYAQJ5EtSOglEwCkbBCAMA2rE6feqEDAgAAAAASUVORK5CYII=","orcid":"","institution":"1.\tDepartment and Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Shengdi","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-03-24 07:29:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4156881/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4156881/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53756689,"identity":"f7ab3536-38f8-440e-a2f0-43b82ee8adc4","added_by":"auto","created_at":"2024-03-29 19:04:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4696602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParabacteroides distasonis supplementation altered gut microbiota composition in APP/PS1 Mice\u003c/strong\u003e (A) Experimental Chronology. (B) Chao1 Index and (C) The Shannon Indices - Depiction for Alpha Diversity. (Wilcoxon matched-pair rank test for longitudinal comparison and Wilcoxon rank sum test for cross-sectional comparison). Beta Diversity evaluated through principal Coordinates Analysis (PCoA) of Bray–Curtis Distance. Each sample was coloured according to the study group; the ANOSIM method was used for comparisons between the groups. \u0026nbsp;(D) LEfSe analysis identifying taxonomic differences in the gut microbiota of the APP/PS1+PBS and APP/PS1+P.distasonis group. OTUs with significant difference that have an LDA score \u0026gt; the threshold value of 2. (E) Cladograms are derived from LEfSe analysis of differential gut microbial taxa. Taxonomic hierarchies were arranged from the inside to the outside (from genus to phylum) in the cladogram. Red and green notes represent differentially abundant taxa between groups. Yellow nodes represent taxa with no significant difference. (n = 5 per group).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/87880f629be7d31ea03acd46.png"},{"id":53756687,"identity":"d82affaf-d7c5-4e92-b422-04d5cd3d0f75","added_by":"auto","created_at":"2024-03-29 19:04:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":700117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParabacteroides distasonis treatment modulates the level of SCFAs. \u003c/strong\u003eThe levels of short-chain fatty acids across the Experimental Groups. (n = 5 per group). Supplementation with P. distasonis significantly increased butyrate levels. Furthermore, compared to the control group, the APP/PS1+P. distasonis group showed a notable reduction in hexanoate levels. Data are eloquently represented as the mean ± SEM. *P \u0026lt; 0.05. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/15189ff80cffc8f42c065a32.png"},{"id":53756691,"identity":"905ab377-52bc-4654-80d6-06ab3de3ea7d","added_by":"auto","created_at":"2024-03-29 19:04:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9769197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProlonged administration of Parabacteroides distasonis enhances working memory and attenuates AD-like pathology in 9-month-old APP/PS1 mice. \u003c/strong\u003e(A) Chronological Framework Illustrating the Novel Object Recognition Assessment. (B) Elaborate Pictorial Depiction of the Y-maze Spontaneous Alternation Evaluation. (C) Recognition index \u0026nbsp;and Discrimination index for the groups presented in A. (D) Histograms show a lower proportion of alternations in the APP/PS1+PBS group , alongside significant recovery evident in the APP/PS1+P.distasonis group during the Y-maze Spontaneous Alternation Test,while no significant difference was noted in total arm entries (n =15 per group). (E) A gallery of images depicting exquisite Immunohistochemical staining of Aβ\u003csub\u003e1–42\u003c/sub\u003e plaques and phosphorylation of tau protein at Ser404 within the hippocampus of mice across all three groups. Scale bars: 200 μm. (F) The histograms delineate the percentage of Aβ\u003csub\u003e1–42\u003c/sub\u003e and p-tau area encountered in the hippocampus and cortex (n = 5 per group). （G）Levels of Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e42\u003c/sub\u003e in the brain tissues of control, APP/PS1+PBS, and APP/PS1+P.distasonis mice were measured by Meso Scale Discovery (MSD) method. (n = 6 per group). Data are eloquently represented as the mean ± SEM. *P \u0026lt; 0.05. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/4d8600ba8fb0b43b10709781.png"},{"id":53756694,"identity":"80aa668c-d44b-4031-8ea2-b3ec7642833d","added_by":"auto","created_at":"2024-03-29 19:04:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16954477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended administration of Parabacteroides distasonis diminishes impairment to the intestinal barrier and mitigates reactive glial activation in APP/PS1 mice. \u003c/strong\u003e(A) Images of representative immunohistochemical staining of ZO-1 and Claudin-1, unveiling the tight junction of intestinal tissue in mice across all three groups. Scale bars: 20 μm. (B) Histograms display the percentage of ZO-1 and Claudin-1 area in the intestines of each distinct group (n = 5 per group). （C）Expression of ZO-1 and Claudin-1 in the intestines were detected by western blotting. (D) Quantification of ZO-1/β-actin and Claudin-1/β-actin in the intestines. (n = 6 per group). (E) Representative immunofluorescence staining of GFAP (green), Iba-1 (red), and DAPI (blue) in the cortex of each group (G) Representative immunofluorescence staining of GFAP (red), Iba-1 (green), and DAPI (blue) in the hippocampus of each group. Scale bars: 100 μm in A and B. (F, H) Quantification of the number of GFAP\u003csup\u003e+\u003c/sup\u003e astrocyte and Iba-1\u003csup\u003e+\u003c/sup\u003e microglia in the cortex (E) and hippocampus (G) of each group. (n = 5 per group.) Data are shown as mean ± SEM. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). Aβ: Amyloid-β;: not detected.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/c26534decd1ae8af02bee668.png"},{"id":53756688,"identity":"d7b76392-7bdb-41e1-897b-ec2152359bcb","added_by":"auto","created_at":"2024-03-29 19:04:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2194846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe administration of Parabacteroides distasonis induces alterations in the immune-inflammatory milieu of APP/PS1 mice. \u003c/strong\u003e(A) Representative flow cytometry plots of the IFN-γ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e, CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and IL-17\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e live cell populations within intestinal tissues of each group in 4-month old mice are shown to the left.\u003cstrong\u003e \u003c/strong\u003eFrequency of Th1, Treg and Th17 cells within CD4+ T cells in intestines of each group. (n = 5 per group; ns, not significant). (B) Representative flow cytometry plots of the IFN-γ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e, CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and IL-17\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e live cell populations in intestines of 9-month old mice are shown to the left of each group. Frequency of Th1, Treg and Th17 cells within CD4\u003csup\u003e+\u003c/sup\u003e T cells cells in intestines of each group. (n = 5 per group; ns, not significant). (C) Frequency of Th1, Treg and Th17 cells within CD4\u003csup\u003e+\u003c/sup\u003e T cells cells in blood and spleens of each group. (n = 5 per group; ns, not significant). Data are shown as mean ± SEM. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/6f9f68e8d258868a0784c85f.png"},{"id":53758116,"identity":"091ff7fb-c91e-49a9-a35c-b2b8a6d78a4f","added_by":"auto","created_at":"2024-03-29 19:12:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2071038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProlonged administration of Parabacteroides led to an enrichment of the Treg cell population within the brain and induced alterations in inflammatory cytokine profiles across multiple regions in 9-month-old APP/PS1 mice. \u003c/strong\u003e(A) Representative flow cytometry plots of the IFN-γ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e, CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and IL-17\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e live cell populations are shown to the left in brains of each group. Frequency of Th1, Treg and Th17 cells within CD4+ T cells cells in brains of each group. (n = 5 per group). (B) Levels of IL-1β, IL-6, IL-10,IL-17,IFN-γ and TGF-β in the brain tissue of the mice were determined using by the Luminex assay (n = 6 per group). (B) The quantification of IL-1β, IL-6, IL-10, IL-17, IFN-γ, and TGF-β levels in the intestines, plasma, and brain tissue of the mice was conducted utilizing the Luminex assay (n = 6 per group). Data are shown as mean ± SEM. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/5703e13ba7bc8b12b9cfc445.png"},{"id":53758117,"identity":"2cd88ea6-722d-4908-9e76-60cf78233b81","added_by":"auto","created_at":"2024-03-29 19:12:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":446107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProlonged gavage with P. distasonis augments the migration of peripheral T cells towards the central nervous system in APP/PS1 mice. \u003c/strong\u003e(A) Representative flow cytometry plots of the IFN-γ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e, CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and IL-17\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e live cell populations are shown to the left in brains of each group. Frequency of Th1, Treg and Th17 cells within CD4+ T cells cells in brains of each group (n = 6 per group). Data are shown as mean ± SEM. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/7b4ab4521e769541250d070f.png"},{"id":56182478,"identity":"97d48091-af83-4ebb-b434-20438227fdcf","added_by":"auto","created_at":"2024-05-09 14:40:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3971116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/75b0fa30-dd20-4945-ad28-cdc6d3cee179.pdf"},{"id":53756692,"identity":"740ed040-42a8-49eb-b664-50f364ec0e20","added_by":"auto","created_at":"2024-03-29 19:04:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":696924,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1Sfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/32b7d5cd3a77c42fc18f98b4.pdf"},{"id":53756696,"identity":"d544c5f3-2ed4-4f30-824f-b57c00dda7f1","added_by":"auto","created_at":"2024-03-29 19:04:42","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":139225710,"visible":true,"origin":"","legend":"","description":"","filename":"graphicabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4156881/v1/61e8ff95f48e592884d3c0af.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTransplantaion of Parabacteroides distasonis mitigated Aβ-associated changes in APP/PS1 mice via gut-brain interactions by attenuating neuroinflammation\u003c/p\u003e","fulltext":[{"header":"1. Background","content":"\u003cp\u003eAlzheimer's disease (AD) is a progressive neurodegenerative illness characterised by cognitive decline\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. It is estimated that between 60 and 70 percent of all occurrences of dementia may be attributed to AD, making it the most prevalent form of dementia among the aged worldwide\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Recent cross-sectional data shows that 9.83\u0026nbsp;million people in China are living with AD\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The hallmark of AD biology is the accumulation of amyloid-beta (Aβ) peptide outside of cells and the aggregation of hyperphosphorylated tau within cells\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Therapeutic efforts are currently focused on targets within this framework that have the potential to substantially alter the clinical course of Alzheimer's disease patients.\u003c/p\u003e \u003cp\u003eSeveral factors including age-related deterioration, degeneration of anatomical pathways, environmental influences, mitochondrial malfunction, immune system dysfunction, and genetic susceptibility have been identified as potential contributors to AD\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Apart from influencing the physiology and metabolism of the host\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, the gut microbiota has been demonstrated to exert an effect on cognitive development and function\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Multiple studies have provided evidence indicating that the presence of intestinal flora may have an impact on the synthesis of various neurotransmitters and neuromodulators. This, in turn, can alter the communication between the gut and the brain, ultimately influencing brain function\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The bidirectional gut-brain axis facilitates the interaction between the gut microbiota and the central nervous system through various channels, including the neuro-immune system, the sympathetic and parasympathetic autonomic nervous system, and the neuroendocrine system\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The intestinal microbiota composition of APP/PS1 mice has been observed to undergo changes upon administration of antibiotics, resulting in a noteworthy rise in the abundance of genus Akkermansia and family Lachnospiraceae. The administration of this treatment is also associated with a decline in the levels of pro-inflammatory cytokines in circulation and a reduction in reactive gliosis surrounding Aβ plaques. Nevertheless, the safety and long-term advantages of this method are still a subject of debate\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The potential of investigating AD through the manipulation of the gut microbiota via targeted microbial transplantation is significant. Akbari et al.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e executed a probiotic intervention study on 60 patients diagnosed with AD, administering Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, and Lactobacillus fermentum. The study findings indicated noteworthy enhancements in MMSE scores and a simultaneous reduction in peripheral inflammatory markers among the participants who received probiotic treatment. Additionally, research has demonstrated that probiotics can rectify microbiota dysbiosis and decrease Aβ accumulation within the brain\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is imperative to acknowledge the significance of the gut microbiota in the investigation of therapeutic approaches for AD.\u003c/p\u003e \u003cp\u003eParabacteroides, a class of gram-negative anaerobic bacteria that frequently inhabit the gut of various species\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Our preceding investigation has found a marked decline in the abundance of Parabacteroides in the fecal microbiota of AD patients compared to their healthy counterparts\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Previous study also demonstrated an inverse correlation between the existence of \u003cem\u003eP.distasonis\u003c/em\u003e and colonic IL-1β levels as well as intestinal tumor burden in Apc1638N mice \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In addition, it is plausible that bacterial elements have the ability to traverse the blood-brain barrier and infiltrate the gut-associated lymphatic tissue (GALT), thereby eliciting responses from T-cells and other immune cells. Research has shown that specific chemicals produced by bacteria have the potential to stimulate the generation of effector-type T cells, thereby exerting an impact on neuroinflammation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eP.distasonis\u003c/em\u003e facilitated the development of IL-10\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003e Tregs and anti-inflammatory CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e T cells in germ-free mice\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, thus, \u003cem\u003eP.distasonis\u003c/em\u003e might hold promise for personalized therapy and nutritional supplements for inflammatory bowel diseases. In addition, extant data suggests that T lymphocytes have the ability to penetrate the brain and engage in inflammatory responses via chemotaxis \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, a process that is intimately linked to the anomalous accumulation of Aβ in the brains of those afflicted with AD. To gain further insights into the impact of \u003cem\u003eP.distasonis\u003c/em\u003e on AD and its underlying mechanism, we performed the study of administering the bacteria to APP/PS1 transgenic mice through gavage.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethics Statement\u003c/h2\u003e \u003cp\u003e All research conducted received approval from The Charles River Laboratories Institutional Animal Care and Use Committee (IACUC) under approval number P2021098. The study was reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals\u003c/h2\u003e \u003cp\u003eThe APP/PS1 mice (B6.Cg-Tg(Thy1-APPSw,Thy1-PSEN1*L166P)21Jckr) were generously contributed by Mathias Jucker. In consideration of the pronounced gender effects noted in prior studies on gut microbiota, this investigation opted for a standardized approach by exclusively utilizing male mice\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Male mice of the APP/PS1 strain, which have a genetic background of C57Bl/6J, were crossbred with female mice of the C57Bl/6J wild-type strain. The male offsprings were subjected to genotyping through the utilisation of polymerase chain reaction. The control group consisted of age-matched male wild-type (WT) littermates lacking the APP/PS1 transgene. The rodents were accommodated in a climate-regulated setting with a temperature range of 20\u0026ndash;22\u0026deg;C and a light/dark cycle of 12 hours each.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3 Microbial Strains\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe strain \u003cem\u003eP.distasonis\u003c/em\u003e 8503 was procured from ATCC (Manassas, VA)and reconstituted in 1.9g Reinforced Clostridial Medium (Becton, Dickinson and Company, 218081) dispensed in 50ml aliquots in a glass bottle supplemented with 0.025g L-Cysteine hydrochloride monohydrate (Solarbio, C0011) and 5ul C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eNNaO\u003csub\u003e4\u003c/sub\u003e (1mg/mL, Sigma-Aldrich, 199303) at 37℃ in an anaerobic chamber (MITSUBISHI, C-33) for 24 hr. The strain was identified by comparing the 16S rRNA gene sequences with the NCBI reference database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, NCBI Reference Sequence: NC_009615.1).\u003c/p\u003e \u003cp\u003eAfter centrifugation (8000\u0026times; g rpm, 15 min at 4 ℃), culture pellets were washed with phosphate buffered saline (PBS; pH 7.2) (G4202, Servicebio, Wuhan, China) maintained in anaerobiosis. Cell pellets were suspended at 10\u003csup\u003e8\u003c/sup\u003e CFU/mL in anaerobic PBS containing 25% glycerol (56-81-5, Sangon Biotech, Shanghai, China).and suspensions were frozen in liquid nitrogen before storage at \u0026minus;\u0026thinsp;80 ℃. The cell suspension for oral administration in treatment group was prepared by suspending the cultured bacterial cells in oxygen-free PBS with a final cell density of 10\u003csup\u003e8\u003c/sup\u003e CFU per mL. A daily administration of suspension solution at a volume of 0.2 mL was implemented. The interventions were administered for a period of one month and six months, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental design\u003c/h2\u003e \u003cp\u003e3-month-old male APP/PS1 mice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;16 per group): (1) AD model (APP/PS1) and \u003cem\u003eP.distasonis\u003c/em\u003e treatment group(APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e); (2) AD model (APP/PS1) and PBS treatment group (APP/PS1\u0026thinsp;+\u0026thinsp;PBS). 3-month-old male WT mice treated with PBS were used as control group (WT\u0026thinsp;+\u0026thinsp;PBS, n\u0026thinsp;=\u0026thinsp;16). All groups had access to food and water ad libitum. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA displays the timeline of the experiment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fecal 16S rDNA gene sequencing\u003c/h2\u003e \u003cp\u003eThe 4-month-old mice were anesthetized with approximately 0.6 mL of liquid isoflurane (Shanghai Yuyan Scientific Instrument Company, Shanghai, China, Cat# 100150) per liter of chamber volume. After anesthesia, fresh colon fecal samples (0.2\u0026ndash;0.3 g each) were collected and stored. DNA extraction utilized the Qiagen QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany, 51504), with concentration and molecular weight assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis. The V3-V4 region of the bacterial 16S ribosomal RNA gene was amplified, sequenced on the Illumina MiSeq platform, and analyzed using QIIME (version 1.9.1) and UPARSE(version 7.1) for quality filtering, de-replication, and clustering. Chimeric sequences were removed by UCHIME. Taxonomic analysis employed the RDP classifier against the Silva (SSU123) 16S rDNA database, and gut microbiota characteristics were assessed using linear discriminant analysis effect size (LEfSe) for significant differences and effect size evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Liquid chromatography-mass spectrometry (LC-MS) for SCFA analysis.\u003c/h2\u003e \u003cp\u003eColonic fecal samples were collected and immediately stored at -80\u0026deg;C, with each sample weighing between 0.5 to 1 gram. To measure short-chain fatty acids (SCFAs) in the fecal matter, we prepared standard solutions containing various concentrations of acetic, propionic, butyric, valeric, and hexanoic acid, along with 4-methylvaleric acid as an internal standard. After mixing each fecal sample with 0.5% phosphoric acid and ethyl acetate, we conducted gas chromatography-mass spectrometry (GC-MS) analysis using an Agilent DB-WAX capillary column. Quality control samples were included intermittently to ensure system consistency. MSD ChemStation software was employed for peak area and retention time extraction, and a standard curve was generated to quantify SCFA content in each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Flow cytometric analysis\u003c/h2\u003e \u003cp\u003eThe animals were anesthetized with isoflurane (Shanghai Yuyan Scientific Instrument Company, Cat# 100150) until respiratory arrest, typically within 2 minutes\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Following euthanasia, blood specimens were collected via cardiac puncture for obtaining peripheral blood mononuclear cells (PBMCs) through gradient centrifugation. Lamina propria lymphocytes (LPLs) were isolated using the Lamina Propria Dissociation Kit (Miltenyi Biotec, 130-097-410), while brain cells were obtained by digesting brain mononuclear cells using the Adult Brain Dissociation Kit (Miltenyi Biotec, 130-607-177). Debris Removal Solution (Miltenyi Biotec, 130-309-198) was used to clear cell debris from the brain tissue post-dissociation. Spleen cells were prepared by gentle mashing and then treated with 1\u0026times;RBC Lysis Buffer (BD Pharmingen, 555899) to eliminate red blood cells. Flow cytometry analysis of surface antigens, including intracellular staining of Foxp3, IL-17, and IFN-γ producing T cells after stimulation, was performed as described\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. For extracellular stains, surface staining was done in staining buffer (BD Pharmingen, 554656). Then, mononuclear cells were labeled with APC-Cy7-anti-mouse CD45 (BD Pharmingen, 561037); Percp-cy5.5-anti-mouse CD45 (BD Pharmingen, 561089); FITC-anti-mouse CD3(BD Pharmingen, 561827); pe-cy7-anti-mouse CD4(BD Pharmingen, 561099); PE-anti-mouse CD4(BD Pharmingen, 561829); APC- anti-mouse CD8(BD Pharmingen, 561093); BB700- anti-mouse CD8(BD Pharmingen, 563061); BV650-anti-mouse IFN-γ (BD Pharmingen, 563854); BV605-anti-mouse CD25 (BD Pharmingen, 563061); PE-anti-mouse FOXP3 (BD Pharmingen, 12-5773-80); BV421-anti-mouse IL17(BD Pharmingen, 566426); BV421-anti-mouse CCR7(BD Pharmingen, 562675); BV786-anti-mouse CCR6(BD Pharmingen, 740840). Stained cells were sorted by BD FACS Celestar flow cytometer (BD Biosciences) and the data were analyzed using FLOWJO software (v10, Tree star, Ashland, OR, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Behavior testing\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Novel object recognition test (NORT)\u003c/h2\u003e \u003cp\u003eThe novel object recognition test (NORT), a common tool for assessing hippocampus-dependent recognition memory, leverages animals\u0026rsquo; natural tendency to explore novel objects over familiar ones\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Conducted in a 40 cm x 40 cm open field arena, the test involves two phases. In the initial phase, animals encounter two identical objects, and after a 10-minute interval, one object is replaced in the subsequent phase(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The data, derived from video footage, is used to calculate the recognition index for novel object exploration, expressed as the time exploring the novel object divided by the sum of times exploring both objects, multiplied by 100%. Recognition index =(time spent with novel object-time spent with familiar object)/total time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Y-maze spontaneous alternation test\u003c/h2\u003e \u003cp\u003eThe Y-maze, a widely used tool for short-term memory assessment in mice, measures spontaneous alternation, reflecting spatial working memory. Utilizing a Y-shaped maze with three arms labeled A, B, and C, mice are allowed to explore freely for 8 minutes(refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Entries and triads are recorded to calculate the percentage of alternation. An entry is noted when all four limbs are within an arm. The alternation percentage, indicative of entering a different arm from the previous two entries, is calculated using the formula: Alternations / (Arm Entries \u0026minus;\u0026thinsp;2) * 100%.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003e At 9 months, the animals underwent behavioral testing and were euthanized afterwards and transcardially perfused with PBS followed by 4% buffered formalin solution. The prefrontal cortex, bilateral hippocampus and intestine of the mice were segregated in cold PBS and postfixed in 4% PFA overnight at 4\u0026deg;C. After 24 hours of fixation, the sample tissues were embedded in paraffin, cut into 3 \u0026micro;m sections. Intestinal tissues were subjected to rabbit ZO-1(1:100, Cell signaling, 13663S) and Claudin-1(1:400, Cell signaling, 13995S) staining. Immunofluorescence staining with rabbit anti-Aβ1\u0026ndash;42 antibody (1:500, Cell signaling, 24090S), anti-GFAP antibody (1:200, Cell signaling, 12389S), anti-AIF-1/Iba1 Antibody(1:50, Cell signaling, 17198S) and anti-phospho-tau(Ser404) antibody(1:400, Cell signaling, 35834S) were performed on the brain sections. Following the incubation with the primary antibody, the slices underwent thorough rinsing with phosphate-buffered saline (PBS) and were subsequently subjected to treatment with secondary antibodies, namely Alexa 488- or Alexa 594-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, Cat# 111-585-003), at a dilution of 1:500 for a duration of 1 hour at a temperature of 37\u0026deg;C in the absence of light. Subsequently, the samples underwent three rounds of rinsing using PBS. The process of nuclear staining was carried out by utilising 4\u0026prime;,6-diamidino‐2‐phenylindole (DAPI, Beyotime, C1005) for a duration of 10 minutes, which was then followed by thorough washing in PBS. The slices were captured using fluorescence microscope (Zeiss-Axio Vert.A1, Germany) or digital slide scanner (Pannoramic DESK, 3DHISTEK, Hungary).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western-blotting\u003c/h2\u003e \u003cp\u003eIntestine tissues were homogenized in Tissue lysate buffer (Absin, Shanghai, China, abs9225) supplemented with 1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA, Cat# P7626) and EASYpack Protease Inhibitor Cocktail (Roche, Basel, Switzerland, Cat# 5892970001) for 30 minutes at 4\u0026deg;C. The supernatant was obtained after centrifugation at 12,000\u0026times;g for 20 minutes at 4\u0026deg;C. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay reagent (Beyotime, Cat# P0010). Subsequently, protein samples were denatured, separated by gel electrophoresis with 4\u0026ndash;15% SDS-PAGE gels (BeyoGel Plus PAGE; Beyotime, Cat# P0520S), and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were probed with primary antibodies against Claudin-1 (1:1000, Proteintech Group, Cat# 13050-1-AP), ZO-1 (1:1000, Proteintech Group, Cat# 21773-1-AP) and β-actin (1:1000, Servicebio, Cat# GB111364) at 4\u0026deg;C overnight. Following this, horseradish peroxidase-conjugated secondary antibodies (Beyotime, Cat# A0208) were applied, and protein bands were visualized using enhanced chemiluminescence (Beyotime, Cat# P0018S) with the Tanon Gel Imaging System (Tanon, Shanghai, China, Cat# 1600). ImageJ software (version 1.53c) was employed for the analysis of the optical density ratio of proteins to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Cytokine Measurement\u003c/h2\u003e \u003cp\u003eThe prefrontal cortex, bilateral hippocampus and intestines of the mice were segregated in cold PBS and immediately stored at -80℃ for subsequent examination. The blood was centrifuged (12,000 g, 4\u0026deg;C, 20 minutes) and the plasma was frozen at 80\u0026deg;C prior to assay. The levels of cytokines, namely IL-1β, IL-6, IL-10, IL-17, and IFN-γ, were quantified in plasma, brain, and intestine tissue lysates. This was achieved by employing a Mouse Premixed Multi-Analyte kit (Magnetic Luminex assay; cat LXSAMSM; R\u0026amp;D Systems) in accordance with the manufacturer's instructions. xPONENT\u0026reg; 4.5 software was used to analyze the samples on the Luminex 200 cytometer and normalized to standard curve average values. The levels of IL-23 and TGF-β in plasma and tissue lysates were evaluated using specific ELISA kits, following the manufacturers\u0026rsquo; instructions. The Meso Scale Discovery (MSD) method was utilised to quantify the concentration of Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e42\u003c/sub\u003e in lysates of brain samples in our investigation. The V-PLEX Aβ Peptide Panel 1 (4G8) (K15199E kits) (MSD, Rockville, Maryland, USA) were utilised to conduct the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eAnalyses were carried out with IBM SPSS Statistics 20.0 and GraphPad Prism (v6.0). All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. All other data were analyzed by one-way ANOVA with Tukey-Kramer post hoc tests. Differences were deemed to be significant if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eEffects of Parabacteroides Distasonis Supplementation on Gut Microbiota Composition in APP/PS1 Mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, we conducted an investigation into the impact of \u003cem\u003eP.distasonis\u003c/em\u003e supplementation on the gut microbiota composition of APP/PS1 mice. Fecal samples were collected following one month of gavage administration, and 16S rDNA gene sequencing was performed. As anticipated, the supplementation of \u003cem\u003eP.distasonis\u003c/em\u003e exhibited a noticeable impact on the gut microbiota composition. Following oral administration, the alpha diversity of the microbiota across the entire cohort remained unchanged. However, when stratified into two groups, the analysis revealed a notable rise in the Chao1 index among APP/PS1 mice receiving \u003cem\u003eP.distasonis\u003c/em\u003e gavage, compared to the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group (p\u0026thinsp;=\u0026thinsp;0.0207, Wilcoxon matched-pair rank test). Conversely, no significant differences were observed in the Shannon index between the APP/PS1\u0026thinsp;+\u0026thinsp;PBS and APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, the ANOSIM analysis provided additional evidence that beta diversity exhibited significant differences among the three groups (ANOSIM, R\u0026thinsp;=\u0026thinsp;0.875 and P\u0026thinsp;=\u0026thinsp;0.001). Subgroup analyses revealed a noticeable alteration in beta diversity between the APP/PS1\u0026thinsp;+\u0026thinsp;PBS and APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e groups (ANOSIM, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;4.0926, p\u0026thinsp;=\u0026thinsp;0.007, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results reinforce the notion that the supplementation of \u003cem\u003eP.distasonis\u003c/em\u003e has a significant impact on the overall microbial composition and diversity in the gut of APP/PS1 mice. Additionally, LEfSe analysis was further applied to identify the bacterial taxa that exhibited significant differences between the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group and the APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e group. The strict version of LEfSe was assigned to robustly identify abundant microbial taxa with a log LDA score above 2.0 that were statistically different(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between biological classes in this study. The major abundant bacterial taxa in the APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e group were \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003ePrevotellaceae\u003c/em\u003e, \u003cem\u003eDesulfovibrionaceae\u003c/em\u003e, \u003cem\u003eDesulfovibrio\u003c/em\u003e, as well as \u003cem\u003eP.distasonis\u003c/em\u003e. In contrast, the major enriched bacterial taxa in the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group were the genus \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eLachnospiraceae\u003c/em\u003e, and \u003cem\u003eCostridia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026amp;E).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of Parabacteroides Distasonis Supplementation on Short-Chain Fatty Acid Levels in the Gut of APP/PS1 Mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe production of short-chain fatty acids (SCFAs) is a complex outcome arising from the dynamic interaction between diet and the gut microbiota within the gut environment\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. To delve into the impact of gut microbiota alterations on SCFA levels, we employed the GC-MS technique to analyze SCFA concentrations in fecal metabolites. The outcomes indicated that while little disparity in gut microbiota metabolites existed between the control group and the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group, supplementation with \u003cem\u003eP.distasonis\u003c/em\u003e significantly elevated butyrate levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, in comparison to the control group, the APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e group exhibited a notable reduction in hexanoate levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLong term P.distasonis administration attenuated learning and memory deficits in APP/ PS1 mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess cognitive function and ascertain the long-term therapeutic effects of \u003cem\u003eP.distasonis\u003c/em\u003e administration over a period of 6 months, we employed the Novel Object Recognition Test (NORT) and the Y-maze spontaneous alternation test. The NORT findings indicated a notable enhancement in object recognition capabilities in the \u003cem\u003eP.distasonis\u003c/em\u003e-treated mice compared to the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group, as reflected in the higher discrimination index (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, we utilized the Y-maze spontaneous alternation test to evaluate spatial working memory. The control group exhibited a significantly higher rate of accurate spontaneous alternation (64.22%\u0026plusmn;2.38%) than the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group (35.79%\u0026plusmn;3.85%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, the administration of \u003cem\u003eP.distasonis\u003c/em\u003e elevated the alternation index of APP/PS1 mice to 54.24%\u0026plusmn;2.84% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and there was no significant difference in the total number of arm entries between the three groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05)( Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results collectively suggested that the prolonged administration of \u003cem\u003eP.distasonis\u003c/em\u003e in the gut has the potential to enhance working memory in APP/PS1 mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLong-term P.distasonis administration attenuates Aβ plaque burden and tau pathology in APP/PS1 mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the impact of \u003cem\u003eP.distasonis\u003c/em\u003e on Aβ plaque deposition in APP/PS1 mice, we utilized immunofluorescence staining to examine the burden of Aβ plaques in APP/PS1 mice following 6 months of gavage treatment. Remarkably, the cortex and hippocampus of \u003cem\u003eP.distasonis\u003c/em\u003e-treated mice exhibited significantly reduced areas of Aβ-positive staining compared to the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026amp;F). To further quantify changes in Aβ levels, MSD immunoassays were employed to measure the levels of Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e42\u003c/sub\u003e in brain tissue. The MSD analysis demonstrated a significant decrease in both Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e42\u003c/sub\u003e levels in brain homogenates from mice treated with \u003cem\u003eP.distasonis\u003c/em\u003e in comparison to the PBS-treated APP/PS1 group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Specifically, phosphorylation of tau protein at Ser404 exhibited a significant increase in the cortex and hippocampus of the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026amp;F, whereas \u003cem\u003eP.distasonis\u003c/em\u003e treatment reversed this pathological alteration in APP/PS1 mice. Collectively, these findings suggest that supplementation with \u003cem\u003eP.distasonis\u003c/em\u003e through gavage could alleviate characteristic pathological changes in the brains of APP/PS1 mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLong-term administration of Parabacteroides distasonis reduces damage to the intestinal barrier and attenuates reactive glial activation and in APP/PS1 mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effect of \u003cem\u003eP.distasonis\u003c/em\u003e on gut barrier permeability in APP/PS1 mice, we assessed gut barrier integrity via immunofluorescence after 6 months of continuous oral gavage in APP/PS1 mice. Tight junction proteins ZO-1 and Claudin-1 were used as indicators of gut barrier integrity, and their expression was examined through immunofluorescence staining. Results showed that the expression of ZO-1 and Claudin-1 per unit area in the intestines of APP/PS1-PBS mice was significantly lower than that of wild-type mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both). Oral gavage with \u003cem\u003eP.distasonis\u003c/em\u003e significantly increased the expression of ZO-1 and Claudin-1 in the intestines of APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). These results suggested that \u003cem\u003eP.distasonis\u003c/em\u003e can repair damaged mechanical barriers in the intestines and reduce abnormal intestinal permeability. We further investigated the neuroinflammatory response in the brains of mice by staining for activated microglia marked by Iba-1 positivity and reactive astrocytes labeled with GFAP. Strikingly, the APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e group exhibited a significant reduction in Iba1-positive microglia and GFAP-positive astrocytes in the cortex and hippocampus regions compared to the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). This suggested that gut transplantation of P.distasonis effectively reduces the proliferation of glial cells and consequently alleviates immune-related pathologies in the brains of APP/PS1 mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdministration of Parabacteroides distasonis modulates T-cell populations and inflammatory cytokine profiles in APP/PS1 mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUtilizing samples of APP/PS1 mice with one-month \u003cem\u003eP.distasonis\u003c/em\u003e administration, we employed flow cytometry to investigate the subtypes of T-helper lymphocytes in the lamina propria of the small intestines, as well as the alterations in T cell populations on peripheral blood, spleen and brain, which play a critical role in maintaining immune equilibrium. Our findings exhibited a noteworthy rise in CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e(Treg) cells within the intestines of APP/PS1 mice treated with \u003cem\u003eP.distasonis\u003c/em\u003e, in comparison to both the control group and APP/PS1\u0026thinsp;+\u0026thinsp;PBS group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Earlier studies have documented the anti-inflammatory role of \u003cem\u003eP.distasonis\u003c/em\u003e in a mouse model of colitis by fostering the proliferation and differentiation of IL10\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003eTreg cells, thereby leading to a considerable anti-inflammatory effect\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.Thus, our flow cytometry outcomes concerning T-cell subsets align with prior reports imply that \u003cem\u003eP.distasonis\u003c/em\u003e might stimulate the proliferation and differentiation of Treg cells in the intestine. Conversely, the proportions of CD4\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003e (Th17) cells, whether in the confines of the small intestine or circulating in the blood, spleen, and brain, remained stoically unaltered between the APP/PS1\u0026thinsp;+\u0026thinsp;PBS and APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e groups (Fig.\u0026nbsp;1SA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVenturing further into our research, we extended our observations to APP/PS1 mice subjected to the prolonged embrace of a 6-month \u003cem\u003eP.distasonis\u003c/em\u003e gavage. Within the small intestines of these mice, a sustained augmentation of Treg cell populations emerged, accompanied by a simultaneous attenuation of Th1 cell populations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). However, no significant differences in Th1, Th17, and Treg cell populations within the spleen were discerned. Notably, there was a significant elevation in the proportion of Treg cells in the peripheral blood of APP/PS1\u0026thinsp;+\u0026thinsp;P. distasonis mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings underscore the profound impact of \u003cem\u003eP.distasonis\u003c/em\u003e administration in fortifying the ranks of Treg cells within the small intestine, both in the short span of one month and the extended saga of 6 months, culminating in a significant surge of Treg cells in the peripheral blood after six months of APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP. distasonis\u003c/em\u003e intervention(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026amp;C).\u003c/p\u003e \u003cp\u003eSubsequently, we conducted an analysis of CD4\u003csup\u003e+\u003c/sup\u003e T cell subpopulations within the brains of mice. Flow cytometry unveiled a conspicuous upsurge in the proportion of Treg cells within the brains of mice treated with \u003cem\u003eP.distasonis\u003c/em\u003e. This profound shift stood in stark contrast to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDoes this administration of \u003cem\u003eP.distasonis\u003c/em\u003e hold within its essence the gift of anti-inflammatory prowess? To unravel this enigma, we delved deeper into the intricacies of inflammatory cytokines inhabiting the same region after long-term gavage. The pro-inflammatory cytokine IL-1β exhibited notably higher levels in the intestines of APP/PS1\u0026thinsp;+\u0026thinsp;PBS mice compare to WT\u0026thinsp;+\u0026thinsp;PBS group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Interestingly, in APP/PS1 mice administered 6-month \u003cem\u003eP.distasonis\u003c/em\u003e, we observed a significant increase in the levels of anti-inflammatory cytokines IL-10 and TGF-β within the small intestine, standing in stark contrast to their counterparts in the APP/PS1\u0026thinsp;+\u0026thinsp;PBS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Additionally, the Th1-inducing cytokine IFN-γ stooped to significantly lower levels in APP/PS1\u0026thinsp;+\u0026thinsp;P. distasonis mice compared to their APP/PS1\u0026thinsp;+\u0026thinsp;PBS counterparts. Furthermore, our multiplex assays uncovered a significant upswing in the TGF-β levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a noteworthy downturn in the pro-inflammatory cytokine IL-1β (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) within the plasma of APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e mice. Oral gavage of \u003cem\u003eP.distasonis\u003c/em\u003e resulted in a significant increase in the secretion of anti-inflammatory cytokines, specifically IL-10 and TGF-β, while concurrently reducing the levels of the pro-inflammatory cytokine IL-1β, but had no significant effect on the Th1-inducing cytokine IFN-γ or the Th17-derived cytokine IL-17 in the brain(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Thus, our findings suggest that changes in inflammatory cytokine profiles corresponded to alterations in Treg cell populations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLong-term gavage with P. distasonis increases the migration of peripheral T cells towards the central nervous system in APP/PS1 mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the intricate interplay of immunoregulation, T cells stationed in the bloodstream serve as vigilant sentinels, receiving signals from inflamed tissues and embarking on migratory journeys to enact their immune-modulating duties\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In order to enter the central nervous system, they first adhere to and halt at the blood-brain barrier or choroid plexus barrier. Subsequently, they traverse through the gaps between endothelial cells and gain entry into the tissue. This process is facilitated by chemokines and cell surface receptors, such as CCR7-CCL20 and LFA-1-ICAM-1 for T cells returning to lymph nodes, and CCR6-CCL20 for T cells crossing the choroid plexus into the central nervous system\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. To explore the mechanisms behind the infiltration of peripheral CD4\u003csup\u003e+\u003c/sup\u003e T cells into the brains of 9-month-old APP/PS1 mice following 6 months of \u003cem\u003eP.distasonis\u003c/em\u003e oral gavage, our study examined the expression of chemokine receptors on CD4\u003csup\u003e+\u003c/sup\u003e T cells in the brain and corresponding chemokine levels. The results revealed a significant increase in CD4\u003csup\u003e+\u003c/sup\u003e T cells expressing the chemokine receptor CCR6 in the brains of APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e mice compared to APP/PS1\u0026thinsp;+\u0026thinsp;PBS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA. Additionally, the expression of CCL20, the ligand for CCR6, was significantly higher in the brains of APP/PS1\u0026thinsp;+\u0026thinsp;P.distasonis mice compared to APP/PS1\u0026thinsp;+\u0026thinsp;PBS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. These findings indicate that peripheral CD4\u003csup\u003e+\u003c/sup\u003e T cells have the capability to accumulate in the central nervous system via the CCR6/CCL20 chemotactic axis, while showing no such tendency via the CCR7/CCL21 chemotactic axis (Fig.\u0026nbsp;1SB).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study, we explored the potential therapeutic effects of \u003cem\u003eP.distasonis\u003c/em\u003e transplantation in the context of AD due to its previously demonstrated efficacy in mitigating various inflammatory conditions such as arthritis, obesity, metabolic dysfunctions, and modulating inflammatory markers\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Our previous investigations\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e has demonstrated a significant decrease in the prevalence of Parabacteroides in both fecal and blood samples from individuals diagnosed with AD compared to the control group (NC). This observation underscores the need to explore the efficacy and underlying mechanism of \u003cem\u003eP.distasonis\u003c/em\u003e application in AD. In the present study, we investigated the effects of gut microbiota transplantation involving \u003cem\u003eP.distasonis\u003c/em\u003e on APP/PS1 mice. Our findings from 16S rDNA sequencing highlight the substantial influence of transplanting \u003cem\u003eP. distasonis\u003c/em\u003e on the composition and diversity of gut microbiota in APP/PS1 mice. Furthermore, this supplementation resulted in a marked alteration in the gut microbiota composition. In the APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e group, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003ePrevotellaceae\u003c/em\u003e, \u003cem\u003eDesulfovibrionaceae\u003c/em\u003e, and \u003cem\u003eDesulfovibrio\u003c/em\u003e emerged as the dominant and prevalent bacterial taxa. In contrast, the genus \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eLachnospiraceae\u003c/em\u003e, and \u003cem\u003eCostridia\u003c/em\u003e were among the top enriched bacterial taxa in the APP/PS1\u0026thinsp;+\u0026thinsp;PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026amp;E). Similarly, a study involving the administration of aloe polysaccharides (APs), primarily composed of mannose polysaccharides, led to a notable increase in the abundance of \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eParabacteria\u003c/em\u003e, which are responsible for the production of short-chain fatty acids (SCFAs) in mouse feces. Conversely, APs reduced the prevalence of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eClostridium\u003c/em\u003e, establishing a positive correlation between the microbiota and SCFAs, with \u003cem\u003eParabacteroides\u003c/em\u003e playing a pivotal role in SCFA production\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eShort-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced through the fermentation of indigestible carbohydrates by gut microorganisms. They play a vital role in promoting gut health by providing energy to intestinal cells, regulating microbiota, enhancing gut function, reducing inflammation and influencing gene expression. Changes in SCFA levels are associated with conditions like obesity, diabetes, and colitis, making them valuable markers for diagnosis and assessment\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. In our study, the addition of \u003cem\u003eP.distasonis\u003c/em\u003e to the gut of APP/PS1 mice led to a notable increase in butyrate levels. \u003cem\u003eP. distasonis\u003c/em\u003e MRx0005 is known for its ability to produce short-chain fatty acids, with a particular emphasis on butyrate, which has demonstrated the capacity to alleviate neuroinflammation in in vitro studies\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Notably, our results also highlighted the prominent role of \u003cem\u003ePrevotellaceae\u003c/em\u003e in the APP/PS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eP.distasonis\u003c/em\u003e group. \u003cem\u003ePrevotellaceae\u003c/em\u003e is closely associated with SCFA synthesis and has been demonstrated to rectify disturbances in gut barrier function and promote butyrate synthesis in colonic macrophages through the PPARα-CYP4X1 axis\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Butyrate serves as a primary energy source for the intestinal mucosa and plays a crucial regulatory role in maintaining gut microbial balance, gut barrier function, and the modulation of various genes involved in lipid metabolism, immunity, inflammation, differentiation, apoptosis, phagocytosis, and clearance of dead cells\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Several studies have also indicated that butyrate exhibits the potential to mitigate inflammatory responses and promotes the differentiation of Treg cells \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Our findings bring to light an intriguing connection. The elevated proportion of Treg cells observed in the small intestine of APP/PS1 mice fed with \u003cem\u003eP.distasonis\u003c/em\u003e may be attributed to the augmented levels of butyrate synthesized by the gut microbiota. By elevating intestinal butyrate levels, the induction and differentiation of Treg cells in APP/PS1 mice are likely to be facilitated. Contrarily, butyrate exhibited a favorable impact, mitigating cognitive impairment, neuronal changes, and BDNF reduction in both in vivo and in vitro models\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. It could potentially contribute as an additional factor to the observed cognitive enhancement following the administration of \u003cem\u003eP. distasonis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, our study demonstrated that prolonged gavage of \u003cem\u003eP.distasonis\u003c/em\u003e led to an increased proportion of Treg cells in the intestine, concurrently curbing the proliferative tendencies of Th1 cells. This culminated in a noteworthy reduction in disturbances to the intestinal barrier, a mitigation of intestinal inflammation, heightened levels of anti-inflammatory cytokines IL-10 and TGF-β within intestinal tissues, and a decrease in IFN-γ concentrations. Moreover, a reduction in the levels of the peripheral pro-inflammatory factor IL-1β within the circulatory system was observed, contributing to an overall alleviation of neuroinflammation and enhancement of cognitive function in APP/PS1 mice. This study highlights the pivotal role of Treg cells in modulating inflammatory responses. Our findings align with previous research showing the detrimental effects of pro-inflammatory cytokines such as IL-1β and tumor necrosis factor (TNF-α) play a critical role in inflammatory bowel disease by contributing to the breakdown of intestinal epithelial barrier function and the expression of tight junction proteins. It is imperative to underscore that the disturbance of gut microbiota equilibrium can compromise intestinal permeability, consequently setting off neuroinflammatory cascades. Prior to immune alterations in the central nervous system, a clear connection exists between imbalances in gut microbiota and the propagation of inflammatory molecules from the peripheral to the central nervous system. These molecules can compromise the integrity of both the intestinal blood barrier and the blood-brain barrier, facilitating the entry of gut-derived substances into the brain, ultimately triggering neuroinflammation. This process serves as a conduit for gut-originating molecules, toxins, and pathogens to infiltrate the brain parenchyma, provoking neuroinflammatory responses\u003csup\u003e[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur research also uncovered potential links between immune cells residing in the CNS and those in the gut. The activation of the immune system in both the gut and the brain is closely associated with reactions to neuroinflammation, responses to brain injuries, and alterations in neurogenesis and neural plasticity.\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. In our one-month-duration experiment using gavage feeding, we observed the favorable influence of \u003cem\u003eP.distasonis\u003c/em\u003e on gut microbiota and immune responses. In particular, the modulation of T cell subtypes, including Th1 cells, Th17 cells, and Treg cells, is of paramount importance. Th1 cells drive inflammation primarily through the production of IFN-γ, while Th17 cells promote cell damage and inflammation via IL-17 production. In contrast, Treg cells execute immunosuppressive functions by releasing anti-inflammatory cytokines, including IL-10 and TGF-β\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. After the extended gavage administration of \u003cem\u003eP.distasonis\u003c/em\u003e, a notable increase in the Treg cell proportion within the brain was observed, in conjunction with a reduction in Aβ deposition and the inhibition of glial cell proliferation. This augmentation significantly facilitated the manifestation of its anti-inflammatory attributes through the expression of cytokines, most notably the prominent IL-10 and TGF-β, thereby effectively mitigating instances of neuroinflammation.\u003c/p\u003e \u003cp\u003eResearch has previously highlighted the role of immune cells, including Treg cells, in the context of conditions like multiple sclerosis, where the gut microbiota stimulates the proliferation and differentiation of active Th1 cells, Th17 cells, and Treg cells within the gut, which subsequently migrate to the CNS and either promote or suppress the onset of inflammation\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. It is essential to understand that immune cells can enter the brain parenchyma through meningeal lymphatic vessels, even in healthy brains\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. The migration of T cells into inflamed tissues is guided by chemoattractants anchored on endothelial cells and the glycocalyx surface. This intricate process consists of multiple navigation signals, including target antigens, chemotactic factors, integrin ligands, and mechanistic markers of the inflammatory microenvironment\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Our findings support the presence of Treg cells in the brain during the chronic inflammatory phase of ischemic stroke mouse models, contributing to neural functional recovery. These brain-resident Treg cells utilize amplification and infiltration mechanisms guided by signaling molecules (IL-2, IL-33, 5-HT7) and chemokines (CCL1, CCL20). They effectively inhibit neurotoxicity and suppress astrocyte proliferation\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.Previous research has suggested that Treg cells release cytokines such as IL-10 and IL-33, which contribute to the promotion of microglial polarization towards the M2 phenotype. M2 microglia, in turn, secrete IL-10 and TGF-β, further facilitating the polarization of Treg cells. This beneficial cycle suppresses the occurrence of neuroinflammatory responses, thereby providing a certain degree of protection\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. The observed increase in brain Treg cells prompts a question: Do they locally multiply? This touches upon the complex immune mechanisms in the central nervous system. In the context of neuroinflammation, the brain hosts resident immune cells, including microglia and astrocytes. They aren't passive; they actively engage in immune responses. What's fascinating is how their interactions with peripheral immune cells, like T cells, impact the intricate web of brain inflammation. While the administration of P. distasonis appeared to markedly mitigate both Aβ and tau pathologies in APP/PS1 mice, its supplementation did not elicit commensurate effects on inflammation and other associated indices. It is conceivable that gastric administration of P. distasonis may engender supplementary neuroprotective mechanisms, thereby enhancing cognitive function. Further elucidation of these mechanisms merits scholarly investigation.\u003c/p\u003e \u003cp\u003eIn summary, the introduction of \u003cem\u003eP.distasonis\u003c/em\u003e into the gut environment appears to play a significant role in ameliorating pathological alterations in a mouse model of AD. This beneficial effect is likely achieved through the modulation of gut microbiota composition, elevation of butyrate levels, stimulation of gut Treg cell proliferation, reduction of intestinal barrier impairment, and augmentation of central CD4\u003csup\u003e+\u003c/sup\u003eT cell chemotaxis, particularly the increased presence of immunosuppressive Treg cells within the brain. These actions collectively contribute to the attenuation of neuroinflammation and enhancement of cognitive function in AD mice. Our research suggests that the supplementation of \u003cem\u003eP.distasonis\u003c/em\u003e could potentially offer an alternative therapeutic approach for both the prevention and treatment of AD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mouse experiments were approved by The Charles River Laboratories Institutional Animal Care and Use Committee (IACUC) under approval number P2021098. The study was reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) \u003csup\u003e[25]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China, (82171401, 81971187 (to SC), (32100798(to YH)), grants from Shanghai Municipal Science and Technology Major Project, No. 2018SHZDZX05 (to SC) and Shanghai Municipal Education Commission, No. 2017-01-07-00-01-E00046 (to SC) and the China Postdoctoral Science Foundation(LCBSHZX017 (to YH)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are not publicly available due to ongoing research and confidentiality concerns. However, summarized data supporting the findings are presented in the article. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Intererts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or other conflicts of interest related to this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJJ was responsible for the design and conception of the research, the collection of data, the analysis and interpretation of the data, as well as the writing of the paper.\u003c/p\u003e\n\u003cp\u003eYH get involved in data collection, analysis, and interpretation .\u003c/p\u003e\n\u003cp\u003eHP\u0026amp;YT: the process of interpreting the data.\u003c/p\u003e\n\u003cp\u003eNX: conceived and designed the experiments.\u003c/p\u003e\n\u003cp\u003eSC\u0026amp;DS: the design and conception of the experimental research, the interpretation of the data and the editing of the paper\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to all those who contributed to this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGrontvedt, G. 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CNS Neurosci Ther[J]. 27, 643\u0026ndash;651.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Parabacteroides distasonis, Alzheimer's Disease, Treg cell, Butyrate, Chemotaxis","lastPublishedDoi":"10.21203/rs.3.rs-4156881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4156881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlzheimer's Disease (AD) is a neuropathological condition marked by cognitive deterioration and chronic neuroinflammation. Previous investigations have unveiled a strong correlation between the gut microbiota and the progression of AD. In this study, our objective is to probe the effects of Parabacteroides distasonis (\u003cem\u003eP.distasonis\u003c/em\u003e), previously found to be conspicuously diminished in AD patients, on the APP/PS1 mice model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of\u0026nbsp; orally administered \u003cem\u003eP.distasonis\u003c/em\u003e on gut microbiota and metabolites, we utilized 16s rDNA sequencing and GC-MS to analyze gut composition and short-chain fatty acids in APP/PS1 mice after one month of \u003cem\u003eP.distasonis\u003c/em\u003e gavage. To investigate the effects of \u003cem\u003eP.distasonis \u003c/em\u003eadministration over a six-month period on APP/PS1 mice, we evaluated cognitive function using novel object recognition and Y-maze tests, assessed intestinal barrier integrity and AD-related pathological features with immunofluorescence, and analyzed immune cell subpopulations in intestine, blood, spleen, and brain tissues via flow cytometry. The Luminex assay was employed to detect inflammatory cytokine secretion in the same regions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-month\u0026nbsp; oral administration of \u003cem\u003eP.distasonis\u003c/em\u003e modulated the gut microbiota, elevated butyrate levels. Six-month oral administration of \u003cem\u003eP.distasonis\u003c/em\u003e improved cognitive function in APP/PS1 mice, reducing Aβ deposition and inhibiting glial cell proliferation. It also amplified Treg cells within the gut, concomitant with the decreased Th1 proliferation and intestinal inflammation. Additionally, we observed the migration of peripheral CD4\u003csup\u003e+\u003c/sup\u003e T cells to the brain through chemotaxis, accompanied by an increase in Treg cells and higher levels of anti-inflammatory factors such as IL-10 and TGF-β in the brain. Collectively, these multifaceted effects contributed to the alleviation of neuroinflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings underscore the potential of transplanting \u003cem\u003eP.distasonis\u003c/em\u003e in alleviating AD-related pathology, suggesting a role for gut microbiota in neuroinflammation attenuation.\u003c/p\u003e","manuscriptTitle":"Transplantaion of Parabacteroides distasonis mitigated Aβ-associated changes in APP/PS1 mice via gut-brain interactions by attenuating neuroinflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 19:04:29","doi":"10.21203/rs.3.rs-4156881/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d5b2ae87-9fa3-4698-8141-2f94991762d8","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-09T14:32:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-29 19:04:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4156881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4156881","identity":"rs-4156881","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}