Ni et al. Journal of Neuroinflammation(2024) 21:166
https://doi.org/10.1186/s12974-024-03160-1

Journal of Neuroinflammation

Open Access

RESEARCH

Intermittent hypoxia exacerbates anxiety
in high-fat diet-induced diabetic miceby inhibiting TREM2-regulated IFNAR1
signaling
Wenyu Ni1†, Yun Niu2†, Sitong Cao2, Chunsun Fan1, Jian Fan1, Li Zhu2* and Xueting Wang2,3*
Abstract
Background Type 2 diabetes mellitus (T2DM) and obstructive sleep apnea (OSA) are mutual risk factors, with bothconditions inducing cognitive impairment and anxiety. However, whether OSA exacerbates cognitive impairment
and anxiety in patients with T2DM remains unclear. Moreover, TREM2 upregulation has been suggested to play aprotective role in attenuating microglia activation and improving synaptic function in T2DM mice. The aim of thisstudy was to explore the regulatory mechanisms of TREM2 and the cognitive and anxiety-like behavioral changes inmice with OSA combined with T2DM.
Methods A T2DM with OSA model was developed by treating mice with a 60% kcal high-fat diet (HFD) combinedwith intermittent hypoxia (IH). Spatial learning memory capacity and anxiety in mice were investigated. Neuronaldamage in the brain was determined by the quantity of synapses density, the number and morphology of brain
microglia, and pro-inflammatory factors. For mechanism exploration, an in vitro model of T2DM combined with
OSA was generated by co-treating microglia with high glucose (HG) and IH. Regulation of TREM2 on IFNAR1-STAT1
pathway was determined by RNA sequencing and qRT-PCR.
Results Our results showed that HFD mice exhibited significant cognitive dysfunction and anxiety-like behavior,accompanied by significant synaptic loss. Furthermore, significant activation of brain microglia and enhanced
microglial phagocytosis of synapses were observed. Moreover, IH was found to significantly aggravate anxiety in the
HFD mice. The mechanism of HG treatment may potentially involve the promotion of TREM2 upregulation, which
in turn attenuates the proinflammatory microglia by inhibiting the IFNAR1-STAT1 pathway. Conversely, a significant
reduction in TREM2 in IH-co-treated HFD mice and HG-treated microglia resulted in the further activation of the
IFNAR1-STAT1 pathway and consequently increased proinflammatory microglial activation.

†

Wenyu Ni and Yun Niucontributed equally to this work.

*Correspondence:
Li Zhuzhulizhou@ntu.edu.cn
Xueting Wangwangxueting@ntu.edu.cn
Full list of author information is available at the end of the article© 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.

Ni et al. Journal of Neuroinflammation(2024) 21:166

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Conclusions HFD upregulated the IFNAR1-STAT1 pathway and induced proinflammatory microglia, leading tosynaptic damage and causing anxiety and cognitive deficits. The upregulated TREM2 inT2DM mice brain exerted
a negative regulation of the IFNAR1-STAT1 pathway. Mice with T2DM combined with OSA exacerbated anxiety via
the downregulation of TREM2, causing heightened IFNAR1-STAT1 pathway activation and consequently increasing
proinflammatory microglia.
Keywords Type 2 diabetes mellitus, Obstructive sleep apnea, Anxiety, Microglia, TREM2, IFNAR1, Cognition
Graphical Abstract

Introduction
Type 2 diabetes mellitus (T2DM) is a chronic metabolic
disease characterized by insulin resistance, accounting
for approximately 90% of all diabetic cases [1]. Cognitive dysfunction is a brain disorder potentially caused
by T2DM, demonstrating characteristic structural brain
abnormalities and reduced learning and memory abilities. More than 60% of people with diabetes experience
mild cognitive impairment, with its prevalence increasing
with age [2]. Anxiety is another brain condition inducedby T2DM. People with diabetes are twice as likely todevelop anxiety or depression as the general population.
Moreover, people with diabetes accompanied by anxiety have a greater mental burden, more complications, asignificantly lower quality of life, and a threefold higher
mortality rate [3, 4]. The possible mechanisms of diabetes-associated cognitive impairment and anxiety include
insulin resistance, inflammation and oxidative stress, andcerebral microvascular and lymphatic dysfunction [5–8].
However, the understanding of most individuals concerning the dangers of T2DM is limited to diabetic eyedisease, diabetic nephropathy, and peripheral neuropathy, with less understanding of diabetes-induced brain
disorders [9]. Therefore, a comprehensive overview of themechanisms of diabetic encephalopathy will enhance thegeneral awareness of the risks linked to T2DM and further improve the clinical treatment.
Obstructive sleep apnea (OSA) is one such T2DM
complication as well as a high-risk factor for T2DM [10].
OSA, a sleep breathing disorder characterized by intermittent hypoxia (IH) and sleep fragmentation, has varied prevalence among patients with T2DM according

to country and region, with an average incidence rateof 56.0% [11]. Both cognitive dysfunction and mooddisorder are common features of OSA, which may be
attributed to cerebral hypoxia, sleep fragmentation,
inflammation, hormone level changes, and altered neurotransmitter concentrations. Although cognitive function impairment has been studied in the context of thecomorbidity of T2DM and OSA [12, 13], the effect ofT2DM complicated with OSA on anxiety and the associated mechanism remain to be elucidated.
Abnormal microglial activation is strongly associatedwith cognitive dysfunction and anxiety. Moreover, brain
microglia have been reported to demonstrate significant activation and increased secretion of proinflammatory factors in T2DM and OSA mice models [14]. The
activated microglia recognize different signals via membrane receptors and perform functions such as phagocytosis and cytokine secretion, which are involved in theneuroprotection and neurodegeneration processes [15,16]. Additionally, the altered activation of the synaptic
remodeling function in microglia causes synaptic loss,
which in turn leads to cognitive impairment and anxiety [17]. The triggering receptor expressed on myeloid
cells-2 (TREM2), an innate immune receptor enrichedin microglial cell membranes, is a known regulator of
microglia polarization and synaptic remodeling [18, 19].
TREM2 participates in clearing apoptotic cells, cellulardebris, and bacteria as well as exerts anti-inflammatory
effects by inhibiting secondary necrosis and the releaseof endogenous proinflammatory signals [20, 21]. Previous studies have indicated that TREM2 exhibits antiinflammatory and neuroprotective effects in Alzheimer’sNi et al. Journal of Neuroinflammation(2024) 21:166

disease (AD) mice. TREM2 mutations promote aberrant
activation of microglia in the brains of AD mice, reducing microglial reactivity to amyloid plaques, and leading to synaptic injury and cognitive impairment [22, 23].
TREM2 is also upregulated in T2DM mice, with TREM2
overexpression inhibiting microglia activation andimproving synaptic transmission and ultrastructure [24].
Although TREM2 have shown neuroprotective effects in
T2DM model mice, its specific mechanism is still unclear.
Furthermore, the function of TREM2 in T2DM concurrent with OSA requires further exploration.
Interferons are pleiotropic cytokines with antiviral,
antitumor, and immunomodulatory properties, demonstrating a crucial role in fighting viral infections [25].
Interferon-α/β receptor (IFNAR) binds to type I interferon and recruits TYK2 and JAK1 to transmit signals,
resulting in the further activation of the signal transducers and activators of transcription 1 (STAT1) signaling[26, 27]. The STAT1 transcription factor is involved inregulating microglia polarization, neuroinflammation,
synaptic plasticity, and cognition [28]. In this study, we
initially screened the genes regulating differential expression in high-glucose (HG)-treated microglia and foundsignificant upregulation in the IFNAR- STAT pathway,
implying that TREM2 was involved in regulating theactivation of HG-treated microglia by altering IFNAR
signaling. Therefore, we hypothesized that the IFNAR1STAT1 pathway might be involved in the developmentof cognitive impairment and anxiety in mice with T2DM
combined with OSA. This study aimed to investigate
the effects and the related molecular mechanism of IHinduced OSA on cognitive dysfunction and anxiety,thereby providing a theoretical basis and a new research
direction for the in-depth exploration of the treatment of
T2DM complicated with OSA.

Materials and methods
Animals and treatments

Male C57BL/6J mice (7-week-old) provided by theExperimental Animal Center of Nantong University
were initially allowed to acclimate for 1 week before
the study experiments. The mice were maintained at
23 ± 2 °C, 45–60% humidity, and a standard 12/12 hlight/dark cycle. HFD mice were fed a 60% kcal fat diet
(D12492), while normal control diet (NCD) mice were
provided a 10% kcal fat diet (D12450B) from the age of
8 weeks to 33 weeks. During the administration of IH
treatment, 28-week-old HFD or NCD mice were placedin a 60 cm × 30 cm × 25 cm chamber. Subsequently,
rapid cycles of oxygen and nitrogen inputs were used
to reduce the oxygen concentration in the chamber to
5% within 30 s, followed by maintaining this concentration for 30 s before rapidly increasing it to 20%. The
next cycle was started after maintaining the oxygen

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concentration at 20% for 30 s. This IH exposure was performed 8 h daily for 28 days between 09:00 a.m. and 5:00
p.m. After 28 days of IH treatment, the mice underwent
behavior tests and were then euthanized by anesthesia.
All the study protocols involving animal experiments
were reviewed and approved by the Animal Care and
Use Committee of Nantong University and the Jiangsu
Province Animal Care Ethics Committee (approval ID:
SYXK[SU]2007-0021).
Behavior tests
Open field test (OFT)

OFTs were performed to assess anxious behavior using
previously reported protocols [29, 30]. Briefly, eachmouse was placed in a 50 cm × 50 cm × 50 cm white polyvinyl chloride box and allowed to move freely for 5 min.
An overhead camera was employed to record and analyze
the movement trajectory and time spent in the peripheral(25 cm from the wall) and central zones (25 cm × 25 cm).
Elevated plus-maze (EPM) test

The EPM test was conducted to determine anxious
behavior according to the earlier described procedure[31]. In this test, each mouse was placed in the middle ofthe four-arm junction of the maze (35 cm × 5 cm) andallowed to move freely for 5 min. The mice trajectories
were then recorded to estimate the number of entries andtime spent in the open arms.
Novel object recognition (NOR) test

Initially, two identical objects, “A” and “B,” with a 3-cm
diameter were placed in a 50 cm × 50 cm × 50 cm box ata distance of 10 cm away from the inner wall of the box.
Next, individual mice were placed in the box, and theirtime spent exploring the objects A and B was recorded
for 5 min. After 1 h or 24 h, the object B was replacedwith a new object having a different appearance. The
individual mice were then placed back in the box andallowed to move freely for 5 min. Finally, the exploration time of the familiar and novel object was recorded.
In the NOR test, the memory capacity of the mice wasobtained using a preference index calculated using thefollowing formula: preference index (%) = TN/(TN + TF)
× 100%, where TN = exploration time of novel object and
TF = exploration time of familiar object. The NOR test
was performed as described in prior studies [32].
Morris water maze (MWM) test

The MWM test for investigating spatial learning-memory
behavior was conducted as previously described [33]. A
150-cm circular pool was divided into four equal quadrants, i.e., northeast, southeast, southwest, and northwest
quadrants. Visual cues were posted on the walls aroundthe maze to facilitate spatial learning of the platform’sNi et al. Journal of Neuroinflammation(2024) 21:166

location. During the experiments, the water was madeopaque with a non-toxic, white pigment and maintained
at a temperature of 21 ± 1 °C. In the training period (4times/day for 5 days), a circular platform of 10 cm indiameter was placed in the middle of the southwest
quadrant at 1.5 cm below the water surface. The individual mice were released into the water and allowed toswim freely for 60 s to find the platform and stay on it
for 20 s. During the probe trials (on the sixth day), theplatform was removed. The individual mice were thenreleased in the northeast quadrant and allowed to swim
freely for 60 s. Video recordings were used to analyze andrecord the swimming trajectory, along with the estimation of the escape latency and the crossing frequency tothe target quadrant.
Cell culture and treatments
Primary microglia culture

Primary microglia were obtained from the cerebral cortices of 2-day-old C57BL/6J mice according to previously
described protocols [34–36]. In short, brain tissue wasdigested with trypsin, and the resulting single-cell suspension was cultured in DMEM-F12 medium (Thermo
Fisher, 11,320,033) at 37 °C in a 5% CO2 humidified
incubator. The DMEM-F12 medium contained 10% fetal
bovine serum (Celligent, CG0430B), GlutaMAX supplement (Thermo Fisher, 35,050,079), 5 ng/ml of granulocyte-macrophage colony-stimulating factor (STEMCELLTechnologies, 78,017), and penicillin/streptomycin(100 U/ml and 100 mg/ml, respectively). After 10 daysof growth, the mixed cell population was dominated byastrocytes and formed a fused trophoblast. Gradually, themicroglia proliferated and floated in the supernatant andwere harvested on the 14th day.
Lentivirus transfection of primary microglia

TREM2 silencing was achieved by transfecting lentivirus
expressing shTrem2 (target sequence: ​G​A​C​C​C​T​C​T​A​G​A​
T​G​A​C​C​A​A​G​A​T) into the trophoblast cells. In this process, after 7 days of primary culture, the trophoblast cells
were incubated with 8 × 107 TU of lentiviruses per 25 cm2
culture flask and allowed to grow until microglia production. The obtained lentivirus transfection efficiency wasapproximately 50% in the harvested microglia.
Construction of shTrem2 BV2 cells

BV2 cells were transfected with shTrem2-expressing lentivirus at multiplicity of infection = 10. The positive cells
were then isolated via flow cytometry at 488 nm and cultured to form single-cell clones.
HG treatment

Initially, cells cultured in DMEM-F12 medium (glucose
concentration = 17.5 mM) were set as a control group. In

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the case of HG treatment, additional glucose was added
to the medium to increase the final glucose concentration
to 50, 75, and 100 mM. Finally, the cells were incubatedin these HG mediums for 6 h to generate in vitro modelsof T2DM.
Anti-IFNAR1 antibody (αIFNAR) treatment

Cells were pretreated with 10 µg/mL of αIFNAR for 1 h,followed by co-treatment of the cells with HG for 6 h.
IH treatment

Cultured cells were placed in the same intermittent
hypoxia chamber used for the experimental mice. In
this chamber, the cells were exposed to cycles of oxygen
concentration fluctuations (i.e., 20% [30 s] to 5% [30 s] ofoxygen) for 6 h.
Microglial endocytosis assay
Synaptosome uptake

Initially, 1 g of fresh brain tissue was well homogenized
and centrifuged at 800 × g for 10 min, followed by aspiration of the supernatant. The supernatant was further centrifuged at 17,000 × g for 20 min, and the precipitate wasresuspended in 0.8, 1.0, and 1.2 mol/l of sucrose gradientbuffers. After centrifugation at 82,500 × g for 2 h, the liquid at the 1.0–1.2 mol/l sucrose interface was collected
and diluted with 10 mM HEPES buffer. After centrifugation at 15,000 × g for 30 min, the obtained synaptosomes
were resuspended in DMEM-F12 medium and thendirectly incubated with the microglia for 30 min. Finally,
the cells were fixed and stained with synaptosome antibodies. Images were acquired using a Leica SP8 confocal
microscope.
Dextran endocytosis

After the specified treatments, primary microglia wereincubated with 100 mg/mL of 40-kDa TRITC-Dextran(AS026, ABclonal) for 30 min. The cells were then fixedand counterstained with DAPI. Images were captured
utilizing a Leica SP8 confocal microscope.
Immunofluorescence staining

Brain sections (Bregma − 1.3 to -1.9 mm) or cultured cells
were fixed in 4% paraformaldehyde and subsequently
permeabilized with 0.3% Triton X-100. Next, the samples
were blocked in 10% donkey serum, followed by incubation with the primary antibodies at 4 °C overnight. The
primary antibody binding was visualized using fluorescently labeled-secondary antibodies. Finally, the samples
were counterstained with DAPI for confocal microscopy.
The antibodies used for immunofluorescence staining arelisted in Table 1.

Ni et al. Journal of Neuroinflammation(2024) 21:166

Table 1 Antibody information
Nameanti-IFNAR1
anti-STAT1
Anti-NeuN
anti-TREM2
anti-Iba1
anti-LAMP1
anti- Synaptophysin
anti-PSD95

Catalog number
16-5945-85
66545-1-Ig
MAB377
AF1729ab5076sc-20,011
1,101,011
3409T

anti-β-actin
Donkey anti-Sheep IgG Alexa
488
Donkey Anti-Rat IgG Alexa 488

66009-1-lg
713-545-003

Donkey Anti-Goat IgG Alexa
488
Donkey Anti-Goat IgG Cy3

705-545-003

Donkey Anti-Mouse IgG Cy3

715-165-150

Donkey Anti-Rabbit IgG Cy3

711-167-003

Donkey Anti-Rabbit IgG Alexa
647
Donkey Anti-Mouse IgG Alexa
647

711-605-152

712-545-153

705-165-003

715-605-151

CompanyeBioscience
Proteintech
MilliporeR&D Systems
Abcam
Santa cruz
Synaptic Systems
Cell Signaling
Technology
Proteintech
JacksonImmunoResearchJacksonImmunoResearchJacksonImmunoResearchJacksonImmunoResearchJacksonImmunoResearchJacksonImmunoResearchJacksonImmunoResearchJacksonImmunoResearch

Circularity coefficient analysis

The circularity coefficient analysis was performed as
described before [37]. Specifically, brain tissue sections(Bregma − 1.3 to -1.9 mm) were labeled with anti-Iba1
antibodies. 10–12 Z-stacks of each mouse were captured
using a 20× objective by a Leica SP8 laser scanning confocal microscope. The obtained images were maximallyprojected to obtain the complete morphology of the cells.
Data on the number of microglia were obtained by manually counting Iba1-positive microglia in 4 pictures of eachmouse. The morphological analysis of microglial cells
was performed by randomly selecting 8–10 microglia/
mouse and using the “shape descriptor” tool of FIJI software. The circularity coefficient is approximately close to
1 indicating increasing cell activation.
NeuN signal analysis

Bain tissue sections (Bregma − 1.3 to -1.9 mm) werelabeled with anti-NeuN antibody. 10–12 Z-stacks of eachmouse was captured using a 20X objective, zoom in 0.75fold, by a Leica SP8 laser scanning confocal microscope.
The obtained images were maximally projected. Dataon the number of neurons were obtained by manuallycounting NeuN signal in 4 pictures of each mouse by FIJI
software.

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PSD95 and nuclear STAT1 signal analysis

Brain tissue sections (Bregma − 1.3 to -1.9 mm) werelabeled with anti-Iba1 or anti-STAT1 (anti-PSD95) antibodies. 10 μm Z-stacks (0.8 μm of each stack) of eachmouse were captured using a 100× objective, zoom in
2-fold by a Leica SP8 laser scanning confocal microscope.
The obtained images were maximally projected to obtainthe complete morphology of microglia. STAT1 intensity
in nucleus of Iba1+ cells or PSD95 intensity in Iba1+ cells
were counted by FIJI software. The average intensity of
10–20 microglia in each mouse was calculated as a data
point.
Synapses number analysis

Brain tissue sections (Bregma − 1.3 to -1.9 mm) werelabeled with anti-Synaptophysin and anti-PSD95 antibodies. 2 μm Z-stacks (0.2 μm of each stack) of eachmouse were captured using a 100X objective, zoom in
2-fold by a Leica SP8 laser scanning confocal microscope.
The obtained images were maximally projected. Signals
from Synaptophysin and PSD95 within 200 nm are considered as intact synapses [38]. The total number of intact
synapses was counted in 4 fields of view per mouse.
Nissl staining

Nissl staining for counting the neurons in the hippocampal CA1 region was performed as described previously[39]. Briefly, brain Sect. (40-mm thick) were soaked intrichloromethane, followed by staining using 1% tar violet. The stained samples were then dehydrated using an
ethanol gradient and xylene transparent. Images wereobtained with a Leica DM4000B microscope.
RNA-seq and qRT-PCR

Initially, purified total RNA was isolated from the brain
tissue or cells using TRIzol buffer. The purified total
RNA was then submitted to OBiO Technology for RNAseq. Differentially Expressed Genes (DEGs) were identified using an empirical Bayes linear model, and a P-value
was calculated for the expression of all genes. A thresholdof P-value < 0.05 and fold-change ≥ 2 was established. The
DEGs were visualized and clustered in a heatmap using
the pheatmap package (version 1.0.12, http://cran.r-project.org/web/packages/pheatmap) in R. The clusterProfiler tool (http://bioconductor.org/packages/release/bioc/
html/clusterProfiler.html) was utilized to carry out Kyoto
Encyclopedia of Genes and Genomes (KEGG) analyses
for the differentially expressed genes that were up- anddown-regulated [40]. The criteria for significance wereset at a P-value < 0.1, adjusted using the Benjamini and
Hochberg method.
First-strand cDNA was synthesized utilizing the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech,
R312-02). Ultimately, gene expression was measured via

Ni et al. Journal of Neuroinflammation(2024) 21:166

qRT-PCR using the AceQ qPCR SYBR Green Master Mix(Vazyme Biotech, Q141-02), which was performed at acycle setting of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for
20 s for 40 cycles. The relative amount of gene expression
was calculated using ΔCt values. The primers employedfor qRT-PCR are presented in Table 2.
Protein isolation and western blot

Brain tissue or cells were lysed in RIPA buffer, and theprotein concentration was estimated using the bicinchoninic acid assay. Proteins were then isolated fromthe remaining lysate and transferred to polyvinylidene
fluoride membranes for hybridization with primary antibodies. Primary antibody binding was detected by HRPconjugated secondary antibodies. The antibodies used forWestern blot are provided in Table 1.
Statistical analysis

FIJI software (National Institutes of Health) was
employed to calculate the fluorescent signal intensities
on the microscopy images and the grayscale values ofthe Western blot protein bands. All data were analyzed
via GraphPad Prism version 8.0 software (GraphpPad).
The Student’s t-test and two-way ANOVA followedby Dunnett’s multiple comparisons procedures wereapplied for statistical assessment. Data were presented as
Table 2 Primer information
Name
Cd86
Fcgr3
Arg1
Mrc1
Il1b
Tnfa
Tgfb
Trem2
Ifnar1
Tyk2
Stat1
Actb
Trem2-OE

Direction
Forward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseSequence (5′—3′)​A​C​G​T​A​T ​T​G​G​A​A​G​G​A​G​A​T ​T​A​C​A​G​C ​T​T​C ​T​G​T​C​A​G​C​G​T ​T​A​C ​T​A​T​C​C​C​G​C​T ​T ​T​G​G​A​C​A​C​C​C​A​G​A​T​G​T ​T ​T​C​A​G​G​T​C ​T ​T​C​C ​T ​T​G​A​G​C​A​C​C ​T​G​G​A​T​C​C​A​T ​T​G​G​C ​T ​T​G​C​G​A​G​A​C​G​T​A​G​A​C​G​C ​T​G​A​A​G​G​T​C ​T​C ​T ​T​C​C​A​T​C​A​C​C​G​T ​T​C​A​C​C ​T​G​G​A​G​T​G​A​T​G​G​T ​T​C ​T​C
​A​G​G​A​C​A​T​G​C​C​A​G​G​G​T​C​A​C​C ​T ​T ​T​T​G​C​C​A​C​C ​T ​T ​T ​T​G​A​C​A​G​T​G​A​T​G​T​G​A​T​G​T​G​C ​T​G​C ​T​G​C​G​A​G​A​T ​T​A​A​G​C​C ​T​G​T​A​G​C​C​C​A​C​G​T​C​G​T​A​G​G​C​A​C​C​A​C ​T​A​G​T ​T​G​G​T ​T​G​T​C ​T ​T ​T​G
​T​G​A​T​A​C​G​C​C ​T​G​A​G​T​G​G​C ​T​G​T​C ​T​C​A​C​A​A​G​A​G​C​A​G​T​G​A​G​C​G​C ​T​G​A​A​A​T​G​A​C​A​C​C​C ​T ​T​G​C ​T​G​G​A​A​C​C​G​C ​T​A​G​A​G​G​T​G​A​C​C​C​A​C​A​G​G​A​C​C​A​A​G​G​C​A​A​G​A​G​C ​T​A​T​G​T​C​C ​T​G
​C​A​G​T​G​C​G​T​A​G​T​C ​T​G​G​A​C​A​T ​T ​T​G​C​G​C ​T ​T ​T​C​C ​T​G​C​A​T​G​G​T​G​T ​T ​T​G​C​G​T​G​T​C​G​C​C​G​T​A​A​C​C​A​C​A​C​A​T​C​C​A​T​A​C​G​G​A​A​A​A​G​C​A​A​G​C​G​T​A​A​T​C ​T​T​G​C​A​C​A​T​G​A​C ​T ​T​G​A​T​C​C ​T ​T​C​A​C​C​A​T​C​C​G​T​A​A​A​G​A​C​C ​T​C ​T​A​T​G​C​C​A​A​C​A​T​G​G​A​G​C​C​A​C​C​G​A​T​C​C​A​C​A​A​C​A​G​C​A​C​C ​T​C​C​A​G​G​A​A​T​C​A​A​G​A​G​G​A​T​C ​T​G​A​A​G​T ​T​G​G​T​G​C​C​C

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mean ± Standard Error of the Mean. The significance levels for all graphs in this study were as follows: *p < 0.05,**p < 0.01, and ***p < 0.001. n.s. indicated no statisticaldifference.

Results
HFD-induced diabetic mice exhibit anxiety-like behavior
and cognitive impairment

We induced T2DM in mice using a 60% kcal HFD toinvestigate whether cognitive impairment may develop
in T2DM mice. As shown in Fig. 1A and s1A, the HFD
mice showed significant weight gain and reduced appetite. Furthermore, the mice exhibited higher fasting glucose (Fig. s1B) and random glucose levels (Fig. 1B) as
well as poorer glucose tolerance compared to the NCD
mice (Fig. 1C and D), establishing that the HFD successfully induced T2DM in the mice. Subsequently, we examined the anxiety-like behaviors of the HFD mice using
OFT and EMP test. In the OFT, the mice demonstrated
reduced entries (Fig. 1E), dwell times (Fig. 1F), and theratio of distance traveled (Fig. 1G) in the central zone of
the field. Consistently, the EPM test results showed that
HFD mice had decreased exploration (Fig. 1H) as well as
lower time spent (Fig. 1I), entries (Fig. 1J), and the ratioof movement distance (Fig. 1K) in the open arm. Neither distance traveled nor average speed of mice during the tests of mice changed in the OFT and EPM tests(Fig. s2). All these results suggested that HFD inducesanxiety-like behavior in HFD-induced diabetic mice. We
also explored the effects of HFD on the spatial learningmemory capacity of the mice via the MWM and NOR
experiments. As depicted in Fig. 1L and M, the HFD
mice exhibited reduced exploration of the target quadrant and significantly poorer learning ability in the water
maze than the NCD mice. In the probe trials, the escapelatency (i.e., the time to locate the target platform quadrant) of the HFD mice was prolonged (Fig. 1N), alongwith significantly reduced entries in the target quadrant(Fig. 1O). In the 1 h-delay test of the NOR experiment,
the HFD mice did not significantly differ from the NCD
mice in the preference index (Fig. 1Q). However, the HFD
mice showed a significantly lower preference index thanthe NCD mice in the 24-h delay test (Fig. 1R), indicating a loss of prolonged memory in the HFD mice. Considering these findings, HFD-induced T2DM mice may
exhibit prominent anxiety-like behavior and cognitive
impairment.
HFD induces microglial activation and synaptic
phagocytosis to trigger neuronal damage in diabetic micebrainsNext, we investigated the morphology of the anxietyrelated basolateral amygdalar complex (BLA) region[41] and the learning memory-related hippocampal

Ni et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 1 HFD mice exhibit elevated blood glucose, cognitive impairment, and anxiety. (A) Weekly body weight of the HFD mice. (B) Random blood glucose
concentration of the mice after 26 weeks of HFD. (C) Glucose tolerance test findings of the mice after 26 weeks of HFD. (D) Area under the curve (AUC)
for the glucose tolerance test results in panel C. (E) Movement trajectory of the mice in the OFT. (F) Dwell times of the mice in the central zone during
the OFT. (G) Percentage of the distance traveled by the mice in the central zone of the open field. (H) Movement trajectory of the mice in the EPM test. (I)Time spent by the mice in the open arm of the maze. (J) Frequency of mice entries in the open arm of the maze. (K) Percentage of distance traveled by
the mice in the open arm of the maze. (L) Swimming trajectory of the mice during the probe period of the MWM test. (M) Escape latency (i.e., the time
to reach the target platform) of the mice during the training period of the MWM test. (N) Escape latency of the mice to reach the target for the first time
during the probe period of the MWM test. (O) Frequency of crossing the target platform area during the probe period of the MWM test. (P) Schematic
diagram of the NOR experiment. During the training stage, the mice are allowed to move freely in a box and familiarize themselves with two identicalobjects. After 1–24 h, one familiar object is replaced with a novel object, and the frequency of explorations of the familiar and novel objects in 5 min ismeasured. (Q) Preference index of the mice for the novel object in the NOR test after a 1-h delay. (R) Preference index of the mice for the novel object in
the NOR test after a 24-h delay. n = 13. *p < 0.05, **p < 0.01 and ***p < 0.001 by Student’s t-test. n.s. indicates no significant difference. HFD, high-fat diet;
OFT, open field test; EPM, elevated plus maze; MWM, Morris water mazeNi et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 2 Upregulation of synaptic phagocytosis and proinflammatory polarization of microglia occur in HFD mice brains. (A) Brain sections of the HFD mice
were probed with anti-NeuN antibody and counterstained with DAPI to obtain microscopy images of the basolateral amygdalar complex (BLA) region.
Scale bar = 200 μm. (B) Brain sections of the HFD mice were labeled with anti-PSD95 and anti-synaptophysin antibodies to acquire microscopy images of
the hippocampal CA1 region. Scale bar = 2 μm. (C) PSD95 in the lysate of mouse brain tissue was detected via Western blot. (D) The number of neurons
in the BLA region in panel A. (E) Colocalization ratio of PSD95 and synaptophysin in panel B. (F) Grayscale values of the bands in panel C (n = 12). (G) Brain
sections of the HFD mice were labeled with anti-LAMP1, anti-PSD95, and anti-Iba1 antibodies to obtain microscopy images of the microglia in the BLA
region. Scale bar = 3 μm. (H) PSD95 intensity in the Iba1+ cells in panel G. (I) Brain sections of the HFD mice were labeled with anti-Iba1 antibodies andcounterstained with DAPI to capture microscopy images of the BLA region. Scale bar = 100 μm. (J) The number of Iba1+ cells in panel I. (K) Circularity coefficients of the Iba1+ cells in panel I were calculated using FIJI software. (L to P) Expressions of Cd86, Tnfa, Il1b, Arg1, and Mrc1 in the BLA and CA1 regions
were measured via qRT-PCR. n = 6, *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t-test. HFD, high-fat diet; BLA, basolateral amygdalar

CA1 region [42]. Our results showed that the number ofneurons was significantly reduced in the BLA region of
the HFD mice (Fig. 2A and D). Although no significant
reduction of neurons was observed in the dentate gyrus(DG) and CA1 region (Fig. s3A to C), the synapses weresignificantly decreased in the HFD mice brains (Fig. 2B
and E, Fig. s3E to H). In line with this finding, reduced
intensity of total brain PSD95 was detected in the HFD
mice (Fig. 2C and F), demonstrating an aberrant synaptic plasticity in the HFD mice brains. Given the regulatory role of microglia in synaptic plasticity [43], we
subsequently examined synaptic uptake by the microglia
in the HFD mice brains. Our results revealed significantly
increased colocalization of PSD95 with LAMP1 in the
Iba1+ cells in the BLA (Fig. 2G and H) and CA1 regions(Fig. s3I and J), indicating that the reduction in synapses

might be related to the enhanced synaptic uptake by the
microglia in the HFD mice brains. In support of thishypothesis, we further observed microglial activation in
the BLA (Fig. 2I to K) and CA1 regions (Fig. s3K to M)
in the HFD mice. Moreover, we verified that the polarization markers Cd86, Arg1, and Mrc1 were significantly
upregulated and that the proinflammatory factors TNF-α
and IL-1β were significantly elevated in the microgliaof the HFD mice brains (Fig. 2L to P). All these results
suggested that HFD induces cognitive impairment bypromoting the proinflammatory activation of microglia,
which in turn enhances the aberrant uptake of synapses.

Ni et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 3 Upregulation of TREM2 inhibits HG-induced proinflammatory polarization of microglia. (A) TREM2 levels in brain tissue lysate were detected using
Western blot analysis. (B) Grayscale values of the TREM2 bands in panel A (n = 12). (C) Trem2 expression in the BLA and CA1 regions was determined using
qRT-PCR (n = 6). (D to G) Expressions of Trem2, Cd86, Tnfa, and Il1b were estimated via qRT-PCR in the primary microglia treated with 50 mM of HG for
specified durations (n = 3). (H) After HG treatment for the indicated durations, primary microglia were incubated with synaptosomes for 30 min. The cells
were then probed with anti-TREM2 and anti-synaptophysin antibodies and counterstained with DAPI. Scale bar = 20 μm. (I) Synaptophysin intensity in the
TREM2+ cells in panel H (n = 7). (J) After HG treatment for 6 h, TREM2-overexpressing (OE) BV2 cells were incubated with 40-kDa TRITC-Dextran for 30 min,followed by fixation and counterstaining with DAPI. Scale bar = 10 μm. (K) TRITC intensity per cell in panel J (n = 10). (L to P) Expressions of Trem2, Cd86,
Arg1, Il1b, and Tnfa were measured by qRT-PCR in TREM2-OE BV2 cells after HG treatment (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 by one-way ANOVA(D to G, I, and K) or two-way ANOVA (L to P). n.s. indicates no significant difference

Upregulated TREM2 inhibits the proinflammatorypolarization of HG-treated microglia

TREM2 is a membrane receptor primarily expressedon microglia in the central nervous system, exhibiting
crucial involvement in synaptic protection, regulating
microglial phenotypic transformation, and anti-inflammatory processes [20, 21]. Similar to a previous study
that demonstrated TREM2 upregulation in the brains of
T2DM mice [24], this study found heightened TREM2
levels in the HFD mice brains (Fig. 3A to C). Furthermore, we treated primary microglia with different glucose concentrations to investigate whether the HG
environment was responsible for TREM2 upregulationand proinflammatory microglial activation. Our results
showed that administrating 50 mM of glucose (HG treatment) caused a significant upregulation of TREM2 andthe proinflammatory markers CD86, TNF-α, and IL-1β

in the microglia (Fig. s4). However, this upregulation
did not exhibit a significant concentration dependence.
Subsequently, we treated the microglial cells with HG
for different durations, revealing that the HG-induced
upregulation of TREM2 and the proinflammatory markers was time-dependent (Fig. 3D to G). Correspondingly,
we observed that HG activated the microglial uptake ofsynapses (Fig. 3H and I), suggesting that the HG extracellular environment contributed to the activation and synaptic phagocytosis activity of the microglia.
Based on the anti-inflammatory functions of TREM2,
we further verified the regulatory role of TREM2 in HGtreated microglia. Our findings indicated that upregulated TREM2 (Fig. 3L) significantly restored HG-induced
excessive phagocytosis in the microglia (Fig. 3J andK), leading to a significant attenuation of the microglia polarization to the proinflammatory type (Fig. 3M

Ni et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 4 TREM2 negatively regulates IFNAR1-STAT1 signaling in HG-treated microglia. (A to D) Total RNA of HG-treated TREM2-OE or shTrem2 BV2 cells was
isolated for RNA-seq (n = 3). (A) A heatmap demonstrating the differentially expressed genes. (B) KEGG pathway analysis depicting the enriched pathways.
(C and D) Expressions of Ifnar1, Tyk2, and Stat1 in the HG-treated TREM2-OE or shTrem2 BV2 cells were detected using qRT-PCR. (E) Brain sections of the
HFD mice were labeled with anti-Iba1 and anti-STAT1 antibodies and counterstained with DAPI, followed by microscopy imaging of the microglia in the
BLA region. Scale bar = 20 μm. (F) Nuclear STAT1 intensity in the Iba1+ cells in panel E (n = 6). (G to I) Expressions of Ifnar1, Tyk2, and Stat1 in the BLA andCA1 regions of the HFD mice were detected using qRT-PCR (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 by two-way ANOVA (C and D) or Student’s t-test(F to I)to P). Moreover, microglial synaptic phagocytosis was
significantly upregulated after the silencing of TREM2(Fig. s5A), with further upregulation detected following
TREM2 silencing in the HG-treated microglia (Fig. s5B
and C). Lastly, silencing TREM2 also exacerbated the
HG-induced proinflammatory activation of the microglia (Fig. s5D to G). All these results demonstrated that
upregulated TREM2 inhibited the further enhancementof proinflammatory activation and synaptic phagocytosis
activity of the HG-treated microglia. Therefore, TREM2

upregulation under HG conditions may be a mechanismof cellular compensation.
TREM2 downregulates IFNAR1 signaling in HG-treatedmicroglia

Further, we investigated the TREM2 mechanism involved
in inhibiting the proinflammatory activation of HGtreated microglia by using RNA-seq to screen for differentially expressed genes in the microglia caused by
the modulation of Trem2. As illustrated in Figs. 4A and
146 genes were significantly altered in the HG-treatedNi et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 5 Inhibition of IFNAR1 attenuates HG-induced proinflammatory polarization of microglia. (A) BV2 cells were co-treated with αIFNAR1 and HG for 6 h,followed by immunofluorescence labeling of STAT1 and counterstaining with DAPI. Scale bar = 10 μm. (B) Nuclear STAT1 intensity in the BV2 cells in panel
A (n = 7). (C to E) Expressions of Stat1, Tnfa, and Il1b in the BV2 cells co-treated with αIFNAR and HG were measured via qRT-PCR (n = 3). (F) BV2 cells wereco-treated with αIFNAR and HG for 6 h, followed by incubation with 40-kDA TRITC-Dextran for 30 min. The cells were then fixed and stained with DAPI.
Scale bar = 10 μm. (G) TRITC fluorescence intensity in each co-treated BV2 cell in panel F (n = 14). (H) shTREM2 and TREM2-OE BV2 cells were co-treatedwith αIFNAR and HG for 6 h, followed by labeling with anti-STAT1 antibodies and counterstaining with DAPI. Scale bar = 10 μm. (I) Nuclear STAT1 intensity
in the shTREM2 and TREM2-OE BV2 cells in panel H (n = 9). (J and K) qRT-PCR was used to estimate Tnfa and Il1b expression in the shTrem2 BV2 cells (J)and TREM2-OE BV2 cells (K) co-treated with αIFNAR and HG (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 by two-way ANOVA. n.s. indicates no significant
difference

microglia, showing a negative association with theoverexpression and silencing of TREM2. Additionally,
we found significant upregulation of the IFNAR-JAKSTAT pathway in the HG-treated microglia, whereas
the upregulation of TREM2 reversed the upregulationof this pathway. Further KEGG analysis for the genes
upregulated after HG treatment revealed that the JAKSTAT, TNF, and adipocytokine signaling pathways were
all significantly activated (Fig. 4B). Furthermore, thisfinding suggested that TREM2 potentially regulated

Ni et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 6 IH exacerbates anxiety-like behavior and neuronal loss in HFD-induced diabetic mice. (A) Flowchart of the OSA model construction. (B) Weekly
body weights of the mice. (C) Glucose tolerance test results of the mice after HFD + IH treatment (n = 4). (D) Movement trajectory of the mice in the OFT.
(E) The frequency of mice entries and their dwell times in the center zone of the open field (n = 9). (F) Movement trajectory of the mice in the EPM test.
(G) Frequency of mice entries and the time spent by them in the open arm of the maze (n = 9). (H) Brain sections of the mice were labeled with antiNeuN antibody and counterstained with DAPI, followed by microscopy imaging of the BLA region. Scale bar = 200 μm. (I) The number of neurons in the
BLA region in panel H (n = 6). (J) Brain sections of the mice were probed with anti-LAMP1, anti-PSD95, and anti-Iba1 antibodies, followed by microscopy
imaging of the microglia in the BLA region. Scale bar = 3 μm. (K) PSD95 intensity in the Iba1+ cells in panel J (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001
by two-way ANOVA

the activation of HG-treated microglia by modulating
IFNAR signaling. Subsequent validation at the transcriptional level confirmed that HG caused the activation of
IFNAR1, Tyk2, and STAT1 and that upregulated TREM2
in the HG-treated microglia significantly inhibited
IFNAR1 pathway activation (Fig. 4C). Conversely, silencing TREM2 exacerbated the activation of the IFNAR1 signaling in the HG-treated microglia (Fig. 4D). Moreover,the HFD mice brains exhibited a significant upregulationof nuclear STAT1 (Fig. 4E and F), along with significantly
upregulated IFNAR1, Tyk2, and STAT1 in the BLA andCA1 regions (Fig. 4G to I). These results suggested thatmicroglial activation in the HFD mice might be related tothe activation of IFNAR1 signaling, whereas the compensatory upregulation of TREM2 was potentially linked tothe inhibition of IFNAR1 activation. This hypothesis was
further corroborated by cellular analysis results, wherein
the overexpression of TREM2 suppressed the nuclear
expression of STAT1, while TREM2 interference further
upregulated STAT1 nuclear expression in HG-treatedmicroglia (Fig. s6).

Ni et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 7 IH and HFD co-treatment promotes proinflammatory microglial polarization in the mouse brain via TREM2-IFNAR1 signaling. (A) Brain sections of
the mice co-treated with IH and HFD were labeled with anti-Iba1 antibodies and counterstained with DAPI, followed by microscopy imaging of the BLA
region. Scale bar = 100 μm. (B) The number of Iba1+ cells in panel A (n = 6). (C) Circularity coefficients of the Iba1+ cells in panel A (n = 6). (D to I) Expressions of Il1b, Tnfa, Trem2, Ifnar1, Tyk2, and Stat1 in the BLA region of the brain tissue were measured using qRT-PCR (n = 5). (J) Brain sections of the miceco-treated with IH and HFD were labeled with anti-Iba1 and anti-STAT1 antibodies and counterstained with DAPI, followed by microscopy imaging of
the microglia in the BLA region. Scale bar = 10 μm. (K) Nuclear STAT1 intensity in the Iba1+ cells in panel J (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 by
two-way ANOVA

Inhibition of IFNAR1 signaling synergizes with TREM2 toco-inhibit the proinflammatory polarization of HG-treatedmicroglia

Next, HG-administered microglia were pretreated with
αIFNAR to ascertain whether the IFNAR1 signaling
pathway promoted the proinflammatory activation of
HG-treated microglia. Our results showed that αIFNAR

significantly inhibited the nuclear expression of STAT1
in HG-treated microglia (Fig. 5A to C), suppressed thesynthesis of proinflammatory factors (Fig. 5D and E),
and inhibited the synaptic phagocytosis activity of HGtreated microglia (Fig. 5F and G), suggesting that inhibiting IFNAR1 reduces the proinflammatory activation of
microglia via diminished STAT1 signaling. Additionally,Ni et al. Journal of Neuroinflammation(2024) 21:166

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Fig. 8 Upregulation of TREM2 inhibits proinflammatory microglial activation induced by IH and HG co-treatment. (A to C) Expressions of Trem2, Stat1,
and Ifnar1 in BV2 cells co-treated with HG and IH were detected via qRT-PCR (n = 3). (D) Primary microglia were co-treated with HG and IH and incubatedwith synaptosomes for 30 min. The cells were then labeled with anti-TREM2 and anti-synaptophysin antibodies and counterstained with DAPI. Scalebar = 20 μm. (E) Synaptophysin intensity in the TREM2+ cells in panel D (n = 6). (F) Expressions of Il1b and Tnfa in TREM2-OE BV2 cells were estimated by
qRT-PCR (n = 3). (G) TREM2-OE BV2 cells were co-treated with αIFNAR, HG, and IH, followed by probing with anti-STAT1 antibodies and counterstaining
with DAPI. Scale bar = 10 μm. (H) Nuclear STAT1 intensity per cell in panel G (n = 9). (I) TREM2-OE BV2 cells were treated with αIFNAR, HG, and IH and thenincubated with 40-kDa TRITC-Dextran for 30 min. Finally, the cells were fixed and stained with DAPI. Scale bar = 10 μm. (J) TRITC intensity per cell in panel
I (n = 9). *p < 0.05, **p < 0.01, and ***p < 0.001 by two-way ANOVA. n.s. indicates no significant difference

we verified that IFNAR1 was involved in TREM2-mediated anti-inflammatory response. Our findings demonstrated that αIFNAR significantly inhibited the nuclear
expression of STAT1 in HG-treated shTrem2 microglia, with relatively greater attenuation of STAT1 nuclear
expression in the HG-treated TREM2-OE microglia(Fig. 5H and I). In line with these results, αIFNAR treatment inhibited the upregulation of proinflammatory factors in HG-treated shTrem2 microglia as well as led to afurther reduction in the proinflammatory factors in HGtreated TREM2-OE microglia (Fig. 5J and K). Considering these findings, the inhibition of the IFNAR1 pathway
may synergize with TREM2 to jointly inhibit the proinflammatory activation of HG-treated microglia.

IH rather than blood glucose exacerbates anxiety-likebehavior and neuronal damage in HFD-induced diabetic
mice

OSA, a common complication of T2DM, is known to
be associated with cognitive impairment. Hence, we
examined whether the OSA model constructed with
IH worsened the spatial learning-memory capacity andanxiety-like behavior of the HFD-induced T2DM mice
(Fig. 6A). Our findings indicated that IH treatment did
not significantly alter the body weight, blood glucose(Fig. 6B and C), and spatial learning-memory capacity of
the HFD mice (Fig. s7). However, IH significantly aggravated the anxiety-like behavior of these mice. In the caseof the OFT, the HFD + IH mice had fewer entries andshorter dwell times in the central zone of the open field
than the HFD mice (Fig. 6D and E). Similarly, the EPM
results demonstrated that HFD + IH mice spent less timeand made fewer entries in the open arm of the maze thanNi et al. Journal of Neuroinflammation(2024) 21:166

the HFD mice (Fig. 6F and G). Neither distance traveled nor average speed of mice during the tests of micechanged in the OFT and EPM tests (Fig. s8). Consistent
with these behavioral results, IH significantly exacerbated the loss of neurons and synapses in the BLA region(Fig. 6H and I, Fig. s9A and B); however, it had no significant effect on the synaptic number in the CA1 region(Fig. s9C and D). Additionally, the localization of PSD95
with LAMP1 was significantly greater in the Iba1+ cells in
the BLA region of the HFD + IH mice brains than in thatof the HFD mice (Fig. 6J and K). All these results suggested that IH induced neuronal loss in the BLA region
and worsened anxiety-like behavior in HFD mice by specifically enhancing the synaptic phagocytosis ability ofthe microglia in the BLA region.
IH increases proinflammatory microglial activation bymodulating TREM2-regulated IFNAR1 signaling

Additionally, we assessed the number and morphologyof brain microglia to verify whether IH aggravates neuronal damage in the BLA region of the HFD mice byincreasing microglial activation. As illustrated in Fig. 7A
to C, IH increased the number and the circularity coefficients of the microglia in the BLA region of the HFD
mice. Furthermore, IH escalated the expression of proinflammatory factors in the HFD mice brains (Fig. 7D and
E). IH also caused TREM2 downregulation in the HFD
mice brains (Fig. 7F). This downregulation of TREM2aligned with the IH-induced amplification of the IFNAR1
pathway expression (Fig. 7G to I) and increased STAT1
nuclear expression (Fig. 7J and K). Therefore, IH potentially exacerbates microglial proinflammatory activationin HFD mice by inhibiting TREM2. We also found thatboth IFN-α and IFN-β were upregulated in the brains of
HFD mice, and IH further exacerbated the elevation of
IFN-α and IFN-β (Fig. s10), which was consistent withthe trend of IFNAR, indicating a complex regulatory
mechanism of the IFN signaling pathway.
We further verified the effect of IH on TREM2IFNAR1 signaling in the HG-treated microglia in vitro.
As depicted in Fig. 8A to C, IH inhibited TREM2 andupregulated the IFNAR1 pathway in HG-treated microglia. IH was also found to heighten the synaptic phagocytosis activity of the HG-treated microglia (Fig. 8D and
E). Conversely, both αIFNAR treatment and TREM2
overexpression reversed the proinflammatory activation(Fig. 8F), nuclear expression of STAT1 (Fig. 8G and H),
and aberrant phagocytosis (Fig. 8I and J) in the HG + IH
microglia. This finding indicated that TREM2 inhibition
by IH leads to the activation of the IFNAR1 signaling
pathway, thereby triggering the proinflammatory activation and increased synaptic phagocytosis activity of
microglia.

Page 15 of 19

Discussion
Mice with T2DM induced by a 60% kcal HFD are typically characterized by weight gain, elevated fasting glucose, decreased glucose tolerance, insulin resistance,
and impaired cognition [24, 44]. Hong Zhuang et al.
reported that the 60% kcal HFD-induced T2DM mice
exhibited an increased number of activated hippocampal microglia, altered microglial morphology, along with
anxiety- and depression-like behaviors as well as memory
impairments [45]. A study by Xiangyu Guo et al. simulated T2DM combined with OSA by administering IH
to KK-Ay mice and revealed that IH inhibited autophagy
and exacerbated apoptosis in the hippocampal neurons
via the HMGB1/TLR4 pathway. Another investigation
by Yu Shi et al. demonstrated that IH significantly aggravated cognitive impairment in KK-Ay mice [12]. Thisfinding was inconsistent with our study results, where
we observed that IH did not worsen cognitive deficits
but significantly heightened anxiety-like behaviors in the
HFD mice. Previous epidemiologic studies have also indicated that OSA significantly increases anxiety in patientswith T2DM [8, 46]. Interestingly, a 14-year continuous follow-up study by Lin Sun et al. highlighted that ahigher proportion of older adults with anxiety symptoms
progressed to cognitive impairment (39.2%) than those
without anxiety symptoms (22.6%), implying that anxiety
might increase the risk of developing cognitive impairment by damaging axons or synapses [47]. Studies havealso shown that anxiety increases the risk of developing
dementia in patients with mild cognitive impairment[48] and may serve as a predictor of cognitive decline inthose with Parkinson’s disease [49]. All these studies suggest that anxiety induces and increases the progression of
cognitive impairment. Moreover, the diabetic condition
of HFD-induced T2DM mice may be milder than that of
KK-Ay mice; therefore, anxiety, which is more sensitive
to environmental changes, is the first symptom of brainrelated changes. We hypothesize that the IH-treated
HFD mice will eventually display cognitive impairment
as the disease progresses. Moreover, we found that the
average speed and movement distance of the HFD mice
did not change during the tests. In the study of Jian Han
et al., HFD mice were subjected to 60 min of OFT andexhibited reduced mobility during the test [50]. We suspected that HFD mice, whose stamina are significantly
compromised due to over-obesity, exhibit significant
under-strength during prolonged exercise.
Our study revealed that abnormal microglial activation was closely associated with anxiety and cognitive deficits in the HFD mice. Earlier clinical studies
have also observed significant microglial activation in
the hypothalamic region of patients with T2DM, alongwith an enhancement in cell number and cytosolic area[51]. Furthermore, neuroinflammation and neurologicalNi et al. Journal of Neuroinflammation(2024) 21:166

damage due to aberrant microglial activation is a distinctive feature of cognitive impairment and anxiety in
T2DM animals, wherein the proinflammatory activation of hippocampal microglia in T2DM has been suggested to exacerbate cognitive deficits and anxiety-likebehaviors through neuroinflammation [42]. Conversely,
inhibiting microglial activation significantly improvesdiabetes-associated cognitive deficits and anxiety-likebehaviors [42, 52]. Microglia have also been demonstrated to induce the release of excessive inflammatory
cytokines via the NF-κB pathway in AD mice, triggering
neuroinflammatory responses, synaptic and neural damage, and cognitive deficits [53]. All these study findings
indicate that abnormal microglial activation may have acentral regulatory role in various cognitive impairments
and emotional disorders associated with diseases such as
T2DM, and the inhibition of proinflammatory microglial activation may be a potential therapeutic target withbroad applications in the treatment of cognitive dysfunction and anxiety.
Additionally, our study highlighted that TREM2 wasupregulated in T2DM mice brains, consistent withprior findings. Earlier studies have shown that TREM2
overexpression improved spatial learning and memory,
upregulated synapse-related proteins to alleviate synaptic transmission and ultrastructure, and inhibited neuroinflammation in T2DM mice [24]. TREM2 has also
been demonstrated to attenuate neuroinflammation andneurological damage via Akt/CREB/BDNF signaling aswell as ameliorate cognitive impairment and anxiety in amouse model of traumatic brain injury [54]. The upregulation of TREM2 in the HFD mice brains may have been
a compensatory response to mitigate neurological injury.
Based on this notion, the increased exacerbation of anxiety in T2DM mice with comorbid OSA may be due to thedecrease in TREM2, which induces a further increase inproinflammatory microglia and additional neurologicaldestruction.
Our study proposes a novel mechanism of TREM2 neuroprotection and identifies for the first time a key regulatory role of TREM2 in the interferon pathway. Given that
IFN-I-mediated immune responses are pivotal in diabetes
development, IFN-I activation not only increases susceptibility to type I diabetes [55] but also exacerbates diabetes progression by initiating and enhancing inflammatory
responses [56]. Conversely, blocking IFNAR has beenshown to disrupt IFN-I signaling and inhibit the onset
and progression of diabetes mellitus [55, 57]. Our study
findings indicated that IFNAR signaling has a regulatoryrole in cognitive and anxiety behaviors in T2DM, suggesting that targeting IFNAR could ameliorate neurological injury in T2DM. Other studies have also reported that
the IFNAR1 pathway is involved in the development ofcognitive impairment and anxiety in several neurologicalPage 16 of 19

diseases [38, 58]. Thus, TREM2 upregulation or IFNAR1
inhibition may serve as an effective treatment for cognitive impairment in patients with various neurological or
non-neurological disorders.
IFN-I is a central coordinator of the immune responsewith antiviral, antitumor, and immunomodulatory properties. IFN-I also mediates the pathogenesis of inflammatory autoimmune diseases and infectious diseases
via the aberrant activation of inflammatory responses[59]. Based on the regulation of the IFNAR1 pathway by
TREM2, we hypothesize that TREM2 modulation may
serve as a therapeutic modality for autoimmune diseases.
Our study represents the first investigation of the interaction between the two membrane receptors TREM2 and
IFNAR1 as a potential mechanism of neuronal injury andprotection. Furthermore, we highlight a critical regulatory role of TREM2 in the interferon pathway, thereby
providing a new perspective on the anti-inflammatory
effects of TREM2 and offering novel ideas for developing interventions for neurodegenerative diseases. Lastly,
we propose a novel mechanism of anxiety exacerbation in
T2DM complicated with OSA and present valuable datafor improving the prevention and treatment strategies.
Microglial IFNAR1 is involved in the developmentof cognitive impairment and anxiety associated withmultiple diseases. In AD mice, long-term blockade of
microglial IFNAR1 reduces cell proliferation and inflammation, thereby attenuating synaptic damage and cognitive impairment [38]. As a downstream of IFNAR1,
STAT1 is involved in regulating synaptic plasticity andcognitive function, modulating microglia polarizationand inflammation. Downregulation of STAT1 attenuates atherosclerosis and cognitive impairment in T2DM
patients [60]. Microglia STAT1 deficiency significantly
reduces synaptic dysfunction and cognitive impairment
associated with Tau accumulation in AD mice [61]. The
above studies suggested an important role of microglial IFNAR1-STAT1 signaling in cognitive impairment.
The present study further suggests the interaction of
TREM2 and IFNAR1 signaling. Ruganzu et al. reported
that overexpression of TREM2 inhibits the JAK/STAT/SOCS pathway to reduce neuroinflammation and attenuate cognitive deficits in AD mice [62], which supports our
findings and underscores the potential significance of ourwork.
Due to the shared mechanisms among Type-1-diabetes, T2DM and AD, researchers proposed AD as “Type3-Diabetes”. Indeed, the association between T2DM and
AD is complex that both are interlinked with impaired
insulin and IGF actions, oxidative stress, mitochondrial
dysfunction, advanced glycation end products, lipid peroxidation, and inflammation [63]. As major symptoms of
OSA, disrupted sleep architecture, intermittent hypoxia
and oxidative stress, intrathoracic and hemodynamicNi et al. Journal of Neuroinflammation(2024) 21:166

changes, and cardiovascular comorbidities, increase
the risk for AD [64]. OSA increases the risk of dementia through the mediation of amyloid-β and downstream
Alzheimer’s disease pathology. Improving OSA may
downregulate amyloid-β load in the brain by affecting
both Aβ release and clearance [65]. TREM2 exerts antiinflammation and neuroprotection, and TREM2R47H
induces aberrant activation of cerebral microglia in AD
mice, exacerbating cognitive deficits [66]. Overexpression of TREM2 reduces activated microglia, Aβ deposition, synaptic and neuronal loss, neuroinflammation, andattenuates cognitive impairment [62]. Thus, the present
study reveals a mechanism by which TREM2 ameliorates
cognitive impairment by targeting IFN signaling, which
could have significant implications for AD research andprevention.

Conclusions
The results of the present study showed that HFD upregulated the IFNAR1-STAT1 pathway and induced proinflammatory microglia, leading to synaptic damage andcausing anxiety and cognitive deficits. TREM2 upregulation is a compensatory cellular response of the brains of
the T2DM mice. The upregulated TREM2 exerts a negative regulation of the IFNAR1-STAT1 pathway, which inturn attenuates neurological damage. Conversely, micewith T2DM combined with OSA may exacerbate anxiety
via the downregulation of TREM2, causing heightened
IFNAR1-STAT1 pathway activation and consequently
increasing proinflammatory microglia.
Abbreviations
BLA	Basolateral amygdalar
DEGs	Differentially Expressed Genes
HFD	High-fat diet
NCD	Normal control diet
HG	High-glucose
OFT	Open field test
EPM	Elevated plus maze
MWM	Morris water maze
IH	Intermittent hypoxia. αIFNAR: anti-IFNAR1 antibody
IFNAR	Interferon-α/β receptor
STAT1	Signal transducers and activators of transcription 1
TREM2	Triggering receptor expressed on myeloid cells-2
T2DM	Type 2 diabetes mellitus
OSA	Obstructive sleep apnea

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03160-1.
Supplementary Material 1
Supplementary Material 2
Acknowledgements
We thank Guijuan Chen and Yuqi Xie, for their preliminary studies in theintermittent hypoxia experiments. We thank Dr. Yapeng Lu, Dr. Dan Wang, andDr. Qianqian Luo for their advice on experimental design. We thank Bullet Edits
Limited for the linguistic editing and proofreading of the manuscript.

Page 17 of 19

Author contributions
Wenyu Ni and Yun Niu carried out experiments, organized the database, andperformed the statistical analysis. Sitong Cao and Xiaoyang Fan performed thestatistical analysis. Chunsun Fan and Jian Fan wrote sections of the manuscript.
Li Zhu contributed to the conception and design of the study and completely
revised the manuscript. Xueting Wang designed the study and wrote themanuscript. All authors participated in reading the manuscript and approved
the submitted version.
Funding
This work was supported by the National Natural Science Foundation of China[grant number 82271914], Science and Technology Project of Nantong City
[grant number JC12022021, JCZ2022077], Qidong Municipal Science andTechnology Project [grant number 2022-32-08] and Program of Nantong
Health Commission [grant number QN2023065].
Data availability
No datasets were generated or analysed during the current study.

Declarations
Ethical approval
All the study protocols of animal experiments were reviewed and approved by
the Animal Care and Use Committee of Nantong University and the Jiangsu
Province Animal Care Ethics Committee (approval ID: SYXK[SU]2007-0021).
Competing interests
The authors declare no competing interests.
Author details
1
Present address: Qidong People’s Hospital, Affiliated Qidong Hospital of
Nantong University, Qidong Liver Cancer Institute, No.9, Seyuan Road,
Chongchuan District, Nantong, Jiangsu 226000, China
2
Institute of Special Environmental Medicine, Co-Innovation Center of
Neuroregeneration, Nantong University, Nantong, China
3
Medical Research Center Affiliated Hospital 2 of Nantong University,
Nantong, China
Received: 20 March 2024 / Accepted: 24 June 2024

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