Journal of Neuroinflammation

He et al. Journal of Neuroinflammation(2024) 21:153
https://doi.org/10.1186/s12974-024-03145-0

Open Access

RESEARCH

Citrobacter rodentium infection impairs
dopamine metabolism and exacerbates thepathology of Parkinson’s disease in mice
Yongtao He1†, Jiayin Zhao1†, Yuanyuan Ma1†, Xin Yan1, Yufei Duan1, Xiaoshuang Zhang1, Hongtian Dong1,
Rong Fang1, Yunhe Zhang1, Qing Li2, Ping Yang2, Mei Yu1*, Jian Fei2,3* and Fang Huang1*
Abstract
Parkinson’s disease (PD) is a prevalent neurodegenerative disorder with indistinct etiology and ill-defined
pathophysiology. Intestinal inflammation involved in the pathogenesis of PD, but the underlying mechanism is
not fully understood. Citrobacter rodentium (C.R) is a gram-negative bacterium that can be used to induce human
inflammatory bowel disease in mice. Here, we investigated whether the proinflammatory effects caused by C.R
infection initiate PD-like injury and/or exacerbate PD pathology and extensively studied the underlying mechanism.
Mice were gavaged once with C.R and monitored for several pathological features at 9 days post infection. The
results showed that C.R delivery in mice induced IBD-like symptoms, including significant weight loss, increased
fecal water content, an impaired intestinal barrier, intestinal hyperpermeability and inflammation, and intestinal
microbiota disturbances. Notably, C.R infection modified dopamine (DA) metabolism in the brains of both male andfemale mice. Subsequently, a single high dose of MPTP or normal saline was administered at 6 days post infection.
At 3 days after MPTP administration, the feces were collected for 16 S rRNA analysis, and PD-like phenotypes andmechanisms were systemically analyzed. Compared with C.R or MPTP injection alone, the injection of C.R and MPTP
combined worsened behavioral performance. Moreover, such combination triggered more severe dopaminergic
degeneration and glial cell overactivation in the nigrostriatal pathway of mice. Mechanistically, the combinationof C.R and MPTP increased the expression of TLR4 and NF-κB p65 in the colon and striatum and upregulated
proinflammatory cytokine expression. Therefore, C.R infection-induced intestinal inflammation can impair dopamine
metabolism and exacerbate PD pathological processes.
Keywords Parkinson’s disease, Citrobacter rodentium infection, Inflammatory bowel disease, Neuroinflammation,
Microbiota disturbances, Neurotransmitter metabolism, TLR4 signaling pathway†

Yongtao He, Jiayin Zhao and Yuanyuan Ma contributed equally tothis work.
*Correspondence:
Mei Yuyumei@fudan.edu.cn
Jian Fei
jfei@tongji.edu.cn
Fang Huang
huangf@shmu.edu.cn

1

Department of Translational Neuroscience, Jing’an District Centre
Hospital of Shanghai, State Key Laboratory of Medical Neurobiology and
MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan
University, 138 Yixueyuan Road, Shanghai 200032, China
2
Shanghai Engineering Research Center for Model Organisms, SMOC,
Shanghai 201203, China
3
School of Life Science and Technology, Tongji University, 1239 Siping
Road, Shanghai 200092, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

He et al. Journal of Neuroinflammation(2024) 21:153

Introduction
Parkinson’s disease is a progressive irreversible neurodegenerative disorder characterized by dopaminergic neuronal death in the substantia nigra (SN) and theaggregation of α-synuclein (α-syn) in surviving neurons.
PD affects more than 10 million people worldwide andthere is currently no effective cure for this disease [1]. PD
patients present with a range of classic movement disorders, including bradykinesia, rigidity, resting tremors,and postural instability [2]. However, some nonmotor
manifestations involving gastrointestinal (GI) dysfunctions such as constipation, dysphagia and nausea often
precede motor symptoms in PD patients [3, 4].
The pathogenesis of PD is obscure, but current evidence strongly supports that intestinal inflammation isa cause [5]. As demonstrated by a study in humans, theexpression of inflammatory cytokines in colonic biopsy
and fecal samples from PD patients was greater than thatin samples from healthy individuals [6, 7]. Moreover,epidemiological studies have shown that the incidenceof idiopathic PD is approximately 50% lower in chronic
users of nonsteroidal anti-inflammatory drugs or cyclooxygenase inhibitors than in age-matched individualwith normal use of these drugs [8]. Inflammatory bowel
disease (IBD), a common type of enteritis, has been confirmed to be a potential risk factor for PD development[9]. For example, epidemiologic studies have reported asignificant association between Crohn’s disease and therisk of developing PD [10, 11]. Genome-wide association
studies have demonstrated that there are several overlapping of gene loci between PD and IBD [12]. In addition, one study showed that the treatment of IBD with
anti-TNF agents reduced PD incidence rates by morethan 78% [10]. However, related experiments are rare inanimal studies. Only one report at present shows thatdextran sulfate sodium (DSS)-evoked ulcerative colitis(UC) can exacerbate lipopolysaccharide-induced damage to the nigral dopaminergic system [13]. Hence, moreevidence is needed to verify the association between
intestinal inflammation and PD and reveal the detailed
underlying mechanisms involved.
C.R is a specific murine pathogenic bacterium thatmainly colonizes the colon, resulting in transient colitis[14, 15]. Multiple strains of adult mice, including outbred
mice (Swiss Webster) and inbred mice (C57BL/6, C3H
and FVB), are susceptible to C.R infection, which induces
attaching and effacing lesions [14, 16, 17]. Proinflammatory states of C.R infection are clearly observed [16].
However, the colonic mucosal inflammation induced
by C.R infection is more moderate than that induced inother colitis models, such as chemical induced models. A
previous report showed more moderate myeloperoxidasescores in C.R-infected mice than in DSS-challenged mice[18], suggesting that C.R infected colitis models could

Page 2 of 26

better mimic human intestinal diseases. Moreover, in
Pink1 deficient mice, infection with C.R triggers PD-like
symptoms by changing the microbial composition [19,20]. The other detrimental effects of microbial disorders
caused by C.R infection on the PD process, however, havenot been fully elucidated.
In this study, we utilized C.R gavage to establish an
intestinal inflammation model and confirmed the induction of IBD-like symptoms through analysis of several
pathological features. Additionally, we investigatedwhether C.R-induced intestinal inflammation impairs
dopamine metabolism and exacerbates the loss of dopamine neurons in an MPTP-based model of PD and elucidated the underlying mechanism.

Materials and methods
Animals

Male and female C57BL/6J mice aged 10–12 weeks
were obtained from Shanghai Model Organisms Center,
INC. The mice were raised (12 h light/dark cycle) understandard conditions (temperature 22 ± 2 °C, humidity
50–60%) with free access to food and water. All animal
experimental procedures were performed following theInstitutional Animal Care and Use Committee of Fudan
University, Shanghai Medical College (IACUC Animal
Project Number: 20190221-141). All operations were
performed under general anesthesia, and all efforts weremade to minimize suffering.
Bacterial culture and infection of mice

C.R (DBS770 strain, chloramphenicol-resistant) was purchased from American Type Culture Collection (ATCC)
and cultured according to previously described methods [21]. Briefly, a single colony was picked after culturing C.R on chloramphenicol MacConkey agar plates(LA8200, Solarbio life sciences, China) for 24 h at 37 ℃.
Then, bacteria were shaken overnight in Luria-Bertani(LB) broth supplemented with chloramphenicol (50 µg/mL) at 200 × g at 37 ℃ and collected the bacteria by centrifugation at 3000 × g for 10 min. Pelleted bacteria were
washed and resuspended in 0.01 M sterile PBS. The concentration of bacteria was assessed by measuring absorbance at an optical density (OD) of 600 nm and seriallydiluted and plated each inoculation culture to confirm
the colony-forming units (CFUs).
Mice were fasted and only given water for 8–12 h
before oral gavage with 4 × 108 CFU in volume of 200 µL.
The mice were allowed ad libitum access to food after andweighted daily after gavage. To monitor bacterial burden, fresh feces were collected from each mouse at 2-day
intervals, weighed, and dissociated in 0.01 M sterile PBS
at a ratio of 0.1 g stool per 1 mL PBS. The supernatant
was collected after centrifugation at 400 × g for 3 min at
4 °C, and plated on chloramphenicol MacConkey agar

He et al. Journal of Neuroinflammation(2024) 21:153

plates after serial dilution. Incubated the plates at 37 °C
overnight to allow colonies to growth, and counted colonies. Final counts were presented as CFU/g and plottedon a log scale in Prism.
To further explore the effector components of C.R, thebacteria were cultured to OD600 around 0.4–0.5 andcentrifuged at 3000 × g for 10 min. The supernatant wascollected and then passed through 0.22 μm polyether-sulfone filters to remove the residual bacterial cells. Finally,
the bacterial pellet was resuspended in sterile PBS andthen sterilized at 121 ℃ for 5 min, to get inactivated bacteria concentration (4 × 108 CFU/ 200 µL).
C.R identification by PCR

Total DNA was extracted from Citrobacter rodentium
and Escherichia coli (ATCC25922) via boiling at 100 ℃
in ddH2O. The primers: 5’-​G​C​T​T​A​A​T​G​G​A​A​T​T​G​G​T​C​A​G​G​C​C-3’ (EspF-F) and 5’-​G​C​G​A​G​A​G​G​G​A​G​T​T​A​A​T​G​A​
C​G-3’ (EspF-R) against Citrobacter rodentium EspF gene
were applied.
Establishment of Parkinson’s disease mouse model

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)(M0896, Sigma-Aldrich, USA) was dissolved in 0.9%
saline to a final concentration 5 mg/mL. Considering thatthe combination of C.R and an acute regimen of MPTP
(four consecutive 10–20 mg/kg injections at 2 h intervals) sharply increased mortality, a single injection ofhigh -dose MPTP (40 mg/kg) was given by intraperitoneal administration, and the control animals identicallywere received the same volume of 0.9% saline. 3 days
after MPTP administration, mice were sacrificed after
performing behavioral tests (treatment time in the study
is depicted in Table S2).
Behavioral tests

All behavioral tests were performed on Day 3 after MPTP
treatment.
Pole test

The mice stood on a rough-surfaced wooden pole (50 cm
in height and 1 cm in diameter) placed in the home cage,and some buffer material was placed at the bottom toprevent falling injury. A training trial was performed
before the experiment. The turning time and time todescend to the bottom of the pole were recorded for eachmouse in 3 trials with an interval of 1 h. The final results
represented the average values of 3 trials.
Rotarod test

Mice were placed on a rotarod apparatus, and then therod revolved at a speed of 32 rpm for up to 300 s. The
time for each mouse to fall off the rod was recorded. Eachmouse performed the test for 3 trials with an interval ofPage 3 of 26

1 h. The final data represented the average values of 3
trials.
Wire-hanging test

The wire hanging test was carried out according to a previously described method with slight modifications [22].
In brief, the mice were suspended on their forelimbs on
a horizontal wire (1.6 mm in diameter, 50 cm long, 30 cm
high between two poles equipped with a platform). The
animals were trained for 2 days with 3 trials per day
before the experiment. The baseline score was 10 points,
and falling from the wire resulted in a one-point deduction, whereas climbing to the platform resulted in the
addition of one point. This test was carried out in 3 trials, and the final scores were an average of scores in the
3 trials.
Open field test (OFT)The animals were placed in an open-field apparatus consisting of a white cube opaque chamber (40 × 40 × 40 cm)
and allowed to freely move for 5 min. All activities wererecorded by a computer system. The total moving distance, mean velocity, frequency of movements and timespent in the central zone were analyzed to evaluate motor
function and emotion.
Rearing test

The mice were placed into a transparent cylinder (25 cm
in diameter, 60 cm high) for 3 min, and the number ofrearing were recorded blindly by an experimenter.
Fecal water and food intake measurements

At 9 days after C.R infection, the animals were placed
individually in a clean and transparent plastic cage for
1 h. The fresh feces were collected into a tube, and thewet weight was measured. The dry weight was determined after 24 h of drying at 85 °C. The water content
percentage was calculated as the wet weight minus thedry weight divided by the wet weight. In addition, foodintake was calculated over a 24-h period before sacrifice.
Measurement of intestinal transmission distance and colon
lengthThe gastrointestinal function was assessed by measurement of intestinal transmission distance [23]. In brief,
at 9 days after C.R infection, animals fasted for 8 h followed by oral administration (10 µL/g) of the suspension
containing 5% activated carbon (934,674, Sigma-Aldrich,USA) and 5% Gum Arabic (G9752, Sigma-Aldrich, USA).
30 min later, the mice were sacrificed and the small
intestine was isolated. Then, the total length of the small
intestine, and the distance from the pylorus to the black
marker were measured, which represented intestinal
transmission distance.

He et al. Journal of Neuroinflammation(2024) 21:153

Meanwhile, the colon length between the end of thececum and the anus was measured.
Intestinal permeability assay

At 9 days after C.R infection, the intestinal barrier integrity was measured by permeability assay using fluorescein isothiocyanate-dextran (FITC)-dextran (FD4,Sigma-Aldrich, USA). In brief, mice fasted for 4 h andthen they were orally given FD4 at 0.6 mg/g body weight.
After another 4 h, mice were euthanized and blood wascollected from the orbital cavity. The sample was kept atroom temperature for 2 h without light and the serum
was collected after centrifugation at 3000 × g for 10 min.
The fluorescence intensity was detected by a fluorescence
spectrophotometer (excitation: 485 nm and emission:
528 nm) (PerkinElmer, USA) and FD4 concentration in
the serum was further calculated according to a standard
curve established in advance.
Enzyme-linked immunosorbent assay (ELISA)The ELISA kits used to detect the level of mouse IL-6
(EMC004.96, Neobioscience, China) in serum and calprotectin (KBR-hlk7175, Keborui, China) in feces at 9days after C.R infection. The operations were conductedaccording to the manufacturer’s instructions. IL-6 andcalprotectin concentrations were expressed in pg/mL andng/g, respectively.
Western blotting

Mouse striatal and colonic proteins were extracted at 3days after C.R plus MPTP administration via lysing in
RIPA buffer (P0013B, Thermo Fisher Scientific, USA)with protease and phosphatase inhibitor (78,425, Thermo
Fisher Scientific, USA). The total protein concentrationof the supernatant was measured by a BCA Kit (23,227,
Thermo Fisher Scientific, USA). After the supernatant
was denatured by boiling for 5 min in 1 × loading buffer, equal amounts of protein samples were subjected to
7.5–12.5% SDS-polyacrylamide gel electrophoresis andtransferred to polyvinylidene difluoride membranes. The
membranes were blocked with 5% non-fat milk in 0.1%
TBST for 1 h at room temperature before incubation
with primary antibodies overnight at 4 °C. After washing with 0.1% TBST, the membranes were incubatedwith an IRDye® 800CW goat anti-mouse or anti-rabbit
IgG (1:20000; 92,632,210, 92,632,211, LI-COR, USA) for
1 h at room temperature. The blots were visualized byan infrared imaging system (Li-Cor, USA) and the relative level of protein was quantified using Image J software(NIH, Bethesda, MD). The primary antibodies used in the
study including: mouse anti-TH antibody (1:1000; T2928,
Sigma-Aldrich, USA), rabbit anti-GFAP antibody (1:1000;
16825-1-AP, Proteintech, USA), rabbit anti-Iba1 antibody(1:1000; ab178846, Abcam, USA) and rabbit anti-COX2

Page 4 of 26

antibody (1:1000; ab179800, Abcam, USA), mouse anti-βactin antibody (1:2000; sc-47,778, Santa Cruz, USA), rabbit anti-DAT antibody (1:1000; ab184451, Abcam, USA),rabbit anti-MAOA antibody (1:1000; ab12675, Abcam,
USA), rabbit anti-MAOB antibody (1:1000; 12602-1-AP,
Proteintech, USA), mouse anti-TLR4 antibody (1:500;
sc-293,072, Santa Cruz, USA), mouse anti-NF-κB p65
antibody (1:500; ab32536, Abcam, USA), mouse antiNF-κB p65 antibody (1:500; ab32536, Abcam, USA), rabbit anti-phospho-NF-κB p65 (Ser536) antibody (1:1000;
93H1, Cell Signaling Technology, USA).
Immunofluorescence and immunochemical staining

At 3 days after MPTP administration, the animals weredeeply anesthetized and perfused through the ascending aorta with 0.9% saline infusion. The brain and colon
were dissected and immediately fixed in a 4% paraformaldehyde (PFA) fixative solution for 24 h. Next, thetissues were dehydrated (in 20% and 30% sucrose overnight at 4 °C, respectively), embedded in OCT and sectioned (30 μm thickness) on a frozen microtome (Leica,
Germany). The sections were permeabilized and blockedwith 0.2% Triton X-100 and 10% normal goat serum in
PBS at 37 °C for 1 h, and subsequently incubated withprimary antibodies as follows: mouse anti-TH antibody(1:1000; T2928, Sigma-Aldrich, USA), rabbit anti-GFAP
antibody (1:1000; 16825-1-AP, Proteintech, USA), rabbit
anti-Iba1 antibody (1:1000; ab178846, Abcam, USA), rabbit anti-C3 antibody (1:1000, ab200999, Abcam, USA),rat anti-CD16/32 antibody (1:300; 553,142, BD Pharmingen, USA), rabbit anti-ZO-1 antibody (1:200; sc-33,725,
Santa Cruz, USA), rabbit anti-Occludin antibody (1:200;
sc-133,256, Santa Cruz, USA), rabbit anti-Claudin 1 antibody (1:200; sc-166,338, Santa Cruz, USA), mouse antiTLR4 antibody (1:200; sc-293,072, Santa Cruz, USA)overnight at 4 °C. The sections were incubated with Alexa
Fluor 488-conjugated, 594 conjugated-, or Cy3-conjugated secondary antibodies (1:1000; A-11,008, A-11,012,
A10522, Invitrogen, USA) for 2 h before image capture.
DAPI solution was performed to stain nuclei.
For immunochemical staining, the sections were incubated with 0.3% H2O2 to reduce endogenous peroxidase
activity, subjected to antigen retrieval with sodium citratesolution (pH = 6.0), and blocked with PBS containing
0.2% Triton X-100 and 10% normal goat serum at 37 °C
for 45 min. Then the sections were incubated overnight at
4 °C with primary antibodies, including mouse anti-TH(1:1000; T2928, Sigma-Aldrich, USA), rabbit anti-GFAP(1:1000; 16825-1-AP, Proteintech, USA), and rabbit antiIba1 antibodies (1:1000; ab178846, Abcam, USA). The
following day, the sections were incubated with a biotin-conjugated goat anti-rabbit or goat anti-mouse IgG
secondary antibody (1:200; PK-6101, PK-6102, Vector
laboratories, USA), and staining was performed using

He et al. Journal of Neuroinflammation(2024) 21:153

an ABC (avidin-biotin complex) system. The reaction
was detected with a DAB substrate kit (1:200; SK-4100,
Vector laboratories, USA). Images were acquired by amicroscope (Olympus DP73, Japan). Optical densityof striatal TH-positive fibers was quantitatively evaluated using Image-Pro Plus 6.0 software (Media Cybernetics, USA) and the density and morphology of striatal
microglia was determined using Image J software (NIH,
Bethesda, MD).
Stereological counting of TH immunoreactivity

To calculate the number of TH-positive cells in the SN
at 3 days after C.R and MPTP combined treatment, stereological cell counting was performed as previously
described [24]. The total number of TH neurons in the
SN was estimated using the Stereo Investigator system (Micro Brightfield, USA) which combined with an
Olympus microscope (Tokyo, Japan). Briefly, a total of
6 Sect. (30 μm thickness/section) at a 5-section interval
between Bregma − 2.80 and − 3.65 mm were chosen. A
specified outline of SN was delimited at 4 × objectives,
and the cells were sampled at 40 × objectives. Stereological counting was analyzed by an experimenter blinded tothe experimental groups. The final data are presented as a
total number of TH neurons.
RNA isolation and quantitative real-time-PCR analysis

Total RNA was extracted from striatum and colon samples with TRIzol isolation reagent (DP424, Tiangen,
China) according to the manufacturer’s instructions, andRNA was reverse-transcribed to cDNA using a highcapacity cDNA reverse transcription kit (KR116, Tiangen, China). The target mRNA amount in the samples
was detected using SYBR-Green (CW3008M, CWBIO,
China) and the relative mRNA expression levels of various genes were determined by quantitative real-timepolymerase chain reaction (qPCR) on QuantStudio 3
Instrument (Applied Biosystems, USA) with the following 3-step thermal cycling protocol: denaturation was
performed at 95 ℃ for 30 s (step1), followed by annealing at 60 ℃ for 10 s (step2) and extension subsequently
occurred at 72 ℃ for of 30 s (step3). The 3-step process
was repeated for a total of 40 cycles. Data collection
occurs at the 72 ℃ stage for 30 s for each of the cycles
across all wells. Normalized to the mRNA expression
level of β-actin, the mRNA expression of other genes wascalculated using the 2−ΔΔCt method. The paired primers
used for amplification are listed in Table S1.
Measurement of neurotransmitters and SCFAs by highperformance liquid chromatography (HPLC)

At 9, 15, 21 and 30 days after C.R infection, the isolated striatum was homogenized in 0.4 M HClO4 andthen centrifuged at 12,000 × g, at 4 °C for 10 min. The

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supernatants were collected to determine the concentrations of neurotransmitters, and 20 µL of the sample
was injected into an Acclaim™ 120 C18 column (Thermo
Fisher Scientific, USA). The concentrations of DA, 5-HT
and their metabolites, including dihydroxyphenylacetic
acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), were quantified by HPLC via
chromatography (ESA, USA) with a 5014B electrochemical detector.
The fresh feces were subjected to ether extraction to
obtain SCFAs. Briefly, approximately 0.3 g of fresh feces
was collected and thoroughly suspended in ddH2O. Aftercentrifugation at 12,000 × g for 10 min, the supernatant
was collected, and 100 µL of concentrated hydrochloric
acid was added. Then, 5 mL of ether was added, and the
mixture was allowed to react for 20 min at room temperature. The resulting organic phase solution was collected
and mixed with 0.5 mL of 1 M NaOH solution. After
thorough mixing, the mixture was extracted for an additional 20 min. The aqueous phase solution was then collected by centrifuging the mixture at 3500 × g for 10 min.
Subsequently, the aqueous phase was filtered througha 0.22 μm organic filter membrane and the concentrations of acetic acid, propionic acid, and butyric acid were
determined using HPLC. The chromatographic conditions involved mobile phase A (0.01% phosphoric acidaqueous solution) and mobile phase B (methanol), withlinear gradient elution. The detection wavelength was
set at 210 nm, the column temperature was 30 °C, andthe flow rate was 1 mL/min. The results are presented inunits of µg/mg of tissue.
Gut microbe 16 S rRNA sequencing and data analysis

Fresh fecal pellets were collected at 9 days after C.R infection or 3 days after MPTP administration, and immediately stored at -80 °C for further analysis. Total genomic
DNA was extracted from fecal samples with a QIAamp
Fast DNA Stool Mini Kit. The DNA concentration wasdetermined with an Equalbit dsDNA HS Assay Kit. The
V3-V4 regions of the microbial 16 S rRNA were amplified
using a panel of proprietary primers aimed at relatively
conserved regions designed by GENEWIZ (Suzhou,
China). All the quantified amplicons were pooled
together at equal concentrations for Illumina MiSeq
sequencing (Illumina, San Diego, CA, USA). Experiments
including DNA extraction, quality assessment, library
construction, and high-throughput sequencing were performed by GENEWIZ.
The raw sequencing data were filtered, and sequences
longer than 200 bp were retained. Only high-quality
sequences without chimeras were used for analysis.
VSEARCH clustering (1.9.6) was used to cluster the highquality sequences with 97% similarity to obtain operational taxonomic units (OTUs). Then representativeHe et al. Journal of Neuroinflammation(2024) 21:153

sequences of OTUs were annotated by comparison to a
database (Silva 132).
α-Diversity indices (ACE index and Shannon index)
were calculated by QIIME (V1.9.1) based on the OTU
results. β-Diversity analysis was performed to investigate
the structural variation in microbial communities acrosssamples using UniFrac distance metrics, and the results
were visualized via principal coordinate analysis (PCoA).
Differences in the UniFrac distances among groups were
determined by analysis of similarities (ANOSIM). LEfSe
was performed to detect differentially abundant taxa at
the phylum, family, and genus levels across groups using
the default parameters.
Histological analysis

After the mice was euthanized at 9 days after C.R infection, the colon was removed and washed once with icecold PBS. The terminal 0.5-cm piece of the colon was
immersed in 4% formaldehyde for 24 h and embedded
in paraffin, after which and 5-micron sections were cut.
The sections were deparaffinized and dehydrated withxylene and decreasing concentrations of ethanol (100%,90%, 80%, and 70% ethanol) for subsequent Hematoxylin
and Eosin (H&E) staining. Images were taken by a microscope (Olympus DP73, Japan). The histological score wasblindly assessed by 2 experimenters. The scoring criteria
were based on published literature [25]. Briefly, histological changes in the intestines, including inflammationand epithelial injury, were graded. Inflammatory infiltration was evaluated based on the severity and extent oflymphocyte and neutrophil infiltration. Epithelial injury
was assessed according to the degree of tissue damage,loss of mucosal glands, and the presence of ulceration or
erosion. Histopathological changes for each parameter
were scored using a scale from 0 to 3, with 0 indicating
no changes, 1 indicating mild changes, 2 indicating moderate changes, and 3 indicating severe changes. The area
involved was rated as follows: 0 = no involvement, 1 ≤ 25%of the section, 2 ≤ 50%, 3 ≤ 75%, and 4 ≤ 100%.
Statistical analyses

Statistical analyses were performed with Prism 7 (GraphPad software, USA). All the data were represented as
the mean ± S.E.M. and assessed for normal distribution
by the Shapiro-Wilk test. Nonparametric test was used
when the data did not fit normal distribution. For experiments involving only two groups, an unpaired t test wasused for comparison. Depending on the number of independent variables, one-way or two-way ANOVA followed by LSD multiple comparison tests were used. The
correlations between different experiments utilized thePearson correlation analysis. Statistically significant differences were defined as p < 0.05.

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Results
C.R infection causes gut microbiota alterations

C.R is a pathogen restricted to mice, and it shares 67% of
its genes with human enteric pathogens [26]. C.R colonies were identified as pink colonies with narrow white
trim on MacConkey agar plates (Fig. S1 A). Specificprimers against the C.R EspF gene were applied for identification [27] (Fig. S1 B). The growth curve of C.R was
established, and the bacterial concentration (CFU/mL) inthe culture was calculated from the OD600 values (Fig.
S1C-D).
Mice were orally administered with 4 × 108 CFU of C.R
after fasting, and different parameters were recorded
and analyzed according to the experimental scheme toconfirm the effectiveness of C.R infection (Fig. 1A). A
significant decrease in body weight was observed after
C.R infection. At 9 days post infection (d.p.i.), the bodyweight of the mice was approximately 5% less than theinitial weight (Fig. 1B). The fecal load of C.R was measured at 2-day intervals. The amount of C.R reachedapproximately 109 CFU/g in feces at 6 d.p.i and remained
relatively constant until the end of monitoring (Fig. 1C).
The fecal water content was monitored at 3-day intervals. Compared with uninfected mice with approximately
58% fecal water content, C.R-infected mice exhibited anincrease in the percentage of water in the feces (63% at
3 d.p.i., 73% at 6 d.p.i., and 68% at 9 d.p.i.) and a maximum at 6 d.p.i. (C.R vs. control: p < 0.0001) (Fig. S2A).
The food consumption of the animals was monitored
at 9 d.p.i., and a notable decrease was observed in C.Rinfected mice compared to the control mice (C.R vs. control: p = 0.0004) (Fig. S2B). The length of small intestinal
transit was evaluated and there was a significant reduction compared to that in the control group (C.R vs. control: p = 0.0375) (Fig. S2C). In contrast, the spleen index
of C.R-infected mice was greater than that of control
group mice (C.R vs. control: p = 0.0017) (Fig. S2D), suggesting an increased immune response. Collectively,
these data confirmed the effectiveness of C.R infection.
To investigate the effect of C.R challenge on the gut
microbiota composition, we characterized changes in
the gut microbiota composition in fecal samples frommice infected with C.R or vehicle solution (control
group) at 9 d.p.i. according to high-throughput sequencing of the V3-V4 region of the 16 S rRNA genes. A significant reduction in α-Diversity was observed in the
C.R-infected mice compared to the control mice basedon the ACE index, an estimate of microbial community
richness (C.R vs. control: p = 0.0016) (Fig. 1D). However,there were no differences between the groups in termsof the Shannon index, which is representative of microbial diversity (Fig. 1E). The extent of similarity of the gut
microbial communities between the control group andthe C.R group was measured using PCoA based on theHe et al. Journal of Neuroinflammation(2024) 21:153

Page 7 of 26

Fig. 1 C.R infection model establishment and analysis of the gut microbiome. (A) The scheme of C.R oral inoculation in the study. (B) The change in bodyweight relative to the initial weight of the mice was analyzed at the indicated time points after infection with C.R. n = 10–26. (C) Bacterial shedding in feces
was monitored at 2-day intervals after infection with C.R. n = 10. (D-E) The analysis of α-Diversity based on 16 S rRNA sequencing predicted gut microbiota
richness according to the ACE index (D) and diversity according to the Shannon index (E). (F-G) PCoA plots based on weighted UniFrac metrics of the gut
microbiota where samples of mice from different groups are highlighted with different colors. PC1 and PC2 explained 49.44% and 14.56%, respectively,of the variance. PC1 and PC3 explain 49.44% and 7.67%, respectively, of the variance. The position and distance of the data points indicated the degreeof similarity in terms of both the presence and relative abundance of the bacterial taxonomies. (H) Percentage stacking chart based on the Bray-Curtis
distance analysis of the differences in the relative abundance of gut microbiota at the phylum level between the groups. (I) Differences in the relative
abundance of gut microbiota at the family level between the groups. (J) Differences in the relative abundances of gut microbiota at the genus level
between the groups. (K-L) Relative abundances of 2 significantly altered bacterial genera: Akkermansia (K), and Lachnospiraceae (L). n = 8–10. **p < 0.01,***p < 0.001, ****p < 0.0001

He et al. Journal of Neuroinflammation(2024) 21:153

weighted UniFrac distance and the Bray-Curtis dissimilarity to identify possible differences between the bacterial components in the gut microbiota. PCoA revealed
that the gut microbiota of the C.R-infected mice was distinct from that of control mice (Fig. 1F-G). These findings
indicated that the composition of the gut microbiota in
C.R-infected mice was significantly different from that incontrol mice.
To further identify the critical bacteria affected by C.R
colonization, we compared the relative abundances of
microbes at various taxon levels between the groups. At
the phylum level (Fig. 1H), the abundance of Firmicutes
was significantly decreased in the C.R group compared tothe control group (C.R vs. control: p < 0.0001) (Fig. S3A).
In contrast, other phyla including Bacteroidetes, Verrucomicrobia, Proteobacteria and Epsilonbacteraeota, had
higher abundances in the C.R group than the control
group (C.R vs. control: p = 0.0420, p = 0.0219, p = 0.0048,
p = 0.0087, respectively) (Fig. S3B-E). Moreover, the ratioof Bacteroidetes to Firmicutes was significantly higher in
the C.R group than in the control group (C.R vs. control:
p = 0.0067) (Fig. 1F). At the family level, as illustrated in
the pie chart (Fig. 1I), the abundances of Bacteroidaceae, Enterobacteriaceae, Erysipelotrichaceae, Lachnospiraceae, and Prevotellaceae after the C.R challenge wasincreased compared to those in the control group, while
the levels of Muribaculaceae, Lactobacillaceae and Clostridiaceae were reduced in the C.R group. As shown in
Fig. 1J, overall significant differences in genera weredetected between the groups. We further selected representative genera and utilized boxplots to display theirdetailed information (Fig. 1K-L and Fig. S3G-N). Compared to those in the control group, the relative abundances of Akkermansia (p = 0.0106) (Fig. 1K), Citrobacter(p = 0.0034) (Fig. S3G), Ruminococcus (p = 0.0035) (Fig.
S3H), Helicobacter (p = 0.0087) (Fig. S3I), Coprobacillus (p = 0.0155) (Fig. S3G) and Parasutterella (p = 0.0117)(Fig. S3K) were significantly increased in the C.R group,
whereas the abundances of the genera Lachnospiraceae (p = 0.0001) (Fig. 1L), Oscillibacter (p = 0.0219) (Fig.
S3L), Clostridium (p = 0.0145) (Fig. S3M), and Roseburia(p = 0.0002) (Fig. S3N) were notably decreased in the C.R
group. Collectively, these results indicated a significantly
altered microbiota profile in C.R-infected mice.
C.R colonization damages the intestinal barrier andinduces intestinal inflammation

C.R is located mainly in the distal part of the colon andcecum during the steady-state phase [28]. To explore thedestructive effect of C.R colonization on the intestine, themorphology and pathology of the 0.5-cm terminal colon
were evaluated at 9 days after infection. Morphologically,
there was an evident lesion indicated by the swelling of
the distal colon of C.R-infected mice, while colons of the

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control mice were normal and filled with feces (Fig. 2A).
Simultaneously, the C.R-infected mice exhibited a significant decrease in colon length (C.R vs. control: p < 0.0001)(Fig. 2B). To better define the basis of these differences,
the terminal colon was examined by H&E staining. The
colon mucosa of the control mice displayed an intact
epithelium and no overt hyperplasia, while C.R-infected
mice showed significant damage, as demonstrated bywidespread epithelial cell sloughing, crypt hyperplasia,
submucosal edema and extensive polymorphonuclear cell
infiltration (Fig. 2C). Correspondingly, when the pathology was scored, the score of tissue damage in the colonof C.R-infected mice was significantly higher than that of
the untreated mice (C.R vs. control: p = 0.0024) (Fig. 2D).
Goblet cell depletion was also detected in the colon tissue (Fig. S4).
At the molecular level, intestinal barrier dysfunction in
the colon was evaluated via immunofluorescence staining for the tight junction proteins zonula occludens-1(ZO-1), Occludin, and Claudin-1 (Fig. 2E). The pattern showed continuous, robust expression of ZO-1 at
the epithelial lining of the control mice while the C.Rinfected mice exhibited a disrupted, discontinuous structure of ZO-1 (Fig. 2E). Semiquantitative analysis of thestaining using an integrity scoring scale demonstrated
a notable decrease after C.R infection (C.R vs. control:
p = 0.0296) (Fig. 2F). Microscopic analysis revealed astronger Occludin staining in the epithelial lining of thecontrol group (Fig. 2E). The integrity scores for Occludin
expression were significantly lower after C.R infection(C.R vs. control: p = 0.0003) (Fig. 2G). Microscopic visualization revealed that Claudin-1 was clearly expressedin the colonic epithelial lining of the control group, andClaudin-1 expression was significantly impacted by C.R
infection (Fig. 2E). The analysis also revealed significantly
lower expression of Claudin-1 in C.R-infected mice thanin control mice (C.R vs. control: p = 0.0093) (Fig. 2H).
Consistently, the mRNA expression of multiple tightjunction-related genes in the colon was significantly
decreased in the C.R-infected group (C.R vs. control:Ocln, p = 0.0004; Cldn3, p = 0.0146; Cldn4, p = 0.0062;
Cldn7, p = 0.0356; Cldn8, p = 0.2302; Cldn12, p < 0.0001;
Tjp1, p = 0.0022; Tjp2, p = 0.0014; Tjp3, p = 0.0176)(Fig. 2I). In addition, an intestinal permeability assay
was conducted in vivo through oral delivery of 4 KDa
FITC-dextran (FD4), and a greater increase in FD4 in theserum represented elevated gut permeability. The results
showed that there was a remarkable increase in FD4
intensity after C.R challenge (C.R vs. control: p = 0.0065)(Fig. 2J). All these data suggested that C.R colonization
caused intestinal barrier impairment.
Immunofluorescence staining revealed a different pattern of GFAP+ enteric glial reactivity in the myentericplexuses of the colon tissue at 9 days post infection

He et al. Journal of Neuroinflammation

Fig. 2 (See legend on next page.)(2024) 21:153

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He et al. Journal of Neuroinflammation(2024) 21:153

Page 10 of 26(See figure on previous page.)
Fig. 2 The intestinal barrier damage and intestinal inflammation induced by C.R infection. (A) The colonic morphology of the control and C.R-infected
mice. The arrows indicate lesion sites in the distal colon. (B) The colon length. n = 5. (C) H&E staining of colons. Scale bar, 200 μm (top) and 20 μm (bottom).
(D) Histopathological scoring of colonic tissue. n = 4–5. (E) Representative immunofluorescence images of the tight junction proteins ZO-1, Occludin,
and Claudin 1 in colon tissue. Scale bar, 100 μm. (F-H) An arbitrary scale of 0–3 (0 = no expression, 3 = continuous normal expression of the barrier) wasused to assess barrier integrity. (F) Integrity scoring data for ZO-1. (G) Integrity scoring data for Occludin. (H) Integrity scoring data for Claudin 1. n = 4.
(I) The expression of nine tight junction proteins in colonic tissue was analyzed by qPCR. n = 4–6. (J) The serum concentrations of FD4. n = 5. (K) The
levels of six inflammatory factors in colonic tissue were analyzed by qPCR. n = 3–5. (L) Representative images of GFAP+ cells in the myenteric plexuses.
Normal: scale bar,100 μm; Magnification: scale bar, 10 μm. The triangle arrow indicates GFAP+ cells in the myenteric plexuses. (M) Optical density analysis
of GFAP expression in the myenteric plexuses. n = 4–5. (N) The level of calprotectin in feces was analyzed by ELISA. n = 7. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001(Fig. 2L). GFAP intensity in the lamina propria of C.Rinfected mice was significantly higher than that in thecontrol mice (C.R vs. control: p = 0.0005) (Fig. 2M). In
addition, the colon of the C.R-infected mice showeda considerably increased mRNA expression of proinflammatory factors, including IL-1β (p = 0.0237), IL-6
(p < 0.0001), IL-12 (p = 0.0004), iNOS (p = 0.0011), and theanti-inflammatory factor IL-4 (p = 0.0005), but decreasedmRNA expression of the TNF-α (p = 0.1587) compared tothe colon of control mice (Fig. 2K). Fecal calprotectin has
been used as an indicator of intestinal inflammation [29].
ELISA data showed that the level of calprotectin in the
fecal pellets of C.R-infected mice was significantly higher
than that in the control mice (C.R vs. control: p < 0.0001)(Fig. 2N). Taken together, these data demonstrated that
C.R colonization induced intestinal inflammation.
C.R infection regulates the metabolism of short-chain fatty
acids in the gut and neurotransmitters in the brain

Mounting evidence has demonstrated that SCFAs are
involved in the regulation of intestinal inflammation [30].
To explore the potential mechanisms of action of C.R
challenge in gut inflammation, the fresh fecal samples
were collected for the evaluation of SCFAs by HPLC. The
results showed that compared to that in the control mice,the concentration of propionic acid (C.R vs. control:
p = 0.0034) (Fig. 3B) and butyric acid (C.R vs. control:
p = 0.0004) (Fig. 3C) exhibited a notable decrease while
the level of acetic acid (Fig. 3A) remained unchanged in
the C.R-infected mice. Moreover, the SCFA receptors
GPR109a and GPR43 were detected in the colon. The
data showed that there was lower expression of GPR109aand GPR43 in the C.R group than in the control group(C.R vs. control: p = 0.0218 and p = 0.0396, respectively)(Fig. 3D-E). In brief, these data suggested that C.R infection affected SCFA metabolism in the gut.
Gut microbiota alterations is linked to the disturbanceof neurotransmitter metabolism [31]. To verify the effectafter C.R infection, the metabolism of tyrosine (Tyr) andtryptophan (Trp) in the striatum was assessed at 9 days
after C.R colonization. Surprisingly, the DA level was
decreased (C.R vs. control: p = 0.0183) (Fig. 3F), while thelevels of DOPAC (C.R vs. control: p = 0.0496) (Fig. 3G),

HVA (C.R vs. control: p = 0.0445) (Fig. 3H) were markedly
increased in the Tyr metabolism pathway.
To further explore the underlying mechanism, the step
limiting enzyme tyrosine hydroxylase (TH), MAOA,
MAOB, catechol-O-methyltransferase (COMT) anddopamine transporter (DAT) were investigated (Fig. 3N).
We found that the expression of TH, DAT and COMT
did not change (C.R vs. control) (Fig. 3Q, O and S),
while MAOA (C.R vs. control: p = 0.0142) (Fig. 3P) and
MAOB (C.R vs. control: p = 0.0142) (Fig. 3R) expression
was markedly increased. In the Trp metabolism pathway, the levels of the metabolites 5-HT and 5-HIAA in
C.R-infected mice were markedly lower than those in thecontrol mice (C.R vs. control: p = 0.0021 and p = 0.0094,
respectively) (Fig. 3L and K). Additionally, the ratios
of DOPAC/DA (C.R vs. control: p = 0.0271) (Fig. 3I),
and HVA/DA (C.R vs. control: p = 0.0368) (Fig. 3J), and
5-HIAA/5-HT (C.R vs. control: p = 0.0131) (Fig. 3M)
were obviously increased, indicating increased turnoverof DA and 5-HT in C.R infection. In addition, similar
trends in Tyr and Trp metabolism were also observed in
the female mice (Fig. S5).
To further explore whether there was a continuous andlong-term effect on neurotransmitter metabolism in the
striatum, the animals were sacrificed at 9, 15, 21 and 30
days after C.R challenge. The results showed that there
was a remarkable decrease in DA in the C.R-infectedgroup in comparison with the control group, with thelowest level being observed at 15 d.p.i. (Fig. 3T). In contrary, the levels of DOPAC and HVA increased during
C.R infection compared to the control group, with thepeaks levels of both metabolites appearing at 21 d.p.i.
(Fig. 3U and V). At the end of monitoring, the DOPAC
and HVA levels returned to baseline (Fig. 3U and V).
The levels of 5-HT and its metabolite 5-HIAA in the C.R
group were lower than those in the control group. After
C.R colonization for 15 days, 5-HT levels were gradually returned to normal (Fig. 3W), while 5-HIAA levels
showed a continuous decline (Fig. 3X).
To further validate the effective components of C.R
gavage, the Luria Broth (LB), supernatant and precipitateof C.R culture medium were given by gavage according
to the manufacturer’s instructions (Fig. S6A). The levels of neurotransmitters and metabolites (DA, DOPAC,He et al. Journal of Neuroinflammation(2024) 21:153

HVA, 5-HT and 5-HIAA) in the striatum were evaluatedby HPLC after 12 days of continuous treatment. Compared with those in LB-treated mice, the five substances
remained unchanged in mice treated with the supernatant or the precipitate (Fig. S6B). Moreover, there was nochange in the expression of the TH and MAOB proteinsafter supernatant and precipitate treatment comparedwith that in the LB-treated group (Fig. S6C-E). Taken
together, these results indicated that only living C.R
delivery disturbs neurotransmitter metabolism.
C.R in combination with MPTP challenge causes motor
deficits

As mentioned in the literature, imbalances in neurotransmitter metabolism and the gut microbiota are causal
factors of PD pathogeny [32]. To evaluate the effects ofintestinal disorders on PD pathogenesis, we established
an infection mouse model by orally giving mice C.R once.
At 6 days post inoculation, the C.R-challenged mice wereadministrated 40 mg/kg MPTP or vehicle (Fig. 4A). At 9
d.p.i. (3 days after MPTP administration), various behavioral tests, including the pole test, the rotarod test, thewire-hanging test and the rearing test, were performed toassess motor function. Overall, there were no significant
behavioral changes in these mice, regardless of treatment
with C.R or MPTP alone compared to the control group(Fig. 4B-J). However, the mice administered C.R and
MPTP combined exhibited significant motor disorders,
including delayed initiation of movement, as indicated bya longer turning time (C.R/MPTP vs. control: p < 0.0001)(Fig. 4C), more time spent climbing down the pole (C.R/
MPTP vs. control: p = 0.0077) (Fig. 4D) and the wholeclimbing process in the pole rest (C.R/MPTP vs. control:
p = 0.0024) (Fig. 4E), the motor deficit characterized byless standing time in the rearing test (C.R/MPTP vs. control: p = 0.0006) (Fig. 4G), poorer muscle force manifested
as a lower score in the wire-hanging test (C.R/MPTP vs.
control: p = 0.0020) (Fig. 4I), and decreased retention timeon the rod (C.R/MPTP vs. control: p = 0.0034) (Fig. 4J) in
the rotarod test.
In addition, the open field test (OFT) was also performed to evaluate locomotion and emotion. The data
showed that there were no marked changes in distances
traveled or the speed of the mice treated with C.R aloneor MPTP alone compared with those of the control mice
(Fig. 4K-M) in the OFT. C.R in combination with MPTP
challenge prominently caused less movement (C.R/
MPTP vs. control: p < 0.0001) (Fig. 4L) and bradykinesia(C.R/MPTP vs. control: p < 0.0001) (Fig. 4M). C.R alone
or C.R and MPTP combined challenge led to shorter
distances in the central area (C.R vs. control: p = 0.0045,
C.R/MPTP vs. control: p < 0.0001) (Fig. 4N) and a lower
frequency in the central area (C.R vs. control: p = 0.0036,
C.R/MPTP vs. control: p < 0.0001) (Fig. 4O) than vehicle.

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The OFT results suggested that C.R infection also participated in emotional regulation. Taken together, these datasuggested that C.R and MPTP induced motor impairments and slight anxiety-like behavior.
C.R infection in combination with MPTP exacerbates theloss of TH-positive dopaminergic neurons and TH protein
expression in the brain

Given that dopaminergic neuronal loss in the nigrostriatal system is the main pathological characteristic of PD,
we examined whether C.R infection also has a destructive effect on the dopaminergic system in the SN andthe striatum. Tyrosine hydroxylase (TH) is an enzyme
involved in DA synthesis; therefore, TH+ cells are dopaminergic neurons. Representative images of immunohistochemical staining of the SN rostral to caudal in thecoronal section are shown in Fig. 5A. By stereologicalcounting analysis of the SN, we found that the number of
TH+ cells decreased by nearly 50% after MPTP exposure
alone (MPTP vs. control: p < 0.0001), while no significant
difference was detected in mice with C.R infection alonecompared to the control mice (Fig. 5B). However, C.R in
combination with MPTP significantly exacerbated theloss of dopaminergic neurons in PD mice (C.R/MPTP
vs. MPTP: p = 0.0006) (Fig. 5B). Consistent with thisobservation, immunostaining and western blotting of thestriatum also revealed marked decreases in the TH+ fiber(MPTP vs. control: p = 0.0004) and TH protein (MPTP vs.
control: p < 0.0001) levels in the MPTP group comparedto those in the control group, while there was no change
in mice challenged with C.R alone compared to the control mice (Fig. 5C-D and E-F, respectively). Nevertheless,there was a more severe decrease in TH+ fibers and TH
protein expression in the C.R/MPTP group compared to
the MPTP-challenged group (C.R/MPTP vs. MPTP: TH+fibers, p = 0.0006; TH protein levels, p < 0.0001) (Fig. 5C-D
and E-F, respectively). MAOB is responsible for the enzymatic degradation of dopamine at axon terminals, often
showing a negative correlation with TH expression.
There were an elevated protein levels of striatal MAOB in
only C.R exposed or only MPTP exposed mice comparedto the control mice (C.R vs. control: p = 0.0392; MPTP
vs. control: p = 0.0012) (Fig. 5E-G). However, C.R in
combination with MPTP significantly increased MAOB
expression in comparison with MPTP alone (C.R/MPTP
vs. MPTP: p = 0.0250) (Fig. 5E-G). Moreover, the level
of DA in the striatum was significantly decreased in the
C.R/MPTP group compared to the MPTP group (C.R/
MPTP vs. MPTP: p = 0.0300) (Fig. 5H), while the levelsof the DA metabolites DOPAC and HVA were not different between the groups (Fig. 5H). The levels of 5-HT and
its metabolite 5-HIAA also did not differ between the
MPTP group and the C.R/MPTP group (Fig. 5H).Taken

He et al. Journal of Neuroinflammation(2024) 21:153

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Fig. 3 C.R infection caused metabolic abnormalities in SCFAs in the gut and in neurotransmitters in the striatum. (A-C) The levels of acetic acid (A), propionic acid (B), and butyric acid (C) in feces at 9 days post infection. n = 6. (D, E) The mRNA levels of GPR109a(D) and GPR43(E) in colonic tissue. n = 5–7.
(F-M) The metabolism of DA and 5-HT. DA (F), DOPAC (G), HVA (H), DOPAC/DA (I), HVA/DA (J), 5-HIAA (K), 5-HT(L), and 5-HIAA/5-HT (M). n = 5–6. (N)Western blotting showing striatal levels of the DAT, MAOA, TH, MAOB and COMT proteins. β-actin served as the loading control. (O-S) Quantification of
the relative expression levels of the DAT (O), MAOA (P), TH (Q), MAOB (R) and COMT (S) proteins. n = 3–6. (T-X) The metabolism of DA and 5-HT at 9, 15,
21, and 30 days post infection. DA (T), DOPAC (U), HVA (V), 5-HT(W), 5-HIAA (X). n = 3–10. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

He et al. Journal of Neuroinflammation(2024) 21:153

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Fig. 4 The combination of C.R and MPTP caused movement impairments in PD mice. (A) The experimental design in the current study. (B-E) Behavioral
changes were assessed in the pole test (B), including the time to turning (C), time to reach the bottom (D) and the total time (E). (F-G) Behavioral changes
were assessed in the rearing test (F) and number of rearing in a 3-minute period (G). (H-I) Behavioral changes were assessed via the wire-hanging test(H) and the total score over 3 min (I). (J) Behavioral changes were assessed in the rotarod test and the retention time on the rod at speed of 32 rpm wasrecorded. (K-O) Behavioral changes were assessed in the open field test; representative traces (K), total moving distance (L), average moving speed (M),moving distance in the center area (N), and frequency in the center (O) are shown. n = 7–12. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

He et al. Journal of Neuroinflammation(2024) 21:153

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Fig. 5 C.R infection aggravated MPTP-induced nigrostriatal system lesions. (A) Immunohistochemical staining showing TH+ cells in the SN. (B) Resultsof stereological counting of TH+ cells in the SN. Scale bar, 100 μm. n = 5–6. (C) Immunohistochemical staining showing striatal TH+ nerve fibers. Scale bar,
200 μm. (D) Densitometric analysis of the relative optical density of the staining. n = 5. (E) Western blotting showing the striatal levels of the TH and MAOB
proteins. β-Actin served as the loading control. (F, G) Quantification of the relative protein expression levels of TH and MAOB. TH: n = 5–7; MAOB: n = 3–4.
(H) The levels of DA, DOPAC, HVA, 5-HT and 5-HIAA in the striatum. n = 4. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

He et al. Journal of Neuroinflammation(2024) 21:153

together, these data confirm that peripheral C.R infection
aggravated the nigrostriatal dopaminergic system lesions.
Activation of microglia in the SN and the striatum isexacerbated after C.R and MPTP combination treatment

Iba1 is a marker of microglia. We first used Western
blotting analysis to evaluate the expression of Iba1 inthe striatum. The results demonstrated that the level of
Iba1 protein was greater in the MPTP group than in thecontrol group (MPTP vs. control: p = 0.0146) (Fig. 6A-B),
and the C.R treatment alone did not affect Iba1 expression (Fig. 6A-B). In the C.R/MPTP group, the Iba1 protein level was significantly increased compared to thatin the MPTP group (C.R/MPTP vs. MPTP: p = 0.0164)(Fig. 6A-B).
Next, we evaluated the number and morphology of
microglia within the striatum via immunohistochemical assays. The results showed that Iba1+ cells in thecontrol group or the C.R group were characterized by asmall number of highly-ramified morphologies (Fig. 6C).
After MPTP intoxication or C.R and MPTP combined
treatment, numerous activated Iba1+ microglia withstouter cell processes and a round shape were observed(Fig. 6C). In terms of quantity, the number of Iba1+ cells
were markedly greater in MPTP-challenged mice thanthe control mice (MPTP vs. control: p = 0.0015) (Fig. 6CD). Conversely, there was no impact on mice challenged
with C.R alone (Fig. 6C-D). However, in the C.R/MPTP
group, the number of Iba1-labeled microglia was significantly higher than that in the MPTP group (C.R/MPTP
vs. MPTP: p = 0.0135) (Fig. 6C-D).
Furthermore, the morphology of the microglia wasanalyzed. There was no obvious difference between the
C.R group and the control group (Fig. 6E-G). Comparedwith those in the control group, the soma of Iba1-labeled
microglia in the MPTP-treated group were enlarged(MPTP vs. control: p = 0.0046) (Fig. 6E), the summed process length (MPTP vs. control: p < 0.0001) (Fig. 6F) andtotal number of endpoints (MPTP vs. control: p < 0.0001)(Fig. 6G) were decreased. Strikingly, in the C.R/MPTP
group, activated microglia exhibited a larger soma
area (MPTP vs. control: p = 0.0002) (Fig. 6E), a shorter
summed process length (MPTP vs. control: p = 0.0034)(Fig. 6F) and fewer endpoints of microglia (MPTP vs.
control: p = 0.0010) than did the MPTP group (Fig. 6G).
Similar results were obtained when microglia in the
SN were analyzed. In the present study, we performed
immunofluorescence staining of the SN region to detect
the activation of glial cells, using Iba1 as a marker ofmicroglia (Fig. 6H). The number of Iba1+ cells was significantly greater in the MPTP group than in the control
group (MPTP vs. control: p < 0.0001) (Fig. 6J). No effecton Iba1+ cells was observed in mice infected with C.R
alone (Fig. 6J). However, more Iba1+ cells were found

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in mice challenged with C.R and MPTP than in micechallenged with MPTP alone (C.R/MPTP vs. MPTP:
p = 0.0033) (Fig. 6J).
To evaluate the classic M1 phenotype of microglia in
the SN, the expression of CD16/32, a typical marker ofthe M1 activation, was analyzed. The data showed that
there were no obvious CD16/32-labeled microglia in
the SN in the control or C.R-infected mice (Fig. 6I and
K). There were more CD16/32-labeled cells in the MPTP
group than in the control group (MPTP vs. control:p < 0.0001) (Fig. 6I and K). In the C.R/MPTP group, thenumber of CD16/32-labeled cells was greater than thatin the MPTP group (C.R/MPTP vs. MPTP: p < 0.0001)(Fig. 6I and K).Collectively, these results illustrated
that peripheral C.R infection significantly exacerbated
MPTP-induced microglial activation in the nigrostriatal
pathway.
Activation of astrocytes in the SN and striatum isexacerbated after C.R and MPTP combined challenge

Astrocytes, another important type of glial cell, play critical roles in homeostasis in the brain. We analyzed astrocyte activation by assessing GFAP expression. Western
blotting data showed revealed greater levels of GFAP
protein in the striata of MPTP-delivered mice than inthose of the control mice (MPTP vs. control: p = 0.0002)(Fig. 7A, B), while C.R alone had no effect on GFAP
expression (Fig. 7A, B). Strikingly, the protein level of
GFAP was dramatically higher in the C.R/MPTP groupthan the MPTP group (C.R/MPTP vs. MPTP: p = 0.0001)(Fig. 7A, B). Moreover, the number of GFAP+ cells inthe striatum was increased, consistent with the protein
expression data (Fig. 7C, D). Specifically, there were more
GFAP+ cells in the C.R/MPTP group than the MPTP
group (C.R/MPTP vs. MPTP: p = 0.0008) (Fig. 7D). In
addition, similar results were acquired in the SN byimmunofluorescence staining and cell counting (Fig. 7E
and G).
To evaluate the neurotoxic effects on the A1 phenotype
of astrocytes in the SN, we analyzed C3, a marker of A1
activation. There were more C3+ cells in the MPTP groupthan in the control group (MPTP vs. control: p = 0.0019)(Fig. 7F and H). In the C.R/MPTP group, the numberof C3-labeled cells was markedly higher than that in the
MPTP group (C.R/MPTP vs. MPTP: p < 0.0001) (Fig. 7F
and H). Collectively, these results demonstrated that
the C.R challenge dramatically exacerbated neurotoxicity-mediated astrocyte activation in the nigrostriatal
pathway.

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Fig. 6 The C.R challenge exacerbated MPTP-induced microglial activation in the striatum and the SN. (A) Western blotting showing striatal levels of Iba1
proteins. β-Actin served as the loading control. (B) Quantification of the relative protein expression levels of Iba1. n = 4–5. (C) Immunohistochemical staining showing Iba1+ microglial cells in the striatum. Scale bar, 40×: 20 μm, 100×: 5 μm. (D) The number of Iba1+ cells. n = 5. (E-G) Morphological analysis of
Iba1+ cells, the soma area (E), the summed process length (F), and the endpoints of microglia (G). n = 5. (H) Immunofluorescence staining of TH (green),
Iba1 (red) and DAPI (blue) in the SN. Scale bar, 100 μm. (I) Immunofluorescence staining of Iba1 (green), CD16/32 (red) and DAPI (blue) in the SN. Scalebar, 200 μm. (J) The numbers of Iba1+ cells in the SN. (K) The ratio of the number of CD16/32+ and Iba1+ cells to the total number of Iba1+ cells. n = 4–5.
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

C.R administration activates the TLR4 signaling pathway
in the colon and the nigrostriatal pathway in the current
model system

C.R is gram-negative bacteria that contain LPS on theirmembrane surface. TLR4 can recognize and respond to

LPS, and its downstream pathways dominate the microbiota-gut-brain axis when gastrointestinal infections or
systemic bacterial infections occur [33]. To examine theactivation status of the TLR4 pathway in the colon andHe et al. Journal of Neuroinflammation(2024) 21:153

Page 17 of 26

Fig. 7 The combination of C.R and MPTP exacerbated neurotoxicity-mediated astrocyte activation in the nigrostriatal pathway at 9 days post infection.
(A) Western blotting showing striatal levels of GFAP protein. β-Actin served as the loading control. (B) Quantification of the relative expression levels of
GFAP protein. n = 5. (C) Immunohistochemical staining showing GFAP in the striatum. Scale bar, 20 μm. (D) The number of GFAP+ cells in the striatum.
n = 4–5. (E) Immunofluorescence staining of TH (green), GFAP (red) and DAPI (blue) in the SN. Scale bar, 200 μm. (F) Immunofluorescence staining of GFAP
(green), C3 (red) and DAPI (blue) in the SN. Scale bar, 50 μm. (G) Number of GFAP+ astrocytes in the SN. (H) The ratio of the number of C3+ and GFAP+ cells
to the total number of GFAP+ cells. n = 3–5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

the nigrostriatal pathway, qPCR, western blotting, andimmunofluorescence staining were performed.
In the colon, the mRNA level of TLR4 was higher in the
C.R (C.R vs. control: p = 0.0033) and C.R/MPTP groups(C.R/MPTP vs. control: p = 0.0104) (Fig. 8A) than thecontrol group. As revealed by Western blotting, except

for the MPTP group, the C.R and C.R/MPTP groups
showed a significantly greater expression of TLR4 andNF-κB p65 protein expression than the control group(C.R vs. control: p = 0.0328 and p = 0.0333, C.R/MPTP vs.
control: p = 0.0010 and p = 0.0002, respectively) (Fig. 8C
and D); while the expression of TLR4 and NF-κB p65

He et al. Journal of Neuroinflammation(2024) 21:153

Page 18 of 26

Fig. 8 The TLR4 signaling pathway was activated in the colon after the C.R challenge. (A) The mRNA level of TLR4 in the colon. n = 4. (B) Western blotting
showing colonic levels of the TLR4 and NF-κB p65 proteins. β-Actin served as the loading control. (C-D) Quantification of the relative protein expression
levels of TLR4 and NF-κB p65. n = 4–6. (E) Immunohistochemical staining showing TLR4 in the colon. Scale bar, 50 μm. (F) The number of TLR4+ cells in thecolon in per field. n = 4. (G) The IL-6 protein level in the serum was analyzed by ELISA. n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

in the C.R group was lower than that in the C.R/MPTP
group (C.R vs. C.R/MPTP: p = 0.0295 and p = 0.0371,
respectively) (Fig. 8C and D). In addition, the immunofluorescence staining results revealed that the numbers of TLR4+ cells in the colon were notably higher in
the C.R and the C.R/MPTP groups than to the control
group (C.R vs. control: p = 0.0001, C.R/MPTP vs. control: p = 0.0005), whereas the number of TLR4+ cells in
the colon of MPTP-injected mice was similar to that incolon of control mice (Fig. 8E and F). In addition, we alsomeasured the levels of IL-6 in mouse serum. The ELISA
results demonstrated that there was a trend toward anincrease in the MPTP group compared to the control
group (Fig. 8G). However, the C.R/MPTP group showed
greater levels of IL-6 than did the MPTP group (C.R/
MPTP vs. MPTP: p = 0.0035) (Fig. 8G).

Similarly, we measured TLR4 expression in the nigrostriatal pathway. In the striatum, the levels of TLR4 (C.R/
MPTP vs. control: p = 0.0125) and the ratio of NF-κB
pp65/p65 (C.R/MPTP vs. control: p = 0.0191) were higher
in the C.R/MPTP group than the control group (Fig. 9AC). The similar trends in TLR4 expression in the SN wereconfirmed by immunofluorescence staining (MPTP vs.
control: p = 0.0018, C.R/MPTP vs. C.R: p = 0.0121, C.R/
MPTP vs. control: p = 0.0078) (Fig. 9D-E). All the evidence supported that combined C.R and MPTP challenge activated the TLR4-NF-κB signaling pathway.
C.R and MPTP combined treatment causes or exacerbates
neuroinflammation and alters the gut microbiota
composition

To confirm the inflammatory process among the different groups, we first measured the corresponding mRNAHe et al. Journal of Neuroinflammation(2024) 21:153

Page 19 of 26

Fig. 9 The TLR4-NF-κB signaling pathway was activated in the nigrostriatal pathway in our model system. (A) Western blotting showing striatal levels of
TLR4, NF-κB p65, and NF-κB pp65 proteins. β-Actin served as the loading control. (B-C) Quantification of the relative expression levels of TLR4 and theratio of NF-κB pp65/p65 proteins. n = 5. (D) Immunohistochemical staining showing TLR4 in the SN. Scale bar, 20 μm. (E) The number of TLR4+ cells in the
SN in per field. n = 4. *p < 0.05, **p < 0.01

expression of inflammatory cytokines in the striatum.
The results displayed a remarkable upregulation in the
MPTP group compared to the control group (MPTP vs.
control: IL-6, p = 0.0131; IL-1β, p = 0.0182; Aif1, p = 0.0132;
S100b, p = 0.0132) (Fig. 10A). However, the levels in the
C.R/MPTP group were even higher than those in the
MPTP group (C.R/MPTP vs. MPTP: IL-6, p = 0.0001;
IFN-γ, p = 0.0055; S100b, p = 0.0379; iNOS, p = 0.0486;
CD86, p = 0.0039; CD68, p = 0.0047; COX2, p = 0.0293)(Fig. 10A and B). However, the mRNA level of BDNF was
lower in the C.R-, MPTP- and C.R/MPTP groups thanthe control group (C.R vs. control: p = 0.0051, MPTP vs.
control: p = 0.0361, C.R/MPTP vs. control: p = 0.0003)(Fig. S7). Importantly, there was much lower expression
in the C.R/MPTP group than the MPTP group (C.R/
MPTP vs. MPTP: p = 0.0248) (Fig. S7). In addition, theexpression of one representative proinflammatory factor(COX2) was measured by western blotting. COX2 protein expression was notably greater in the MPTP groupthan in the control group (MPTP vs. control: p = 0.0241).
Compared to that in the MPTP group, the level of COX2
in the C.R/MPTP group was further increased (C.R/
MPTP vs. MPTP: p = 0.0417), while the COX2 level in the
C.R group remained unchanged (Fig. 10C). Additionally,
the TLR4 antagonist TAK242 was used to prevent theactivation of TLR4, and we found that TAK242 had noeffect on striatal DA metabolism (Fig.S8).
To investigate the effect of C.R challenge on MPTPinduced PD pathology, we compared the composition of
the gut microbiota between the groups receiving MPTP
and C.R plus MPTP treatment at 9 d.p.i using 16 S rRNA
sequence analysis. We observed a significant increase in
α-Diversity, as assessed by the ACE index, in the C.R.
plus MPTP-treated group compared to the MPTP group(MPTP vs. C.R/MPTP: p = 0.0109) (Fig. S9A). However,no significant differences were found in the Shannon
index between the groups (Fig. S9B). PCoA and BrayCurtis dissimilarity revealed a distinct gut microbiotaHe et al. Journal of Neuroinflammation(2024) 21:153

Page 20 of 26

Fig. 10 Effect of C.R infection or C.R plus MPTP administration on PD pathology. (A-B) The mRNA levels of inflammatory factors in the striatum wereanalyzed. n = 4. #p < 0.05, the MPTP group versus the control group; *p < 0.05, **p < 0.01, ***p < 0.001, the MPTP group versus the C.R/MPTP group. (C) Western blotting showing striatal levels of COX2. β-Actin served as the loading control. (D) Quantification of the relative expression levels of COX2. n = 4–5,
*p < 0.05, **p < 0.01, ***p < 0.001. (E) C.R content in the colon were negatively correlated with the levels of the striatal DA. (F) The levels of calprotectin in
the feces were positively correlated with TLR4 protein expression in the striatum detected by western blotting. (G) The ZO-1 expression in the colon was
inversely correlated with TLR4+ cell numbers in the SN region. (H) The relative abundance of Parabacteroides was negatively correlated with TH+ neuron
numbers in the SN region. (I) The relative abundance of Lachnospiraceae was inversely correlated with TH+ neuron numbers in the SN region. (J) The
performances of the wire-hanging test were significantly positively the relative abundances of Lactobacillus

He et al. Journal of Neuroinflammation(2024) 21:153

composition for C.R/MPTP-treated mice compared to
MPTP-treated mice (Fig. S9C-D). Furthermore, to identify the specific bacteria impacted by C.R colonization in
MPTP-treated mice, we conducted a comparative analysis of microbial relative abundances at various taxonomic
levels. At the phylum level (Fig. S9E), the abundance of
Bacteroidota and Firmicutes showed a significant decline
in the C.R/MPTP group compared to the C.R group,
whereas Proteobacteria, in contrast, exhibited a higher
abundance in the C.R/MPTP group than the MPTP
group (Fig. S9E). At the family level, as illustrated in thepie chart (Fig. S9F), the abundances of Citrobacter, Parabacteroides, Marvinbryantia, and Blautia were higher in
the C.R/MPTP group than the MPTP group, while thelevels of Muribaculaceae, Lactobacillus, and Lachnospiraceae were lower in the MPTP group. As shown in Fig.
S9G, significant differences between the groups were also
detected at the genus level. To present detailed information, we selected representative genera and used boxplots(Fig. S9H-K). Compared to the MPTP group, the relative abundance of Lactobacillus (p = 0.0399) (Fig. S9H)
and Lachnospiraceae (p = 0.0427) (Fig S9I) was notably
decreased in the C.R/MPTP group whereas the relative abundance of Citrobacter (p < 0.0001) (Fig. S9J) andParabacteroides (p = 0.0015) (Fig. S9K) showed a significant increase following C.R plus MPTP treatment. These
results collectively demonstrated a significantly alteredmicrobiota profile in C.R/MPTP-treated mice.
To gain deeper insights into the role of the microbiotagut-brain axis in the pathogenesis of PD associated with
C.R infection, we conducted correlation analysis on aseries of experimental data. Our findings revealed a significant inverse correlation between C.R colonizationin the colon and DA level in the striatum (r = -0.6645,
p = 0.0361) (Fig. 10E). A positive correlation was observed
between fecal calprotectin expression and TLR4 protein expression in the striatum (r = 0.9387, p = 0.0017)(Fig. 10F). Furthermore, a significant negative correlation
was identified between the ZO-1 integrity score and thenumber of TLR4+ cells in the SN (r = − 0.7947, p = 0.0327)(Fig. 10G). Additionally, a strong negative correlation was
found between the relative abundance of Parabacteroides
and the number of TH+ neurons in the SN (r = − 0.8272,
p = 0.0217) (Fig. 10H). The study also revealed a negative
correlation between the relative abundance of Lachnospiraceae and the number of Iba1+ cells in the striatum(r = − 0.8851, p = 0.0460) (Fig. 10I). Furthermore, a significant positive correlation was observed between therelative abundance of Lactobacillus and performance inthe wire-hanging test (r = 0.9328, p = 0.0022) (Fig. 10J).
Collectively, these correlation results provide strong evidence for the involvement of the microbiota-gut-brain
axis in the development of PD, especially when C. R
infection is present.

Page 21 of 26

Discussion
There is increasing evidence that intestinal inflammation
is a high risk factor for developing PD [34]. However, thedetailed mechanism remains unknown. In the present
study, we demonstrated that C.R infection induced an
IBD-like pathology. Notably, neurotransmitter metabolism was also affected by C.R infection. Additionally,
C.R infection further exacerbated MPTP-induced motor
deficits, the impairment of the nigrostriatal dopaminergic system, glial cell activation and neuroinflammation inmice (Fig. 11).
C.R infection, which closely mimics IBD-like disease, iswidely used to establish a murine infection model [21].
After the animals were infected with C.R, astrocytes
were activated in the colon plexuses, similar to the findings in another colitis model [35]. Many inflammatory
markers in the C.R-challenged colon displayed increased
expression. In addition, the level of calprotectin was significantly increased in the feces of the C.R-infected mice,
consistent with the findings in IBD patients [36], suggesting the development of intestinal inflammation after C.R
colonization.
Intestinal inflammation also affects intestinal barrier function [37]. In agreement with a previous report,
we observed that the C.R challenge caused a significant increase in FD4 intensity in the serum. H&E staining of the colon tissues revealed an immune infiltrationand epithelial damage caused by C.R infection. In addition, our work showed that the expression of key tightjunction- proteins (ZO-1, Occludin, and Claudin-1) incolonic tissues was markedly decreased after C.R colonization. Moreover, at the mRNA level, nine tight junction
molecules in the colonic tissues were analyzed, and consistent results was obtained. These phenomena suggested
increased colonic permeability [35, 38, 39].
C.R infection impacts the intestinal microbiota composition [40, 41]. The α-Diversity of the gut microbiota
significantly decreased while the microbial diversity did
not change in C.R-infected mice, which was consistent
with previous studies [19]. At the moment, the C.R strain
plausibly exhibited dominance and engaged in competition with other microorganisms for resources and habitat, consequently impacting microbial abundance rather
than diversity. However, unlike other reports, C.R infection greatly disturbed bacterial communities as indicatedby β-Diversity in the present data [19]. This discrepancy
may be due to differences in the genetic background,feeding conditions and diets of the mice [19, 41]. At thephylum level, in accordance with the observations in PD
patients, C.R challenge significantly reduced the relative
abundance of Firmicutes and increased the levels of Bacteroidetes, Proteobacteria, Verrucomicrobia and Epsilonbacteraeota [42]. It has been reported that increases inProteobacteria can be a consequence of gut inflammation

He et al. Journal of Neuroinflammation(2024) 21:153

Page 22 of 26

Fig. 11 Diagram of C.R infection impairs dopamine metabolism and exacerbates the pathology of Parkinson’s disease[43]. Notably, the ratio of Bacteroidetes to Firmicutes
increased which was associated with increased susceptibility to C.R [44]. At the family level, the relative abundance of Enterobacteriaceae increased after the C.R
challenge, which is similar to observations in PD patients,
where this increase in Enterobacteriaceae abundance

was positively associated with the severity of postural
instability and gait difficulty [45]. At the genus level, we
noted a modest increase in Akkermansia abundance
after challenge with C.R; Akkermansia is a gram-negative
mucin-degrading microbe associated with anti-inflammatory immune signatures in humans [46]. Akkermansia

He et al. Journal of Neuroinflammation(2024) 21:153

considered a signature of the fecal microbiota of PD
patients compared to that of healthy individuals [47]. A
greater proportion of Akkermansia was also observed ina chronic stress-induced mouse model of PD [35]. Additionally, increased levels of Helicobacter pylori and Roseburia have also been reported to be associated with PD[42, 48]. The abundances of Ruminococcus and Parasutterella, which are closely associated with Crohn’s disease,are obviously increased, which was in consistent with ourdata [49, 50].
Gut microbiota disorders, such as alterations in SCFAs,
cause metabolic abnormalities in the gut. One of theFirmicutes, Lachnospiraceae which can produce SCFAs
showed a significant decrease in abundance in C.Rinfected animals [51]. Acetate, propionate, and butyrate
are the primary SCFA molecules produced from gut bacterial fermentation and are endowed with anti-inflammatory properties. In fecal samples from C.R-infected
mice, the levels of butyric acid and propionic acid wereprominently decreased, in line with the findings in PD
patients, while the acetic acid level remained unchanged,
which was different from the findings that observed in
PD patients [52]. C.R infection caused obvious intestinal inflammation, which may be related to the abnormal
metabolism of SCFAs. In addition, the levels of GRP109aand GPR43, which mainly respond to SCFAs, were significantly decreased after the C.R challenge, further supporting the insufficiency of SCFAs in the gut. In addition,
SCFAs are highly implicated in intestinal barrier integrity
and intestinal permeability [53]. Within our model system, intestinal barrier impairment and high permeability
were observed after the C.R challenge, and SCFAs suchas propionic acid and butyric acid were altered, which
may be a plausible underlying mechanism [54, 55].
Gut microbiota disorders affect neurotransmitter metabolism in the brain. After the administration of C.R, we found that the levels of DA and 5-HTin the striatum decreased with increasing levels of thedownstream metabolites DOPAC, HVA, and 5-HIAA,
respectively, with no differences between the sexes, suggesting increased turnover of dopamine and 5-HT. The
rate of turnover recovered at 30 days post infection, andthe complete clearance of C.R in the gut may also bepresent at this timepoint [28]. The decrease in DA may
be partially attributed to the upregulation of MAOA and
MAOB, which are two critical enzymes involved in themetabolism of DA [56, 57].
We chose a single high-dose MPTP to induce PD-like
pathology, and we observed dopaminergic degeneration,
which was consistent with previous reports. However, themotor behaviors of MPTP-treated mice were unchanged
compared to those of normal saline-treated mice 3 days
after neurotoxicity. In accordance with earlier reports,
the behavioral performance in our study may return toPage 23 of 26

normal in mice 3 days after MPTP administration [58,59]. C.R infection potentiated the MPTP-induced loss of
dopaminergic neurons in the SN and dopaminergic fibers
in the striatum. Consequently, we were able to observethat animal behavioral performance appeared to be mostsensitive to the combined effects of C.R and MPTP, suggesting that the sensorimotor system could be highly
impacted by the gut-originated proinflammatory state,what has been observed in other studies [13, 60, 61]. C.R
challenge alone had no obvious effect on the movement
performance of the animals, consistent with the findings of other studies [20]. However, inconsistent with one
report, in the present study, anxiety-like behavior wasobserved at 9 days after C.R infection [62]. The mood disorders may be attributed to the dysfunction of 5-HT systems. We found there was abnormal metabolism of 5-HTin the C.R-infected mice, and a similar phenomenon was
also reported in a depression model [63].
In the current study, C.R infection potentiated the
MPTP-induced effects on microglial and astrocyte activation in the nigrostriatal pathway. First, the combinationof C.R and MPTP changed the state of microglia from“ramified” to “stouter”, and polarized microglia to the M1
proinflammatory phenotype (manifested by the excessive increase in CD16/32-positive cells) and astrocytes
to the A1 phenotype (demonstrated by the large increasein C3-marked astrocytes). Second, there were increased
expression levels of proinflammatory molecules (IL-6,
IFN-γ, S100b, iNOS, CD86, CD68 and COX2) in the striatum of the C.R/MPTP mice. The expression of COX2
protein was also upregulated. Third, the ELISA results
showed increased serum levels of the cytokine IL-6.
These data clearly confirmed that C.R infection exacerbated the MPTP-mediated inflammatory response. The
concept of immune training has been proposed by previous studies showing that peripherally applied inflammatory stimuli can induce acute immune training in thebrain, which effectively acts as an important modifierof neuropathology [64]. Our results implied that there
may be an immune training response to preestablish C.R
infection in the brain. The precise mechanism of immune
memory in response to bacterial stimulation still needs tobe confirmed in future studies.
C.R infection exacerbated MPTP-induced PD pathology at the gut microbiota level in the present study.
Cotreatment with C.R. and MPTP markedly decreased
the relative abundance of the Bacteroidetes and Firmicutes phyla while increasing the abundance of Proteobacteria. At the family level, there was a noticeable
increase in the relative abundance of Citrobacter andParabacteroides, suggesting a significant presence ofintestinal inflammation. Furthermore, the combination of
MPTP and C.R resulted in a significant reduction in therelative abundance of Lactobacillus and Lachnospiraceae

He et al. Journal of Neuroinflammation(2024) 21:153

genera, both of which are known for producing SCFAs[51]. However, this combination treatment led to a modest increase in the abundance of Citrobacter and Parabacteroides, which are gram-negative bacteria characterized
by a high abundance of LPS on their outer membrane[51, 65]. LPS can act as a stimulator of the TLR4 pathway, potentially contributing to the exacerbation of PD
pathology. According to our data, TLR4 expression was
indeed upregulated in the colon and brain after C.R and
MPTP combined treatment. Furthermore, intestinal barrier damage and high permeability were observed in thecolon after C.R delivery. This finding prompted us to
speculate that bacteria and/or bacterial molecules mightleak into the circulation. This leakage contributes to systemic inflammation, leading to BBB disruption and neurodegeneration in the central nervous system. Herein, we
hypothesized that certain molecules, such as LPS, mightreach the SN and striatum regions and locally stimulate the TLR4 pathway, thus initiating or increasing theexpression of proinflammatory cytokines. Therefore, the
TLR4-NF-κB signaling pathway might be involved in ourmodel system. However, further in-depth studies are necessary to verify the significance of this signaling axis. To
further validate the significance of the microbiota-gutbrain axis in our study, we performed correlation analysis on various representative results. The alterations in
the gut microbiota caused by C.R. infection were closely
associated with the concentrations of DA, the expressionof TLR4, the activation of microglia, and the density of
dopaminergic neurons in the nigrostriatal pathway. Thesestrong correlations suggest that microbiota disorders
induced by C.R. infection play a crucial role in the pathogenesis of PD through the microbiota-gut-brain axis.

Conclusions
In conclusion, our study further elucidates how C.R
infection affects PD. One mechanism is that C.R infection-induced microbiota imbalance disturbs dopamine
metabolism, particularly its turnover rate. The othermechanism is that the IBD-like symptoms caused by C.R
infection exacerbate MPTP-induced PD pathology inmice. The IBD-like model induced by C.R infection is an
ideal model for studying the relationship between intestinal inflammation and PD.
Abbreviations
C.R	Citrobacter Rodentium
IBD	Inflammatory Bowel DiseaseUC	Ulcerative Colitis
CD	Crohn’s Disease
SCFAs	Short Chain Fatty Acids
BBB	Blood-brain Barrier
LPS	Lipopolysaccharide
SN	Substantia Nigra
DSS	Dextran Sulphate Sodium
CFUs	Colony-forming Units
RT-qPCR	Real-time Quantitative PCR

Page 24 of 26

C3	Complement Component 3
GI	Gastrointestinal Dysfunctions
DA	Dopamine
D.p.i	Days Post Infection
MAOA	Monoamine Oxidase A
MAOB	Monoamine Oxidase B

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03145-0.
Supplementary Material 1
Acknowledgements
We thank the members of Huang laboratory for their valuable input, and
Dr. Chao. Zhong for valuable suggestions. We thank the Shanghai Model
Organisms Center for providing animals and the Instrument platform ofInstitutes of Brain Science, Fudan University.
Author contributions
F.H., M.Y. and J.F. conceived the study, supervised the research, reviewed andrevised the manuscript, and contributed to financial support. Y.H. designed
the study, performed the experiments, collated and analyzed the data, andprepared the manuscript. Y.M. performed the HPLC analysis. J.Z., X.Y, Y.D., X.Z.,
H.D., R.F. and Y.Z helped performing the experiments. Q.L. and P.Y. conducted
the histological analysis. The authors read and approved the final manuscript.
Funding
This work was supported by grants from the National Natural Science
Foundation of China (32271003, 31970908), Shanghai Municipal Science andTechnology Major Project (No.2018SHZDZX01) and ZJLab, Shanghai Center
for Brain Science and Brain-Inspired Technology, the innovative research teamof high-level local university in Shanghai and the Open Project of State Key
Laboratory of Medical Neurobiology (SKLMN2003).
Data availability
The data and materials generated during the current study are not publicly
available but are available from the corresponding author upon reasonable
request.

Declarations
Ethics approval and consent to participate
All experiments involving animals were performed in accordance with
Institutional Animal Care and Use Committee of Fudan University, Shanghai
Medical College.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 25 June 2023 / Accepted: 29 May 2024

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