(2024) 21:176
Wang et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03166-9

Journal of Neuroinflammation

Open Access

RESEARCH

Herpes simplex virus 1 accelerates
the progression of Alzheimer’s diseaseby modulating microglial phagocytosis
and activating NLRP3 pathway
Zhimeng Wang1,2†, Jing Liu3†, Jing Han1,2, Tianyi Zhang4, Shangjin Li4, Yanfei Hou1, Huili Su1,
Fangping Han1 and Conggang Zhang1,2*

Abstract
Accumulating evidence implicates that herpes simplex virus type 1 (HSV-1) has been linked to the development
and progression of Alzheimer’s disease (AD). HSV-1 infection induces β-amyloid (Aβ) deposition in vitro and in vivo,
but the effect and precise mechanism remain elusive. Here, we show that HSV-1 infection of the brains of transgenic
5xFAD mice resulted in accelerated Aβ deposition, gliosis, and cognitive dysfunction. We demonstrate that HSV-1
infection induced the recruitment of microglia to the viral core to trigger microglial phagocytosis of HSV-GFP-positive
neuronal cells. In addition, we reveal that the NLRP3 inflammasome pathway induced by HSV-1 infection played a crucial role in Aβ deposition and the progression of AD caused by HSV-1 infection. Blockade of the NLRP3 inflammasome
signaling reduces Aβ deposition and alleviates cognitive decline in 5xFAD mice after HSV-1 infection. Our findings
support the notion that HSV-1 infection is a key factor in the etiology of AD, demonstrating that NLRP3 inflammasome activation functions in the interface of HSV-1 infection and Aβ deposition in AD.

†

Zhimeng Wang and Jing Liu contributed equally to this work.

*Correspondence:
Conggang Zhang
cgzhang@tsinghua.edu.cn
1School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084,
China
2
State Key Laboratory of Membrane Biology, Tsinghua‑Peking Center
for Life Sciences, Beijing Frontier Research Center of Biological Structure,
SXMU‑Tsinghua Collaborative Innovation Center for Frontier Medicine,
Tsinghua University, Beijing 100084, China
3
State Key Laboratory of Stem Cell and Reproductive Biology, Instituteof Zoology, Chinese Academy of Sciences, Beijing 100101, China
4
School of Pharmaceutical Sciences, IDG/McGovern Institute for Brain
Research, Tsinghua‑Peking Joint Center for Life Sciences, Tsinghua
University, Beijing 100084, China

Introduction
Alzheimer’s disease (AD) is the most common neurodegenerative disorder, resulting in progressive memory
loss and dementia. It exists in two forms: a genetic etiology of early-onset familial AD (fAD) caused by mutations of genes, such as APP, PSEN1 and PSEN2 genes;
and a sporadic form of late-onset AD (LOAD) caused
by a complex multifactorial etiology [1–3]. The deposition of senile plaques enriched with β-amyloid (Aβ)
peptides is a pathological hallmark of both fAD and
LOAD [4]. Aβ is primarily produced in neurons throughthe sequential processing of APP by β-secretase/
BACE1 and γ-secretase/Presenilin [5, 6]. Traditionally, Aβ has been identified as the primary cause of thedisease in fAD, known as the “Aβ cascade hypothesis”,
where increased production of toxic Aβ species initiates
a series of progressive changes that ultimately lead to© 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 theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, 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.

Wang et al. Journal of Neuroinflammation(2024) 21:176

neurodegeneration [7, 8]. However, the precise mechanism of Aβ production and clearance in LOAD is notclear.
Mounting evidence suggests that pathogens are
involved in the progression of AD pathogenesis [9–11].
The AD pathogen hypothesis suggests that viral or microbial infection of the central nervous system (CNS) may
act as triggers to induce a pathological cascade, leading toaccumulation of Aβ [12–14]. Several pathogens, including Chlamydophila pneumoniae [15], Borrelia spirochetes[16], Candida glabrata [17], human herpesviruses 6 and
7 (HHV6 and HHV7) [13] and herpes simplex virus 1(HSV-1) [11, 18, 19], have been linked to LOAD. Amongthese pathogens, HSV-1 has emerged as one of the leading pathogens and has been proposed as a potential risk
factor in AD pathogenesis [20, 21]. Independent cohort
studies have revealed that HSV-1 is strongly associatedwith neurodegenerative diseases, especially AD [20,22]. It has been established that HSV-1 is present in thebrains of a high proportion of elderly normal subjects
and Alzheimer’s disease (AD) patients [23, 24].
HSV-1 is a neurotropic double-strand DNA virus that
establishes a latent infection in the sensory neurons oftrigeminal ganglia of humans [25]. HSV-1 brain infection
leads to the activation of glial cells, especially microglia
cells [26]. Microglia cells, the resident innate immune
cells in the central nervous system, play essential roles
in healthy and neurodegenerative diseases [27]. In physiological conditions, resting microglia have ramified processes that constantly survey the microenvironment,
assisting neuronal development, synaptic pruning andsupport neuronal survival [28, 29]. In response to pathogen infections or neurodegenerative disorders, reactive
microglia mediate phagocytic uptake and the secretionof inflammatory cytokines and chemokines [30–32].
HSV-1 infection activates the cGAS-STING signaling
pathway and NLRP3 inflammasome pathway in microglia
to fight against viral invsion [33–36]. In the other hands,
the pathogen-mediated innate immune response also
could cause neuroinflammation, which is an important
hallmark of neurodegenerative diseases. However, themechanism of microglia activation and innate immune
signaling in AD pathology upon HSV-1 infection remains
largely unclear. Therefore, a comprehensive study of
microglia, Aβ deposition, and neuroinflammation after
HSV-1 infection might deepen our understanding of AD.
In this study, we administrated HSV-1-GFP into 5xFAD
mice and investigated the effects of HSV-1 infection
on Aβ deposition and cognitive function. The results
revealed that HSV-1 infection led to Aβ deposition andcognitive deficits in 5xFAD mice. Following HSV-1 infection, microglia were recruited to the viral core but not
Aβ deposition, enhancing their phagocytic uptake ofPage 2 of 24

viruses, thereby leading to the accumulation and deposition of Aβ. Moreover, the NLRP3 inflammasome signaling was activated after HSV-1 infection and drove
the accumulation of Aβ aggregates. Administration of
MCC950 sodium, a selective small-molecular inhibitorof the NLRP3 inflammasome, reduced Aβ deposition
and ameliorated cognitive decline in 5xFAD mice following HSV-1 infection. Together, our findings support theconcept that HSV-1 infection and NLRP3 inflammasome
pathway are connected to the development and progression of AD pathology.

Materials and methods
Transgenic mice and mouse infection model

The 5xFAD transgenic mice (B6SJL-Tg (APPSwFlLon,
PSEN1*M146L*L286V) 6799Vas/Mmjax, 34840-JAX)
were originally obtained from the Jackson laboratory andmaintained on the C57BL/6 background. For behavioral tests, 3-month-old male 5xFAD mice were used. For
other in vivo experiments, age and sex-matched mice
were used. Mice were housed with free access to waterand food under a 12 h/12 h light–dark cycle. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Tsinghua
University.
For behavioral tests, 3-month-old male 5xFAD micereceived bilateral injections of HSV-1-GFP viron suspension at coordinates AP, − 2.0; ML, ± 1.5; DV, − 2.0. This
injection was carried out using a 5 μL syringe equippedwith a 30-gauge needle attached to a digital stereotaxic
apparatus and an infusion pump, administrated at a rateof 0.2 μL/min. Following the completion of each injection, the needle was retained in place for 5 min before
gradual withdrawal. 21 days after the injections, the mice
were subjected to a battery of behavioral tests.
In the case of other in vivo experiments, we employed
the approach involved by Eimer and coauthors [13]. In
brief, age and sex-matched 5xFAD mice received unilateral injections of HSV-1-GFP viron suspension at
the coordinates AP, − 2.0; ML, − 1.5; DV, − 2.0 (rightside) using a 5 μL syringe fitted with a 30-gauge needle
attached to a digital stereotaxic apparatus and an infusion pump, administrated at a rate of 0.2 μL/min. Sterile
PBS was injected into the contralateral side of the brain
as controls (left side). Subsequently, the mice were either
perfused for immunostaining or euthanized for western
blotting at the indicated time points.
Cell culture

BV2 cells, HT22 cells and Vero cells were cultured in
Dulbecco’s modified Eagle medium (DMEM) (Gibco)
supplemented with 10% (v/v) fetal bovine serum(FBS) (Gemini), 100 U/ml penicillin and 100 μg/ml

Wang et al. Journal of Neuroinflammation(2024) 21:176

streptomycin. All these cells are grown at 37 °C in ahumidified atmosphere containing 5% ­CO2.
Primary microglial cells were derived from P0-P5 WT
mice as previously described [4, 37]. Briefly, neonatal
cortex was collected, trypsinized for 10 min, and filtered through a 70 μm filter. Cells were then cultured in
DMEM medium containing 10% FBS and 1% penicillin/
streptomycin for 2–3 weeks. The medium was replacedwith fresh cell culture medium every three days. The
microglia were isolated by shaking (200–220 rpm, 3 h)after 2–3 weeks of primary cultivation. The medium
containing detached microglia was collected and seeded
into PDL-coated 12-well or 24-well plates. After the cells
attached, the medium was replaced with fresh cell culture
medium.
Virus production

Herpes simplex virus 1-GFP (HSV-1-GFP, KOS Strain,
generously provided by Dr. Daxing Gao, University of
Science and Technology of China), was employed in
the innate immune studies [38]. The virus was amplified in Vero cells as described in previous paper [39, 40].
The supernatant containing the virus was harvested andcentrifuged at 5000 rpm/4 °C. The viral suspension was
filtered through a 0.22 μm filter and purified by ultracentrifugation as 20,000 rpm/2 h. The concentrated virus
was aliquoted and stored at − 80 °C.
Immunofluorescence

Mice were anesthetized with 5% chloral hydrate andperfused with cold PBS, followed by 4% paraformaldehyde (Aladdin, PH 7.4). Subsequently, mouse brains
were harvested and coronally sectioned into 40 μm-thickserial sections. The brain sections were then washedthree times with PBS and blocked for 2 h with a blocking buffer (3% BSA containing 0.1% Triton X-100) at
room temperature, followed by overnight incubation
with primary antibodies against MOAB2 (1:1000, Cat #ab126649, Abcam), Iba1 (1:1000, Ca t# 019-19741, Wako,
PRID: AB_839504), GFAP (1:1000, Cat # 13-0300, Invitrogen, PRID: AB_86543), CD3 (1:1000, Cat #ab135372,
PRID: AB_2884903), F4/80 (1:500, Cat #ab300421,
PRID: AB_2936298), ASC (1:500, Cat #AG-25B-0006,
Adipogen, PRID: AB_2490440), CD68 (1:1000, Cat#ab53444, Abcam, PRID: AB_869007), NLRP3 (1:1000,
Cat #AG-20B-0014, Adipogen, PRID: AB_2490202) at
4 °C. After washing, the sections were labeled with fluorescent secondary antibodies conjugated to Alexa Fluor
568/594/647 in blocking buffer containing DAPI (Invitrogen). The slides were then observed with a fluorescencemicroscopy (ZEISS LSM780 microscopy or OLYMPUS
FV3000RS-BX microscopy).

Page 3 of 24

For the Aβ and HSV-GFP phagocytosis in vitro assay,
HT22 cells were infected with HSV-1-GFP for 24 h,then washed and suspended with PBS. BV2 and primary microglial cells were incubated with 1 μg/ml
Alexa 555-labeled Aβ1-42 (Anaspec), or 1 μg/ml Alexa555-labeled Aβ1-42 plus HSV-1-GFP from HT-22 neuronal cells for 4 h. Subsequently, cells were fixed with 4%paraformaldehyde for 20 min at room temperature andstained with DAPI. The cells were then imaged with the
Zeiss LSM780.
Thioflavin S staining

Thioflavin S staining was used to label the Aβ plaques.
Briefly, brain sections were stained with 0.1% Thioflavin S(Thio-S, sigma) in the dark for 8 min in 50% ethanol, followed by two washes with 50% ethanol and three washes
with PBS. Subsequently, the sections were mounted for
imaging.
Microscopy and image analysis

All imaging was performed using an OLYMPUS
FV3000RS-BX and ZEISS 780 Confocal laser scanningmicroscope with × 4, × 10, × 20, × 40 and × 63 objectives. For Aβ plaques quantification, ten views fromthe hippocampus of five mice and the entire brain wereassessed. For microglia, astrocyte and inflammasome
markers quantification, Iba1-, GFAP-, CD68-, ASC-, andNLRP3-positive cells were counted and then calculated
as the ­Iba1+, ­GFAP+, ­CD68+, ­ASC+ and ­NLRP3+ cells
divided by DAPI cells. To assess Aβ plaque and HSV-GFP
phagocytosis by microglia cells, confocal single-plane
images of lysosomes (CD68), Aβ and microglia (Iba1)
were separately isolated, and the areas of colocalization­(CD68+Aβ+GFP+ or ­Iba1+Aβ+GFP+) were measured by
ImageJ software. Image processing and analysis were performed by Image J software or Imaris software (Bitplane,
Switzerland), as appropriate. Imaris were used for 3D
reconstruction of confocal images to analyze microglia
morphology and colocalization of Aβ plaque with CD68,
GFP with CD68, Aβ plaque with Iba1, and GFP with Iba1.
Immunoblotting

Mouse brain tissues were homogenized and lysed in
RIPA lysis buffer (P0013B, Beyotime) supplemented
with PMSF (ST505, Beyotime) on ice for 30 min. Subsequently, the lysates were separated on SDS-PAGE gelsand transferred to PVDF membranes. The membranes
were blocked in 5% skim milk in TBS-T for 2 h and incubated with specific primary antibodies against Caspase1(p20) (1:1000, Cat #AG-20B-0042, Adipogen, PRID:
AB_2490248), beta Amyloid (APP, 1:1000, Cat #51-2700,
Thermo Fisher, PRID: AB_2533902), presenilin1/PS-1(1:1000, Cat #ab76083, Abcam, PRID: AB_1310605),

Wang et al. Journal of Neuroinflammation(2024) 21:176

ADAM10 (1:1000, Cat #ab124695, Abcam, PRID:
AB_10972023), BACE1 (1:1000, Cat #ab2077, Abcam,
PRID: AB_302817), p-TBK1 S­ er172 (D52C2) (1:1500, Cat#5483, Cell Signaling Technology, PRID: AB_10693472),
TBK1/NAK (1:1500, Cat #3504, Cell Signaling Technology, PRID: AB_2255663), p-STING S­ er366 (1:1500, Cat#85735, Cell Signaling Technology, PRID: AB_2732796),
STING (D2P2F) (1:1000, Cat #13647, Cell Signaling
Technology, PRID: AB_2732796), GAPDH (14C10)(1:3000, Cat #2118, Cell Signaling Technology, PRID:
AB_561053) overnight at 4 °C. After three washes with
TBS-T, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature.
Immunoreactive bands were imaged using the Automatic
Chemiluminescence Imaging System (Tanon 5200).
RNA isolation and quantitative real‑time PCR (qRT‑PCR)
5

PBS or HSV-GFP (1 × ­10 PFUs/hippocampus) was
administered into 3-month-old 5xFAD mice. Three andseven days after HSV-1 infection, control and HSV1-infected hippocampi from the same mice were collected and homogenized in TRIZOL reagent to isolate
total RNA. Subsequently, cDNA was reverse transcribedusing the One-Step gDNA Removal and cDNA Synthesis
Kit (Mei5Bio). Relative mRNA expression of IL6, IL-1β,
TNFα, IFNβ, IFNγ, IL10, and CCL5 was determined byquantitative real-time PCR.
Real-time PCR was performed using SYBR Green
Master Mix with the following primers: IL6 (Fp: 5′-TAC​
CAC​TTC​ACA​AGT​CGG​AGGC-3′; RP: 5′-CTG​CAA​GTG​CAT​CAT​CGT​TGTTC-3′), IL-1β (Fp: 5′-TGG​ACC​
TTC​CAG​GAT​GAG​GACA-3′; Rp: 5′-GTT​CAT​CTC​
GGA​GCC​TGT​AGTG-3′), TNFα (Fp: 5′-GGT​GCC​TAT​
GTC​TCA​GCC​TCTT-3′; Rp: 5′-GCC​ATA​GAA​CTG​
ATG​AGA​GGGAG-3′), IFNβ (5′-GCC​TTT​GCC​ATC​
CAA​GAG​ATGC-3′ Rp: 5′-ACA​CTG​TCT​GCT​GGT​
GGA​GTTC-3′), IFNγ (Fp: 5′-CAG​CAA​CAG​CAA​GGC​
GAA​AAAGG-3′; Rp: 5′-TTT​CCG​CTT​CCT​GAG​GCT​
GGAT-3′), IL10 (Fp: 5′-CGG​GAA​GAC​AAT​AAC​TGC​
ACCC-3′; Rp: 5′-CGG​TTA​GCA​GTA​TGT​TGT​CCAGC3′), CCL5 (Fp: 5′-CCT​GCT​GCT​TTG​CCT​ACC​TCTC3′; Rp: 5′-ACA​CAC​TTG​GCG​GTT​CCT​TCGA-3′) and
β-actin (Fp: 5′-CAT​TGC​TGA​CAG​GAT​GCA​GAAGG3′; Rp: 5′- TGC​TGG​AAG​GTG​GAC​AGT​GAGG-3′).
The expression levels of genes were normalized to β-actin
and quantified by the ΔΔCT method.
Behavioral tests

Mice were housed in groups of 3–5 animals on a 12:12 hlight–dark cycle. 3-month-old male 5xFAD mice, whichwere infected with sterile PBS or HSV-1-GFP, were used
for all the behavioral tests. Videos were recorded andPage 4 of 24

analyzed by the Smart V3.0.03 software (Panlab, Barcelona, Spain).
Y‑maze test

Y-maze spontaneous alteration test was carried out toassess short-term spatial memory. Mice were allowed toacclimate to the testing room for 48 h. The Y-maze apparatus comprised three opaque arms at 120° angles fromeach other. Each mouse was gently placed in the distal
part of the maze and allowed to explore freely for 5 min.
Detailed recordings were made of their movements andthe total number of arms entered. Spontaneous Alteration [%] was defined as the ratio of consecutive entries
into 3 different arms divided by the total arm entries
minus 2.
Morris water maze test

A circular water tank (diameter: 120 cm) was filled withwater to a depth of 25 cm, and the water was madeopaque with non-toxic white paint. A 13-cm in diameterround platform was hidden 1 cm beneath the surface of
the water at the center of a given quadrant of the watertank. Mice received training for five consecutive days,
with each session comprising four trails starting from different sites. For each trail, the mouse was gently released
from the wall of the tank and allowed to explore, locateand stand on the platform for a duration of 20 s, within
the 60-s trail period. Following the completion of training, a probe test was performed 24 h later. During theprobe test, the platform was removed and task performance including swimming tracks, speed, time spent andentries into the platform were recorded for analysis.
Flow cytometry

For the Aβ and HSV-GFP phagocytosis assay, HT22 cells
were infected with HSV-1-GFP for 24 h, then washed andsuspended with PBS. BV2 and primary microglial cells
were incubated with 1 μg/ml Alexa 555-labeled Aβ1-42, or
1 μg/ml Alexa 555-labeled Aβ1-42 plus HSV-1-GFP fromHT-22 neuronal cells for 4 h. Subsequently, cells were
harvested and analyzed using a FACSAria (BD Bioscience). These experiments were repeated three times andanalyzed using FlowJo v10.4 (Tree Star).
Drug treatment

PLX3397 (Selleck, Cat# S7818) was employed for thepharmacological depletion of brain microglia cells.
Briefly, mice were fed with PLX3397-formulated AIN76A diet (290 mg/kg) or normal AIN-76A diet ad libitum[41], and the treatment continued until the mice wereeuthanized.
MCC950 sodium (MedChemExpress, MCE, Cat# HY12815A) is a selective NLRP3 inhibitor. Mice received

Wang et al. Journal of Neuroinflammation(2024) 21:176

MCC950 (10 mg/kg, dissolved in PBS with 2% DMSO)
or vehicle (PBS with 2% DMSO) via intraperitoneal injection every second day after HSV-1 infection for 21 days.
ELISA assay

PBS or HSV-GFP (1 × ­105 PFUs/hippocampus) was
injected into 1-month-old, 3-month-old, and 6-monthold 5xFAD mice. Seven days later, hippocampi werecollected, homogenized, and diluted in PBS. The concentration of IL-1β was assessed using ELISA kits according
to the manufacturer’s instructions (EK0394, BOSTER
Biological Technology).
Statistical analysis

All statistical analyses were performed using Prism 8.0(GraphPad Software). Datasets were analyzed for p-values using either unpaired Student’s two-tailed t tests or
ANOVA multiple comparison post hoc tests; all data arepresented as means ± SEM. Statistical significance wasrepresented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001,****p ≤ 0.0001, n.s.: p > 0.05.

Results
HSV‑1 infection exacerbates Aβ plaque depositionand cognitive decline

To investigate the correlation between HSV infection
and Aβ plaque deposition, we administrated HSV1-GFP virus into the hippocampus of 3-month-old
transgenic AD (5xFAD) mice, using phosphate-buffered saline (PBS) as a vehicle control (Fig. 1A). Sevendays after stereotaxic injection of HSV-1-GFP, resultsof Aβ immunostaining revealed a dramatic increaseof amyloid plaques in the whole brain (Fig. 1B and C).
Thio-S staining of brain slices confirmed a significant
increase in both the number and size of Aβ plaques in
3-month-old 5xFAD mice infected with HSV-GFP compared to those injected with PBS (Fig. 1D and E). The

Page 5 of 24

Aβ protein amounts in the hippocampus of 3-monthold 5xFAD-HSV mice were notably higher compared to
3-month-old 5xFAD-Ctrl mice (Fig. 1F). Accumulationand deposition of Aβ may trigger neurotoxic effects,
neuronal loss, and degeneration, ultimately leading
to cognitive impairments. To assess the detrimental
effects of HSV-1 infection on amyloid deposition andits association with cognitive dysfunction in 5xFAD
mice, we performed Y-maze and Morris water-maze
tests in 3-month-old 5xFAD mice following virus injection. At 3 months of age, 5xFAD mice did not exhibit
significant deficits in spatial learning and memory(Fig. 1G–I). However, HSV-1 infection resulted in significant impairments in spatial learning and memory,
characterized by reduced spontaneous alterations without a decrease in total arm entries (Fig. 1G), prolonged
escape latency time during the training trails and theprobe trail, and a lower number of platform crossings(Fig. 1H and I). Swimming speeds were comparable
across all groups of animals, thus excluding impairments in motor function (Fig. 1I).
To further confirm that HSV-1 facilitate Aβ accumulation, we also administrated HSV-1-GFP virus into the
hippocampus of 1-month-old and 6-month-old 5xFAD
mice and observed a significant increase in the number and size of Aβ plaques in 1-month-old 5xFAD-HSV
mice (Fig. 2A and B) and 6-month-old 5xFAD-HSV mice
(Fig. 2C and D). Of note, Aβ deposition was alreadyclearly detectable in 6-month-old 5xFAD-Ctrl mice,
whereas the density and signal intensity of Aβ plaques
were stronger in 6-month-old 5xFAD-HSV mice (Fig. 2E
and F). The Aβ protein amounts in the hippocampus
also increased in both 1-month-old 5xFAD-HSV mice
(Fig. 2G) and 6-month-old 5xFAD-HSV mice (Fig. 2H).
Together, these data indicate that HSV-1 infection can
exacerbate β-amyloid deposition and contribute to spatial and learning memory deficits in 5xFAD mice.

(See figure on next page.)
Fig. 1 HSV-1 infection exacerbates Aβ deposition and cognitive decline in 5xFAD mice. A Three-month-old 5xFAD mice were injected with 1 × ­105
PFUs of HSV-GFP or PBS in the hippocampus of the brain. B Representative images of Aβ (MAOB2, red) in the whole brain of 3-month-old 5xFAD
mice seven days after HSV-GFP infection. Scale bars, 200 μm, 100 μm and 50 μm. C Quantification of Aβ plaques and plaque area in the brainof 3-month-old 5xFAD mice infected with PBS or HSV-GFP. (n = 5 mice per group). D Representative images of Thioflavin S staining (gray)in the whole brain of 3-month-old 5xFAD mice seven days after HSV-GFP infection. Scale bars, 200 μm, 100 μm and 50 μm. E Quantification of Aβplaques and plaque area in the brain of 3-month-old 5xFAD mice infected with PBS or HSV-GFP. (n = 5 mice per group). F Three-month-old 5xFAD
mice were randomly divided to three groups: control group (no treatment); Vehicle group (PBS treatment) and HSV-GFP group. Mouse hippocampi
were collected and homogenized for immunoblotting of Aβ seven days post-infection. The intensities of Aβ were quantified by ImageJ software. G
Three-month-old 5xFAD mice were randomly divided to two groups: control group (PBS treatment) and HSV-GFP group. Y-maze test was performed
to measure the total number of arm entries and spontaneous alteration (%) at 21 days post-infection. (n = 9 mice per group). H, I Morris water maze
was performed to evaluate the spatial learning and memory of 5xFAD mice infected with HSV-GFP. During the training phase, each group showed
improved latency to the platform, but compared with the control group, the HSV-GFP group exhibited a significant delay to locate the platform (H).
In the probe test, there was no significant difference in swimming speeds between the two groups of the mice. Compared to the control group,
the 5xFAD-HSV group exhibited a significantly longer latency to locate the platform, but fewer target crossings (I). (n = 9 mice per group). Data arepresented as means ± SEM. n.s.: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

Wang et al. Journal of Neuroinflammation

Fig. 1 (See legend on previous page.)(2024) 21:176

Page 6 of 24

Wang et al. Journal of Neuroinflammation(2024) 21:176

HSV‑1 infection promotes microglia cell activationin 5xFAD mice

The proliferation and activation of glial cells are prominent features of virus infection [42, 43]. Iba1 and GFAP
are widely used as immunohistochemical markers for
microglia and astrocytes, and their expression increases
upon glial cell activation. Indeed, we observed increasing expression of Iba1 and GFAP in the hippocampus
of 5xFAD mice infected with HSV-1, regardless of theirage (Fig. 3). Interestingly, we observed distinct microglia morphology that may represent different activation
states following HSV-1 infection. In the viral core, microglia often displayed an activated “rounded” morphology,
while in the periphery of the viral core, microglia displayed an activated “ameboid” morphology characterized by shorter processes, larger cell bodies and reduced
microglia volumes (Fig. 4A). In contrast, microglia exhibited a ramified morphology in 5xFAD-Ctrl mice (Fig. 4B
and C). Collectively, our data suggest that HSV-1 infection enhances gliosis and microglial activation in 5xFAD
mice.
HSV‑1 infection induces enhanced microglial phagocytosisof HSV‑GFP‑positive neuronal cells

The production and aggregation of Aβ are attributed tothe imbalance between Aβ production and clearance[44]. Having observed significant Aβ plaque depositionin 5xFAD mice after HSV-1 infection, we next investigated whether APP processing was involved in Aβ production. To test this hypothesis, we examined a series ofkey factors involved in Aβ production. In comparison to
5xFAD control mice, there was no significant change inthe expression levels of APP, PS1, or the APP processing
secretases ADAM10 and BACE1 in the HSV-infected
5xFAD mice (Figure S1). These results demonstrate that
HSV-1 infection exacerbates Aβ burden without altering
APP expression and processing, both in young and aged
5xFAD mice.
Given the significant accumulation of Aβ plaques withunchanged Aβ production in 5xFAD mice after HSV-1
infection, we next sought to explore whether HSV-1

Page 7 of 24

infection affects Aβ clearance. Since microglia play a
crucial role in phagocytizing and degrading extracellular Aβ in the brain of AD mice, we then performed
immunostaining assay using anti-Aβ antibody along with
anti-Iba1 antibody to visualize the colocalization of Aβand microglia. Confocal images showed that microglia
clustered around the Aβ plaques in 5xFAD-Ctrl mice
(Fig. 5A). However, we observed a significant decreasein the number of Iba1 + microglia near Aβ plaques anda significant increase in the number of Iba1 + microglia
near the viral core in 5xFAD mice following HSV-1 infection (Fig. 5A and B).
To further elucidate whether the enhanced recruitment of microglia around the viral core affects Aβengulfment and clearance, we performed immunostaining of CD68, a phagocytic marker for microglia. Compared with the control group, HSV-1 infection markedly
upregulated CD68 expression in all age groups of 5xFAD
mice (Fig. 5C and D, Figure S2A and S2B). Quantitative
analysis of the colocalization area of CD68 with Aβ or
HSV-GFP revealed a significant increase in G
­ FP+CD68++
microglia and a significant decrease in Aβ CD68+ microglia in 5xFAD-HSV mice (Fig. 5E–G, Figure S2C), Thissuggests that microglia preferentially phagocytize HSVGFP-positive neuronal cells after HSV-1 infection, but
not Aβ plaques, leading to Aβ accumulation in 5xFADHSV mice. To further characterize this microglia-HSV-1
module, we examined the effects of HSV-1 infection on
Aβ uptake in cultured BV2 and primary microglial cellsby flow cytometry. BV2 and primary microglial cells
were incubated with Alexa 555-labeled Aβ1-42 or Alexa555-labeled Aβ1-42 plus HSV-1-GFP from HT-22 neuronal cells for 4 h. Consistent with our in vivo data, weobserved a decrease in Aβ1-42 uptake and an increase in
GFP-positive phagocytosis upon incubation with GFPpositive cell debris (Fig. 6A, B, Figure S3A-B). In addition,
we confirmed this phenomenon using an immunostaining assay, which consistently showed that BV2 andprimary microglial cells preferred to phagocytize GFPpositive cell debris (Fig. 6C–E, Figure S3C-D). Together,
these results suggest that HSV-1 infection enhances the(See figure on next page.)
Fig. 2 HSV-1 infection exacerbates Aβ deposition in 1-month-old and 6-month-old 5xFAD mice. A and B Representative images (A)and quantification of Aβ plaques and plaque area (B) in the whole brain and hippocampus of 1-month-old 5xFAD mice seven days after HSV-GFP
infection. Scale bars, 200 μm, 100 μm and 50 μm. (n = 5 mice per group). C and D Representative images (C) and quantification of Aβ plaquesand plaque area (D) in the whole brain of 6-month-old 5xFAD mice seven days after HSV-GFP infection. Scale bars, 200 μm, 100 μm and 50 μm.
(n = 5 mice per group). E Representative images (E) and quantification of Aβ plaques and plaque area (F) in the whole brain of 6-month-old 5xFAD
mice seven days after HSV-GFP infection. Scale bars, 200 μm, 100 μm and 50 μm. (n = 5 mice per group). G Immunoblotting and quantificationof Aβ protein levels in the hippocampus of 1-month-old 5xFAD mice (Ctrl), 5xFAD mice infected with PBS (Vehicle) or 5xFAD mice seven days
after HSV-GFP infection. (n = 3 mice per group). H Immunoblotting and quantification of Aβ protein levels in the hippocampus of 6-month-old
5xFAD mice (Ctrl), 5xFAD mice infected with PBS (Vehicle) or 5xFAD mice seven days after HSV-GFP infection. (n = 3 mice per group). All data arepresented as means ± SEM. n.s.: p > 0.05, *p ≤ 0.05, **p ≤ 0.01

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Wang et al. Journal of Neuroinflammation(2024) 21:176

recruitment of microglia to the viral core and decreases
their phagocytic uptake of Aβ plaques, consequently
leading to Aβ accumulation and deposition both in vivo
and in vitro.
Microglia are indispensable to control Aβ plaqueaggregation after HSV‑1 infection

Our results indicated that microglia may be not able toperform normal Aβ clearance following HSV-1 infection. To further confirm the role of microglia in Aβ
pathology, we employed the CSF1R inhibitor, PLX3397,
to deplete microglia [45, 46]. 5xFAD mice were fed with
PLX3397-formulated diet for 14 days and then injectedwith PBS or HSV-GFP for another 7 days (Fig. 7A). After
PLX3397 treatment, both microglia numbers and theirphagocytic ability, as assessed by Iba1 and CD68 staining, were substantially reduced in both 5xFAD-Ctrl
mice and 5xFAD-HSV mice (Fig. 7B–E, Figure S4). To
determine if microglia are responsible for HSV-1-mediated plaque aggregation, we depleted microglia with
PLX3397, followed by the administration of HSV-1 tothe hippocampus. Consistent with the previous findings,
treatment of 5xFAD mice with PLX3397 led to a reduction in Aβ plaque deposition (Fig. 7F, G and Figure S5).
However, compared to 5xFAD-HSV mice in the absenceof PLX3397 treatment, the plaque load was markedly
increased in 5xFAD-HSV mice treated with PLX3397(Fig. 7F, G, Figure S5), indicating that microglia play an
indispensable role in controlling HSV-1-mediated Aβplaque aggregation. Furthermore, we examined the protein amounts of Aβ in 5xFAD mice treated with PLX3397
and injected with HSV-1 and found that the amountsof Aβ were significantly increased in 5xFAD-HSV micetreated with PLX3397 (Fig. 7H, I). Together, these results
indicate that microglia play an indispensable role inmaintain the homeostasis of HSV-1-GFP and Aβ plaqueaggregation in 5xFAD-HSV mice.
NLRP3 inflammasome is activated after HSV‑1 infection
in 5xFAD mice

Our data suggested that depletion of microglia did notallow to establish a precise role for Aβ deposition andPage 9 of 24

HSV-1 infection in AD pathogenesis. Neuroinflammation, an important hallmark of AD, is associated withAβ deposition and pathogen invasion [47–49]. To further define the regulation mechanism of HSV-1 and Aβ,
we characterized the innate immune signaling pathways
induced by HSV-1. Recent reports support the hypothesis that cGAS-STING pathway or NLRP3 inflammasome signaling may act as a driver of neuroinflammationin the brain [50, 51]. To investigate whether the cGASSTING pathway or the NLRP3 inflammasome pathway is
involved in HSV-1-mediated AD pathology, we injected
HSV-1 virus into the brains of 5xFAD mice for the indicated time periods and found that HSV-1 infection promotes Aβ accumulation at day 3 and day 7 (Fig. 8A andB), consistent with our findings in Fig. 1.
Next, we examined the protein markers of signalingcascades of NLRP3 inflammasome and the cGAS-STING
pathway in the hippocampus of 5xFAD mice for the indicated time periods. We observed that phosphorylationlevels of TBK1 and STING were upregulated in the earlystages (6 h–24 h) following HSV-1 infection (Fig. 8C and
D). Additionally, NLRP3 and cleaved-caspase1 protein
expression levels were upregulated on day 3 and day 7
after HSV-1 infection (Fig. 8C and D). Further immunostaining assay confirmed that the NLRP3 inflammasome was activated in the hippocampus of 3-month-old
5xFAD mice at day 7 after HSV-1 infection (Fig. 8E). Thisphenomenon was also observed in 1-month-old 5xFAD
mice and 6-month-old 5xFAD mice at day 7 after HSV-1
infection (Figure S6). Subsequently, IL-1β production
was also assessed by ELISA assay in 1-, 3-, and 6-monthold 5xFAD mice infected with PBS or HSV-GFP. We
observed increased IL-1β levels in 1-, 3-, and 6-monthold 5xFAD mice after HSV-1 infection (Figure S6C, S6E
and S6H).
We then performed immunofluorescence double
staining of NLRP3 along with the microglia marker
Iba1, astrocyte marker GFAP, and observed that NLRP3
is mostly expressed in microglia and to a lesser extent
in GFP-positive neuronal cells (Fig. 8F and Figure S7AB). In addition, we detected significantly more ASC
specks in 5xFAD mice after HSV-1 infection (Fig. 8G),

(See figure on next page.)
Fig. 3 HSV-1 infection leads to gliosis in 5xFAD mice. A Representative images of microglia (Iba1, red) and astrocytes (GFAP, magenta)
in the hippocampus of 1-month-old 5xFAD mice seven days after HSV-GFP infection. Scale bars, 20 μm. B Quantification of ­Iba1+ microglia
and ­GFAP+ astrocytes in the hippocampus of 1-month-old 5xFAD mice seven days after HSV-GFP infection. (n = 5 mice per group). C Representative
images of microglia (Iba1, red) and astrocytes (GFAP, magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP
infection. Scale bars, 20 μm. D Quantification of ­Iba1+ microglia and ­GFAP+ astrocytes in the hippocampus of 3-month-old 5xFAD mice seven days
after HSV-GFP infection. (n = 5 mice per group). E Representative images of microglia (Iba1, red) and astrocytes (GFAP, magenta) in the hippocampusof 6-month-old 5xFAD mice seven days after HSV-GFP infection. Scale bars, 20 μm. F Quantification of ­Iba1+ microglia and ­GFAP+ astrocytes
in the hippocampus of 6-month-old 5xFAD mice seven days after HSV-GFP infection. (n = 5 mice per group). All data are presented as means ± SEM.
*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001

Wang et al. Journal of Neuroinflammation(2024) 21:176

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Fig. 3 (See legend on previous page.)primarily expressed in microglia (Figure S7C) and
responding to Aβ accumulation after HSV-1 infection(Figure S8). These results indicate that the expression
pattern of the NLRP3 inflammasome is consistent withAβ accumulation. Together, our data show that the
NLRP3 inflammasome is activated after HSV-1 infection, and its expression trend is consistent with Aβ
accumulation.

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Fig. 4 HSV-1 infection promotes microglia activation in the viral core in 5xFAD mice. A Representative images of ­Iba1+ microglia in the coreand periphery of hippocampus seven days after HSV-GFP infection. Dashed-white frames are magnified to illustrate the representative morphologyof microglia. Original magnification × 40, scale bars; 20 μm. Zoom-in images with a scale bar equal to 10 μm. B Three-dimensional reconstructionof ­Iba1+ microglia in the core and periphery of the hippocampus seven days after HSV-GFP infection. Scale bars, 5 μm. C and D Quantificationof microglial morphology (C) and processes (D) in the 5xFAD mice seven days after HSV-GFP infection. (n = 5 mice per group). All data are presented
as means ± SEM. ****p ≤ 0.0001(See figure on next page.)
Fig. 5 HSV-1 infection induces enhanced microglial phagocytosis of HSV-GFP-positive neuronal cells. A Representative images of microglia(Iba1, gray) and Aβ plaques (magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection. Right panels show
three-dimensional reconstructed enlarged images of yellow dotted boxes, illustrating engulfed GFP-positive cells or Aβ plaques by microglia. Scalebars, 10 μm and 3 μm. B Quantification of engulfed Aβ plaques or HSV-GFP-positive cells by microglia, indicated by the co-staining of Aβ and Iba1
(Aβ+Iba1+) or GFP and Iba1 ­(GFP+Iba1+). (n = 4 mice per group). C and D Representative images (C) and quantification (D) of CD68-positive cells
in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection. Original magnification × 40, scale bars; 20 μm. Zoom-in images
with a scale bar equal to 10 μm. (n = 5 mice per group). E Representative images of phagocytic microglia (CD68, magenta) co-stained with Aβ(red) in the hippocampus of 3-month-old 5xFAD mice (Ctrl) or 5xFAD mice infected with HSV-GFP (HSV). Original magnification × 63, scale bars;
10 μm. Zoom-in images with a scale bar equal to 5 μm. F Representative images of phagocytic microglia (CD68, blue) co-stained with Aβ plaques(magenta) in the core of the hippocampus infected with PBS or HSV-GFP. Three-dimensional reconstructed enlarged images of dashed-whiteor dashed-yellow frames show engulfed GFP-positive cells or Aβ plaques in the lysosomes (blue). Original magnification × 63, scale bars; 10 μm.
Zoom-in images with a scale bar equal to 5 μm and 3 μm. G Quantification of engulfed GFP-positive cells or Aβ plaques in F. (n = 4 mice per group).
All data are presented as means ± SEM. ***p ≤ 0.001, ****p ≤ 0.0001

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Fig. 6 HSV-1 infection induces enhanced microglial phagocytosis of HSV-GFP-positive neuronal cells in vitro. A Representative FACS dot plotsof engulfed GFP-positive cell fragments or 555-labed Aβ in BV2 microglial cells are shown. These experiments are repeated three times. B
Quantification of engulfed GFP-positive cell fragments or 555-labed Aβ in the BV2 microglial cells as measured by flow cytometry. C Representative
images of microglial phagocytosis of GFP-positive cell fragments or 555-labed Aβ after uptake for 4 h in cultured BV2 microglial cells. Theseexperiments are repeated three times. Scale bars; 20 μm. D Representative images of microglial phagocytosis of GFP-positive cell fragments
or 555-labed Aβ after uptake for 4 h in cultured BV2 microglial cells. These experiments are repeated three times. Scale bars; 5 μm. E Quantificationof internalized 555-labed Aβ using ImageJ software. (n = 10 per group). All data are presented as means ± SEM. **p ≤ 0.01, ****p ≤ 0.0001

Wang et al. Journal of Neuroinflammation(2024) 21:176

Intracranial HSV-1 infection disrupts the blood–brain
barrier (BBB) and leads to the infiltration of periphery
immune cells [52, 53]. Macrophages and T cells infiltrate
and play a variety of key roles in the immune defense system in neurodegenerative diseases, CNS injury, and CNS
infections. Activated microglia, macrophages, and T cells
secrete a series of cytokines (e.g., IL-6, IL-1β, TNFα, andIL-10) and chemokines (e.g., CCL5, CCL2, and MCP-1)
to exert different functions [54–58]. To investigate theinflammatory responses after HSV-1 intracranial infection, we performed immunostaining assays using antiCD3 and anti-F4/80 antibodies along with anti-Iba1
antibody and observed a significant infiltration of T cells
and macrophages (Figure S9A-D). Subsequently, we isolated total RNA from the non-infected and HSV-infectedhippocampi of 3-month-old 5xFAD mice three or sevendays after HSV-1 infection. The results showed that
inflammatory genes significantly increased three days
after HSV-1 infection (Figure S9E-F). Overall, our data
reveal that macrophages and T cells infiltrate and secrete
inflammatory factors after HSV-1 infection in 5xFAD
mice.
Blockade of NLRP3 inflammasome signaling reduces Aβ
deposition and ameliorates cognitive deficits in 5xFAD
mice after HSV‑1 infection

Our data support a positive correlation between NLRP3
inflammasome activation and Aβ accumulation following HSV-1 infection. To further define if the NLRP3
inflammasome signaling drives Aβ accumulation and
AD pathology after HSV-1 infection, we administrated
MCC950 sodium, a selective small-molecular inhibitor of the NLRP3 inflammasome, to inhibit inflammasome activation [59, 60]. 5xFAD mice received MCC950
sodium via intraperitoneal injection every second day for
21 days after HSV-1 infection (Fig. 9A). First, we examined the expression levels of the NLRP3 inflammasome
through western blot and immunostaining assay andobserved a significant inhibition of NLRP3 inflammasome activity after MCC950 treatment following HSV-1
infection (Fig. 9B–E). Next, we evaluated the effects of
MCC950 treatment on Aβ deposition. We found that

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MCC950 treatment significantly reduced the total Aβburden in 5xFAD mice after HSV-1 infection (Fig. 10A–
C). We further examined the Aβ expression amounts byimmunoblotting assay. Consistent with the decreased
Aβ deposition in imaging data, the expression amountsof Aβ were also decreased in 5xFAD mice treated with
MCC950 after HSV-1 infection (Fig. 10D and E). To further investigate whether inflammasome inhibition could
affect uptake of HSV-1-infected cells as well as Aβ, we
performed an immunostaining assay using an anti-CD68
antibody along with an anti-Aβ antibody to visualize thecolocalization of Aβ, HSV-GFP and CD68. Consistent
with our data in Fig. 5, microglia preferentially phagocytize HSV-GFP-positive cells after HSV-1 infection, but
not Aβ plaques. However, MCC950 treatment did notaffect the phagocytic preference of microglia for Aβ or
HSV-GFP-positive cells (Figure S10). Overall, inhibition
of NLRP3 inflammasome activity did not alter the phagocytic preference of microglia but reduced Aβ deposition.
Then, we assessed the social learning and memoryof mice through Y-maze and Morris water-maze tests.
Compared with 5xFAD control mice, 5xFAD-HSV mice
showed a reduction in spontaneous alterations in the
Y-maze test and were less efficient at finding the hidden platform (Fig. 10F–H), indicating a deficit in social
learning and memory after HSV-1 infection. However,
MCC950 treatment significantly reversed these deficits
in 5xFAD mice after HSV-1 infection (Fig. 10F–H). The
improvement of these phenotype upon MCC950 treatment was not affected by the total number of arm entries(Fig. 10F) and the swimming speed of mice (Fig. 10H).
Collectively, these results suggest that MCC950 treatment to inhibit NLRP3 signaling can ameliorate AD
pathology, including Aβ deposition and impaired cognitive function.

Discussion
AD is the most common neurodegenerative disorder inthe world, and HSV-1 is one of the most common DNA
viruses globally. In this study, we characterized the roleand regulation mechanism of HSV-1 in 5xFAD mice.
Microglia are essential in the modulatory network of(See figure on next page.)
Fig. 7 Depletion of microglia increases Aβ plaque deposition and reduces microglial phagocytosis after HSV-1 infection. A Scheme for PLX3397
administration and HSV-GFP injection in 5xFAD mice. B and C Representative images (B) and quantification of Iba1-positive microglial cells (C)in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection following 21 days treatment with PLX3397. Scale bars,
50 μm. (n = 5 mice per group). D and E Representative images (D) and quantification of CD68-positive microglial cells (E) in the hippocampusof 3-month-old 5xFAD mice seven days after HSV-GFP infection following 21 days treatment with PLX3397. Scale bars, 50 μm. (n = 5 mice
per group). F and G Representative images (F) and quantification of Aβ plaques and plaque area (G) in the whole brain of 3-month-old 5xFAD
mice seven days after HSV-GFP infection following 21 days treatment with PLX3397. Scale bars, 200 μm, 100 μm and 50 μm. (n = 5 mice per group).
H and I Immunoblotting and quantification of Aβ protein in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection
following 21 days treatment with PLX3397. Data are presented as means ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

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Wang et al. Journal of Neuroinflammation(2024) 21:176

HSV-1 infection and Aβ deposition. In addition, we
combined viral infection, immunology, biochemistry,cell imaging, and animal approaches to confirm for the
first time that HSV-1 infection induced the activationof NLRP3 inflammasome signaling to drive Aβ deposition and AD pathology (Fig. 11). Our results suggestthat NLRP3 inflammasome may drive the developmentof AD in the HSV-1 infection condition, and inhibition
of NLRP3 signaling have potential to prevent Aβ accumulation and cognitive decline of AD.
Despite considerable efforts, elucidating the pathogenesis of AD is challenging due to its complex andundefined etiology. Growing evidence implicates thatviral or microbial infection of the central nervous system (CNS) are potential pathological factors in the
etiology of sporadic AD. The AD pathogen hypothesis
suggests that various pathogens act as triggers, initiating a pathological cascade that leads to the accumulation of Aβ [11–14]. According to this hypothesis, Aβ
peptides have been shown to possess antimicrobial
and antiviral effects and are produced by the CNS as adefense mechanism. Viral or microbial infection resultin rapid seeding and accelerated β-amyloid deposition [13]. In this process, these pathogens also activate glial cells and induce a pro-inflammatory innate
immune response, ultimately leading to progressive
neurodegeneration and dementia [61]. However, theprecise mechanism between viral infections and AD
remains unclear. In this study, we utilized the approach
introduced by Eimer and coauthors [13]. We administrated HSV-1-GFP into the hippocampus of 1-monthold, 3-month-old and 6-month-old 5xFAD mice andobserved the aggregation and deposition of Aβ in thewhole brain following HSV-1 infection. Moreover, we
also observed a significant accelerated cognitive dysfunction in 3-month-old 5xFAD mice following HSV-1
infection. These results are consistent with the previous
reports in HSV-1-induced Aβ deposition and suggest
HSV-1 infection leads to Aβ deposition and cognitive
deficits in AD mouse model.

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Microglia are the resident innate immune cells in the
CNS and have been shown to play major roles in internalization and degradation of Aβ [62]. They are the primary phagocytes in the CNS for uptake and proteolytic
clearance of both soluble and fibrillary forms of Aβ [63].
Inflammatory responses influence the activation statusof microglia and subsequently regulate their ability to
uptake and degrade Aβ [63]. HSV-1 brain infection leads
to persistent activation of microglia, which results in the
upregulated expression of type I interferon (IFN) in acGAS-STING-dependent manner to exert antiviral function [49, 64]. However, abnormal activation of microglia
may also lead to dysregulated microglia function, including phagocytosis and inflammation [42, 65–67]. Here,
we observed that microglia were activated after HSV-1
infection. In the viral core, microglia often displayed an
activated “rounded” and “amoeboid” morphology characterized by shorter processes, larger cell bodies andreduced microglia volumes. Interestingly, we observedthat microglia were recruited to the viral core andengulfed HSV-1-GFP-positive neuronal cells, demonstrated by CD68 and Aβ coimmunostaining. Our results
demonstrate that the recruitment of microglia to the viralcore increased the phagocytic ability to viruses following HSV-1 infection. In this case, no enough microglia
would be recruited to clear Aβ, causing Aβ deposition.
Our results indicate that microglia modulate their phagocytic preferences, enhancing their uptake of viruses after
HSV-1 infection and reducing Aβ uptake, ultimately
leading to Aβ accumulation and deposition. In recent
years, with the fast development of techniques, growing research on microglia has remarkably revealed theirphagocytic roles in neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD),
Huntington’s disease (HD), frontotemporal dementia(FTD), and amyotrophic lateral sclerosis (ALS) [37, 68–70]. Microglia have a high phagocytic capacity and canclear pathological protein aggregates (Aβ, α-synuclein,
and TDP43) [71]. Nevertheless, excessive extracellular
protein aggregate release and autophagy impairment(See figure on next page.)
Fig. 8 NLRP3 inflammasome is activated after HSV-1 infection in 5xFAD mice. A Representative images of Aβ (red) and Thio-S (gray)in the hippocampus of 3-month-old 5xFAD mice infected with PBS or HSV-GFP over the indicated time periods. B Quantification of Aβ plaquesin the hippocampus of 3-month-old 5xFAD mice infected with PBS or HSV-GFP over the indicated time periods. (n = 5 mice per group). C Western
blot analysis of NLPR3 inflammasome protein markers and cGAS-STING signaling pathway protein markers from the hippocampi over the indicatedtime periods upon HSV-GFP infection. D Quantification of the band intensity in C using ImageJ software. (n = 3 mice per group). E Representative
images and quantification of NLRP3-positive cells (magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection.
Original magnification × 40, scale bars; 20 μm. Zoom-in images with a scale bar equal to 10 μm. (n = 5 mice per group). F Representative images
of NLRP3 (gray) co-stained with microglia (Iba1, red) and astrocytes (GFAP, magenta) in the hippocampus of 3-month-old 5xFAD mice sevendays after HSV-GFP infection. Scale bars; 14 μm. G Representative images and quantification of ASC-positive cells (magenta) in the hippocampusof 3-month-old 5xFAD mice seven days after HSV-GFP infection. Original magnification × 40, scale bars; 20 μm. Zoom-in images with a scalebar equal to 10 μm. (n = 5 mice per group). Data are presented as means ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

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Fig. 9 MCC950 treatment inhibits NLRP3 inflammasome activation after HSV-1 infection in 5xFAD mice. A Timeline illustrating the intraperitoneal
injection of MCC950 into 3-month-old 5xFAD mice following HSV-GFP infection. B Western blot analysis of NLRP3 inflammasome protein
markers from the hippocampi 21 days after vehicle or MCC950 injection into the 5xFAD mice following HSV-GFP infection. C Quantificationof the band intensity in B using ImageJ software. (n = 3 mice per group). D Representative images of NLRP3 (magenta) co-stained with Iba1
(red) in the hippocampus of 3-month-old 5xFAD mice treated with MCC950 for 21 days post HSV-GFP infection. Scale bars, 20 μm. E
Quantification of NLRP3-positive cells in D using ImageJ software. (n = 5 mice per group). Data are presented as means ± SEM. *p ≤ 0.05, **p ≤ 0.01,
and ****p ≤ 0.0001(See figure on next page.)
Fig. 10 Blockade of the NLRP3 inflammasome signaling reduces Aβ plaque deposition and ameliorates cognitive deficits after HSV-1 infection. A
Three-month-old 5xFAD mice were administrated with either MCC950 or vehicle for 21 days following HSV-GFP infection. Coronal sections were
stained with Aβ (red) and Thio-S (gray). Representative images of the whole brain are shown. Scale bars; 600 μm. B Representative images of Aβ(red) and Thio-S (gray) staining in the hippocampus of 3-month-old 5xFAD mice treated with MCC950 for 21 days post HSV-GFP infection. Scalebars, 50 μm. C Quantification of Aβ plaques and Aβ area in A using ImageJ software. (n = 5 mice per group). D and E Three-month-old 5xFAD
mice were administrated with MCC950 for 21 days following HSV-GFP infection. Subsequently, immunoblotting and quantification of Aβ protein
levels from the hippocampi are presented. (n = 3 mice per group). F Three-month-old 5xFAD mice were randomly divided into three groups:
control group, HSV-GFP group (vehicle) and HSV-GFP + MCC950 group. Y-maze test was performed to measure the total number of arm entries
and spontaneous alteration (%) at 21 days post MCC950 injections. (n = 8 mice per group). G and H Morris water maze was performed to evaluate
the spatial learning and memory of 5xFAD mice treated with vehicle or MCC950 following HSV-GFP infection. During the training phase, each group
showed improved latency to the platform, but MCC950 treatment significantly improved the latency to the platform compared to the HSV-Veh
group (G). In the probe test, although MCC950 did not significantly change swimming speed, it ameliorated the impaired latency to the platform
and increased the number of target crossings in 5xFAD mice after HSV-GFP infection (H). (n = 8 mice per group). Data are presented as means ± SEM.
n.s.: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

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Fig. 11 Schematic of HSV-1 infection accelerates the progression of Alzheimer’s disease by modulating microglial phagocytosis and activating
NLRP3 pathway

both contribute to pathological progression and neurodegeneration. Thus, enhancing microglial phagocytosis isconsidered a promising strategy for the therapy of neurodegenerative diseases.
Microglia execute their roles as the first line of defense
against CNS infection through their immune response
mediated by immune receptors [72, 73], including NLRmediated responses such as the NLRP3 inflammasome[35, 36], and nucleic acids receptor-mediated responses,
such as RIG-1 and cGAS [34, 38, 74]. Previous studies have found that NLRP3 is activated in AD mousemodels and AD patients, contributing to the progression of AD pathology. NLRP3 inflammasome deficiency
resulted in decreased deposition of Aβ and reduced tau
hyperphosphorylation, ultimately delaying the progression of AD pathology [51, 75]. In this study, we observedthat the NLRP3 inflammasome signaling was activatedon day 3 and 7 after HSV-1 infection, with its activation
trend being correlated with Aβ accumulation. Therefore, we speculated that the NLRP3 inflammasome
was involved in the accumulation of Aβ. To testify thishypothesis, we employed a selective small-molecularinhibitor (MCC950) of the NLRP3 inflammasome signaling to inhibit inflammasome activation and revealed that
MCC950 treatment significantly prevented Aβ deposition and alleviated the cognitive impairments induced by

HSV-1 infection. These findings suggested that NLRP3
inflammasome activation mediates HSV-1-induced Aβpathology in 5xFAD mice. Other selective NLRP3 inflammasome inhibitors, such as OLT1177 (Dapansutrile),
exert therapeutic effects in the experimental autoimmune encephalopathy (EAE) mouse model and attenuate the infiltration of CD4 T cells [76]. Stavudine (D4T),
stimulates Aβ phagocytosis by macrophages to eliminate
Aβ [77]. Overall, previous studies and our work indicatethat patients with AD and HSV-1 infection may potentially benefit from NLRP3-targetted treatment strategies.
As a typical DNA virus, HSV-1 could activate cGASSTING DNA sensing pathway [38, 78]. In addition, previous study reveals that HSV-1 infection could induce
releases of mitochondrial DNA (mtDNA) into cytosol
to activate cGAS-STING signaling. Indeed, we detected
strong activation of cGAS-STING signaling at early stage
after HSV-1 infection in this study. Recent studies showthat abnormal activation of cGAS-STING signaling pathway is a driven-factor of aging and neurodegeneration[79]. Of note, the aberrant cGAS-STING activation are
also involved in AD [80–83]. The mtDNA release intocytosol of the microglial could activate cGAS-STING
signaling to contribute to AD pathogenesis in 5xFAD
mice [81]. Tau can also activate cGAS-STING pathway
by inducing microglia mtDNA leakage [82]. Consistently,

Wang et al. Journal of Neuroinflammation(2024) 21:176

cGAS deletion or inhibitors of cGAS (or STING inhibitor) could restrict cGAS-STING pathway and mitigate
AD progress in different AD disease models [80, 81].
Therefore, we hypothesize that, in AD, distinct sourcesof innate immune components can be activated in different time course to promote neuroinflammation. We
propose that distinct innate immune signaling pathways,
including cGAS-STING pathway and NLRP3 inflammasome, may contribute to neuroinflammatory phenomena
and AD pathogenesis with distinct mechanisms. Further studies are required to investigate how these innate
immune pathways are regulated and cooperates witheach other during the pathological phenomena of AD.
Aβ proteins, herpesviruses, and innate immunity are
all ancient (at least 300 million years old). Understanding
the underlying regulatory mechanisms in brain and neurodegenerative disorders would be very interesting andvaluable. While previous reports have suggested a potential link between HSV-1 and Aβ accumulation throughthe use of AD mouse model and 3D human brain model[11, 13, 67, 84], the cognitive impairment and the precise modulatory mechanism behind it remain unclear. In
this study, we demonstrate that HSV-1 infection leads toAβ deposition, activation of NLRP3 inflammasome, andaccelerated cognitive deficits in 5xFAD mice. Our data
reveal that microglia and the NLRP3 inflammasome play
crucial role in AD pathology induced by HSV-1 infection. Our results provide some information to unravel themechanism of HSV-1-induced AD pathogenesis, suggesting new therapeutic approaches for this multifactorial
and devastating neurodegenerative disease.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03166-9.
Additional file 1: Figure S1. HSV-1 infection does not alter APP processing. (A and B) Immunoblotting (A) and quantification (B) of APP, PS1,
ADAM10 and BACE1 proteins in the hippocampus of 1-month-old 5xFAD
mice (Ctrl), 5xFAD mice infected with PBS (Vehicle) or 5xFAD mice sevendays after HSV-GFP infection (HSV-GFP). (n = 3 mice per group). (C andD) Immunoblotting (C) and quantification (D) of APP, PS1, ADAM10 and
BACE1 proteins in the hippocampus of 3-month-old 5xFAD mice (Ctrl),
5xFAD mice infected with PBS (Vehicle) or 5xFAD mice seven days after
HSV-GFP infection (HSV-GFP). (n = 3 mice per group). (E and F) Immunoblotting (E) and quantification (F) of APP, PS1, ADAM10 and BACE1
proteins in the hippocampus of 6-month-old 5xFAD mice (Ctrl), 5xFAD
mice infected with PBS (Vehicle) or 5xFAD mice seven days after HSVGFP infection (HSV-GFP). (n = 3 mice per group). Data are presented asmeans ± SEM. n.s.: p > 0.05.
Additional file 2: Figure S2. Enhanced microglial phagocytosis inducedby HSV-1 infection in 5xFAD mice. (A) Representative images andquantification of CD68-positive cells (magenta) in the hippocampus of
1-month-old 5xFAD mice seven days after HSV-GFP infection. Original
magnification × 40, scale bars; 20 μm. Zoom-in images with a scale bar
equal to 10 μm. (n = 5 mice per group). (B) Representative images andquantification of CD68-positive cells (magenta) in the hippocampus of
6-month-old 5xFAD mice seven days after HSV-GFP infection. Original

Page 21 of 24

magnification × 40, scale bars; 20 μm. Zoom-in images with a scale bar
equal to 10 μm. (n = 5 mice per group). (C) Representative images ofphagocytic microglia (CD68, magenta) co-stained with Aβ (red) in the
hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP
infection. Scale bars; 10 μm.
Additional file 3: Figure S3. HSV-1 infection induces enhanced phagocytic of HSV-1-positive cell debris in primary microglia. (A) Representative FACS dot plots showing engulfed GFP-positive cell fragments or
555-labeled Aβ in primary microglia. (B) Quantification of engulfed GFPpositive cell fragments or 555-labeled Aβ in (A). (n = 3 mice per group).
(C) Representative confocal images of microglial phagocytosis of GFPpositive cell fragments or 555-labeled Aβ after 4 h uptake in cultured
primary microglia. Scale bars, 10 μm. (D) Quantification of internalizedAβ using ImageJ software. (n = 10 per group). Data are presented asmeans ± SEM. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
Additional file 4: Figure S4. Microglia depletion attenuates microglia
activation and phagocytosis. (A) Representative images of phagocytic microglia (CD68, magenta) co-stained with Iba1 (red) in the
hippocampus of 1-month-old 5xFAD mice seven days after HSV-GFP
infection following 21 days treatment with PLX3397. Scale bars, 50 μm.
(B) Quantification of phagocytic microglia (­ CD68+ cells) in (A) using
ImageJ software. (n = 5 mice per group). (C) Representative images ofphagocytic microglia (CD68, magenta) co-stained with Iba1 (red) in the
hippocampus of 6-month-old 5xFAD mice seven days after HSV-GFP
infection following 21 days treatment with PLX3397. Scale bars, 50 μm.
(D) Quantification of phagocytic microglia (­ CD68+ cells or I­ba1+ cells)in (C) using ImageJ software. (n = 5 mice per group). Data are presented
as means ± SEM. ****: p ≤ 0.0001.
Additional file 5: Figure S5. Microglia depletion increases Aβ plaquedeposition in 6-month-old 5xFAD mice after HSV-1 infection. (A) Representative images of Aβ plaques and plaque area in the whole brain of
6-month-old 5xFAD mice seven days after HSV-GFP infection following
21 days treatment with PLX3397. Scale bars, 200 μm. (B) Representative
images of Aβ plaques (red) in the hippocampus of 6-month-old 5xFAD
mice seven days after HSV-GFP infection following 21 days treatment
with PLX3397. Scale bars, 50 μm. (C) Quantification of Aβ plaques andplaque area in the whole brain of 6-month-old 5xFAD mice seven days
after HSV-GFP infection following 21 days treatment with PLX3397.
(n = 5 mice per group). Data are presented as means ± SEM. *: p ≤ 0.05.
Additional file 6: Figure S6. The NLRP3 inflammasome is activated in
1-month-old and 6-month-old 5xFAD mice following HSV-1 infection.
(A and B) Immunoblotting (A) and quantification (B) of NLRP3 inflammasome protein markers from the hippocampi of 1-month-old 5xFAD
mice seven days after HSV-GFP infection. (n = 3 mice per group). (C)The expression of IL-1β is determined by ELISA assay in 1-month-old
5xFAD mice seven days after HSV-GFP infection. (n = 3 mice per group).
(D) Representative images of NLRP3 (magenta) co-stained with Iba1
(red) and quantification of NLRP3-positive cells in the hippocampus
of 1-month-old 5xFAD mice seven days after HSV-GFP infection. Scalebars, 20 μm. (n = 5 mice per group). (E) The expression of IL-1β isdetermined by ELISA assay in 3-month-old 5xFAD mice seven days after
HSV-GFP infection. (n = 3 mice per group). (F and G) Immunoblotting(F) and quantification (G) of NLRP3 inflammasome protein markers
from the hippocampi of 6-month-old 5xFAD mice seven days after
HSV-GFP infection. (n = 3 mice per group). (H) The expression of IL-1β isdetermined by ELISA assay in 6-month-old 5xFAD mice seven days after
HSV-GFP infection. (n = 3 mice per group). (I) Representative images
of NLRP3 (magenta) co-stained with Iba1 (red) and quantification of
NLRP3-positive cells in the hippocampus of 6-month-old 5xFAD miceseven days after HSV-GFP infection. Scale bars, 20 μm. (n = 5 mice pergroup). Data are presented as means ± SEM. n.s.: p > 0.05, ***: p ≤ 0.001,****: p ≤ 0.0001.
Additional file 7: Figure S7. NLRP3 is mostly expressed in microglia
in 5xFAD mice following HSV-1 infection. (A) Representative imagesof microglia (Iba1, red) co-stained with NLRP3 (magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection. Scale bars, 20 μm. (B) Representative images of NLRP3 (magenta)

Wang et al. Journal of Neuroinflammation(2024) 21:176

co-stained with GFP in the hippocampus of 3-month-old 5xFAD miceseven days after HSV-GFP infection. Original magnification × 40, scale bars;
20 μm. Zoom-in images with a scale bar equal to 10 μm, 5 μm. (C) Representative images of microglia (Iba1, red) co-stained with ASC (magenta) in
the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP
infection. Scale bars, 20 μm.
Additional file 8: Figure S8. ASC specks responding to Aβ accumulationin 5xFAD mice following HSV-1 infection. (A) Representative imagesof ASC (magenta) co-stained with Aβ (red) in the hippocampus of
3-month-old 5xFAD mice seven days after HSV-GFP infection. Original
magnification × 40, scale bars; 20 μm. Zoom-in images with a scale bar
equal to 10 μm. (n = 4 mice per group). (B) Representative images of ASC(magenta) co-stained with Aβ (red) in the hippocampus of 6-month-old
5xFAD mice seven days after HSV-GFP infection. Original magnification × 40, scale bars; 20 μm. Zoom-in images with a scale bar equal to
10 μm. (n = 4 mice per group).
Additional file 9: Figure S9. HSV-1 infection induces T cell and macrophage infiltration and dramatically induces inflammatory response.
(A) Representative images of microglia (Iba1, red) co-stained with T cells(CD3, magenta) in the hippocampus of 3-month-old 5xFAD mice threedays after HSV-GFP infection. Scale bars, 50 μm. (B) Quantification of C
­ D3+
cells in the hippocampus of 3-month-old 5xFAD mice infected with PBS
or HSV-GFP (1 × ­105 PFUs/hippocampus). (n = 4 mice per group). (C) Representative images of microglia (Iba1, red) co-stained with macrophages
cells (F4/80, magenta) in the hippocampus of 3-month-old 5xFAD mice
three days after HSV-GFP infection. Scale bars, 50 μm. (D) Quantificationof F4/80+ cells in the hippocampus of 3-month-old 5xFAD mice infectedwith PBS or HSV-GFP (1 × ­105 PFUs/hippocampus). (n = 4 mice per group).
(E and F) HSV-GFP viruses were administered into 3-month-old 5xFAD
mice. Hippocampi from non-infected and HSV-infected mice were collected three and seven days post-infection and homogenized for gene
expression analysis by real-time PCR. (n = 3 mice per group). Data are presented as means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Additional file 10: Figure S10. MCC950 treatment does not affect thephagocytic preference of microglia following HSV-1 infection in 5xFAD
mice. (A) Representative images of phagocytic microglia (CD68, blue)
co-stained with Aβ plaques (magenta) in the hippocampus of 5xFAD micetreated with MCC950 for 21 days following HSV-1 infection. Three-dimensional reconstructed enlarged images of dashed-white or dashed-yellow
frames show engulfed Aβ plaques or GFP-positive cells in the microglia.
Scale bars, 5 μm. (B) Quantification of engulfed Aβ plaques or GFP-positive
cells in (A). (n = 4 mice per group). Data are presented as means ± SEM.
****p ≤ 0.0001.
Acknowledgements
We are grateful for the technical support provided by Baoyang Hu.
Author contributions
Z.W, and J.L. planned the study under the guidance of C.Z., performed theexperiments, analyzed and organized the data, and wrote the manuscript; J.H.,
Y.H., H.S. and F.H. helped organize the data and revised the manuscript; T.Z.
and S.L. provided the virus injection apparatus and helped in virus injection;
C.Z. designed and supervised this study.
Funding
This work is supported in part by the National Natural Science Foundation ofChina (Project approval number: 32070875 and 82241074 to C.G. Zhang). We
thank Grants form Tsinghua-Peking Center for Life Sciences, Tsinghua University Initiative Scientific Research Program and Dushi Program.
Availability of data and materials
No datasets were generated or analysed during the current study.

Page 22 of 24

Declarations
Ethics approval and consent to participate
All experimental animal procedures were approved by the Institutional Animal
Care and Use Committee of Tsinghua University.
Competing interests
The authors declare no competing interests.
Received: 1 May 2024 Accepted: 3 July 2024

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