Syage et al. Journal of Neuroinflammation(2024) 21:157
https://doi.org/10.1186/s12974-024-03153-0

Journal of Neuroinflammation

Open Access

RESEARCH

Cystatin F attenuates neuroinflammationand demyelination following murine
coronavirus infection of the central nervous
systemAmber R. Syage1, Collin Pachow2, Kaitlin M. Murray1, Caden Henningfield1, Kellie Fernandez1, Annie Du1,
Yuting Cheng2, Gema Olivarria1, Shimako Kawauchi3, Grant R. MacGregor4, Kim N. Green1 and Thomas E. Lane1,5*Abstract
Background Cystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting theseverity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination.
How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has
not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a wellestablished model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in
host defense, demyelination and remyelination.
Methods Wildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic
JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays
and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation
were determined by flow cytometry and 10X genomics chromium 3’ single cell RNA sequencing (scRNA-seq). Spinal
cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal
damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy(EM) and calculation of g-ratios.
Results JHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F
is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMVinfected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal
damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enrichedfrom spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cellchemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1)
transcripts in CD8 + T cells from Cst7-/- mice compared to controls.

*Correspondence:
Thomas E. Lane
tlane@uci.edu
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
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Syage et al. Journal of Neuroinflammation(2024) 21:157

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Conclusions Cystatin F is not required for immune-mediated control of JHMV replication within the CNS. However,
JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelinationand impaired remyelination. The increase in disease severity was associated with elevated expression of T cellchemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea thatcystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation
and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.
Keywords Cystatin F, Coronavirus, Microglia, Demyelination, Remyelination

Introduction
The human demyelinating disease, Multiple Sclerosis(MS), is a devastating neurodegenerative disease that
affects millions of people worldwide. Though current
therapies have been very successful in slowing the progression of disease in patients with the Relapsing-Remitting MS, there are currently few treatment options forindividuals with progressive forms of MS, highlighting afundamental need for discovery of treatments that cantarget inflammation and promote repair from within the
CNS. To recapitulate the hallmark immune-mediatedmyelin damage seen in MS, we performed intracranial(i.c.) inoculation of C57BL/6J mice with the neurotropic
JHM strain of mouse hepatitis virus (JHMV), which
results in viral replication in glial cells with minimal
infection of neurons [1, 2], leading to an acute encephalomyelitis and subsequent demyelination. Innate immune
responses, including expression of type I interferon andchemokines that attract myeloid cells to the CNS, areimportant in limiting viral replication as well as allowing
virus-specific T cells access to the brain parenchyma [3].
Both CD4 + and CD8 + T cells are recruited into the CNS
of JHMV-infected mice by T cell chemotactic chemokines CCL5, CXCL9 and CXCL10 and these cells control
viral replication through secretion of interferon-g (IFNg) and cytolytic activity, respectively [4–11]. Antibodysecreting cells (ASCs) are also capable of responding
to CXCL9 and CXCL10 and aid in host defense by preventing viral recrudescence [12, 13]. Nonetheless, sterile
immunity is not achieved, and the majority of animals
that survive the acute stage of disease develop immunemediated demyelination in which both virus-specific T
cells and macrophages amplify the severity of white matter damage associated with hind-limb paralysis [1, 2, 14,
15].
Under non-disease conditions, microglia function
to maintain tissue homeostasis [16] and are importantfor maintaining overall myelin health through regulation of myelin growth and preservation of structural
integrity [17, 18]. Microglia serve various functions
under neuroinflammatory conditions by influencing
antigen presentation, which either amplifies or restricts
disease progression via secretion of regulatory factors that, in turn, control the activation state of inflammatory immune cells [19, 20]. Moreover, microglia areconsidered the immune cell of the CNS and are important in aiding in host defense in response to viral infection [21, 22]. Depletion of microglia via administration ofsmall molecule antagonists specific for colony stimulating factor 1 receptor (CSF1R) results in increased susceptibility to viral-induced demyelinating disease [23–26].
Following microglia depletion, mice infected with JHMV
exhibit an increase in mortality, associated with impaired
control of viral replication within the CNS, that correlates with reduced expression of MHC class II on
antigen-presenting cells (APCs) and muted CD4 + T cellanti-viral immune responses [27]. Further, the severity ofspinal cord demyelination was increased while remyelination was dramatically reduced [28]. These findings support a role for microglia in regulating immune responses
within the CNS in response to microbial infection as well
as restricting the severity of white matter damage. In support of this, several recent reports demonstrated thatmicroglia influence expression of genes and pathways
associated with immune responses and myelin repair,in response to JHMV infection of the CNS [28–30]. We
have shown that microglia depletion results in a dramatic reduction in transcripts encoding cystatin F (Cst7)
that correlated with both increased demyelination andimpaired remyelination [30].
Cystatin F is papain-like lysosomal cysteine proteaseinhibitor expressed by immune cells, including macrophages, cytotoxic T-lymphocytes (CTLs), and by
microglia within the CNS, under conditions where demyelination and remyelination are concurrently occurring[31, 32]. Cystatin F is synthesized as an inactive dimer,
which is converted to a monomeric form to enable inhibition of target cathepsins. Cystatins are generally found toact on cathepsins intracellularly or extracellularly; however, cystatin F is somewhat unusual in that it is secreted
in an inactive form, only becoming active following internalization by neighboring cells, where it regulates cathepsin activity within the endosomes and lysosomes of thoseneighboring cells. The broad expression of cystatin F in
immune cells raises the possibility that it has an important function in the immune response. Furthermore,
microglial expression of cystatin F is triggered by thephagocytosis of myelin debris suggesting that cystatin F
plays a role in response to demyelination [32]. Expression
patterns of cystatin F overlap in demyelinating regions

Syage et al. Journal of Neuroinflammation(2024) 21:157

undergoing remyelination in pre-clinical mouse models
of demyelination and in plaques from patients with multiple sclerosis (MS), garnering new interest in cystatin F
for its potential impact on neuroinflammation, immunemediated demyelination, and remyelination [31–34].
In this study, we set out to determine if cystatin F is
involved in this process of modulating white matter damage and repair in a viral model of demyelinating disease
and employed a novel Cst7 knockout (Cst7-/-) mouse toassess the response to JHMV infection. Findings fromthis study indicate that the absence of cystatin F did
not affect T cell-mediated control of JHMV replication
within the CNS, arguing that this pathway is not critical in anti-viral effector responses. However, infiltrationof T cells into the CNS of JHMV-infected Cst7-/- mice
was increased early during chronic disease which correlated with increased expression of transcripts encoding
T cell chemoattractant chemokines CXCL9 and CXCL10
accompanied by more severe demyelination, impaired
remyelination, and elevated axonal damage. These results
indicate a potential role for cystatin F as a mechanism
influencing the severity of neuroinflammation, affecting both white matter damage and repair, in response toviral-induced neurologic disease.

Materials and methods
Generation and genotyping of Cst7 null-mutant mice

To generate Cst7-null mutant mice (B6-Cst7em1Aduci/J,
Jackson Laboratory stock number #037727), Alt-R Crispr
RNAs (TMF1131–5’-​C​C​T​G​C​A​T​T​T​C​C​C​C​A​G​C​C​A​T​G-3’,
TMF-1132–5’-​ A ​ A​ G ​ C ​ A​ G ​ A ​ A​T​ G ​ G ​ C ​ C ​ A​ G ​ C ​ C ​ A​ C ​ A-3’)
and tracrRNA plus CAS9 protein (HiFi Cas9 nuclease
V3, Integrated DNA Technologies (IDT), Coralville, IA)
as a ribonucleoprotein (RNP) were microinjected into
C57BL/6J zygotes (Jackson Lab Stock # 000664) to generate double stranded DNA breaks within exon 1 of Cst7.
G0 founder mice were screened by PCR to identify animals having a deletion allele of Cst7 produced by removalof DNA in the vicinity of the CAS9 cut sites, followedby non-homologous end joining (NHEJ) repair. G0 animals containing a prospective deletion allele were backcrossed with C57BL/6J mice, and N1F1 heterozygous
mice were sequenced to determine the mutant allele.
The Cst7-null allele (Cst7-null) has a deletion of 168 bp
of contiguous DNA from nucleotide position 150,412,438
to 150,412,605 inclusive (nucleotide position from mouse
genome build GRCm39, Ensembl release 107). The deletion begins one nucleotide upstream of the translation initiation ATG codon in exon 1 and extends 100
nucleotides into intron 1, i.e., exon 1 ATTTCCCCAGCcatgtggctgg…………catctttctTAGGACACTC
intron1, where the uppercase sequence is retained, and thelowercase sequence is deleted. N1F1 heterozygous
Cst7em1Aduci mutant mice were backcrossed to produce

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N2F1 heterozygotes, which were crossed to produce
experimental Cst7-/- mice and wildtype (WT) controls(Cst7+/+). Oligonucleotides for PCR-based genotyping
were purchased from IDT (Coralville, IA). Cst7em1Aduci
genotyping was performed using a common primer set toamplify both the Cst7 wildtype allele and the Cst7em1Aduci
null allele (For 5′- ​C​C​C​A​A​G​T​C​C​T​G​A​A​G​A​T​G​A​A​G​C​G− 3′ and Rev 5′- ​A​G​G​G​T​C​T​T​T​C​T​C​A​G​G​G​T​T​C​C​A − 3′).
Amplification was performed in a 10 µl reaction volumecontaining 2.5 ul diluted DNA, 1x New England Biolabs (NEB) PCR buffer, 0.75mM MgCl2, 0.2mM dNTPs,
0.25 μm each primer and 0.0375u Taq polymerase (NEB)
for 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C
for 30 s. The wild-type allele produced a 344 bp productwhile the Cst7-null allele produced a 176 bp product. All
mice used in this study were on an inbred or co-isogenic
C57BL/6J background.
Viral infection of mice

Cystatin F knock-out (Cst7-/-) mice were generated by
the University of California, Irvine transgenic mousefacility. Five to eight-week-old male and female Cst7-/mice and control (Cst7+/+) mice were infected i.c. with
1200 plaque forming units (PFU) of JHMV in 30µL ofsterile Hanks balanced salt solution (HBSS), and animals
were euthanized at days 7, 14, and 21 p.i. Clinical disease
in JHMV-infected mice was evaluated using a previously
described scale. To determine viral titers within brains,
experimental animals were sacrificed at defined times
p.i., with brains isolated and homogenized, and plaqueassays performed on the DBT astrocytoma cell line as
described previously [35]. Spinal cords of JHMV-infected
controls and Cst7-/- mice were homogenized, with RNA
extraction, cDNA synthesis, and qPCR for comparison of
JHMV mRNA levels performed as previously described[36] Primer sequences used were: GAPDH – forward
sequence, ​A​A​C​T​T​T​G​G​C​A​T​T​G​T​G​G​A​A​G​G; reverse
sequence, ​G​G​A​T​G​C​A​G​G​G​A​T​G​A​T​G​T​T​C​T: JHMVMatrix glycoprotein– forward sequence, ​T​C​A​A​C​C​C​C​G​
A​A​A​C​A​A​A​C​A​A​C​C; reverse sequence, ​G​G​C​T​G​T​T​A​G​T​
G​T​A​T​G​G​T​A​A​T​C​C​T​C​A . qPCR was performed using the
Bio-Rad iQ5 and iTaq™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA). Reactions were 10µL, andthe machine was set to run for 1 cycle (95℃ for 3 min),followed by 40 cycles (95℃ for 10 s, then 55℃ for 30 s).
Ct values for each sample were normalized to an internal control (GAPDH), yielding the dCt values; lower
dCT values indicate higher mRNA levels while higher
dCT values are indicative of lower mRNA levels as morecycles of amplification are required to detect signal. All
animal studies were reviewed and approved by the University of California, Irvine Intuitional Animal Care and
Use Committee.

Syage et al. Journal of Neuroinflammation(2024) 21:157

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Cell isolation and flow cytometry

Immunofluorescence staining

Flow cytometry was performed to identify inflammatory cells entering the CNS using established protocols[37]. In brief, single cell suspensions were generated
from tissue samples by grinding with frosted microscope slides. Immune cells were enriched via a 2-step
Percoll cushion (90% and 63%), and cells were collected
at the interface of the two Percoll layers. Before staining
with fluorescent antibodies, isolated cells were incubatedwith anti-CD16/32 Fc block (BD Biosciences, San Jose,CA) at a 1:100 dilution. Immuno-phenotyping of cells
was performed using commercially available antibodies specific for the following cell surface markers: CD4(Invitrogen, 11-0042-82), CD8a (Invitrogen, 17-008182), CD11b (Abcam, ab24874) and CD45 (BioLegend,
103,114; 103,130). Cells were simultaneously incubatedwith LIVE/DEAD Aqua Dead Cell Stain (Invitrogen,
L34966). The following flow cytometric gating strategies
were employed for inflammatory cells isolated from the
CNS: macrophages/myeloid cells (CD45hiCD11b+) andmicroglia (CD45loCD11b+); FITC-conjugated rat antimouse CD4 and a PE-conjugated tetramer specific for the
CD4 immunodominant epitope present within the JHMV
matrix (M) glycoprotein, spanning amino acids 133–147(M133-147 tetramer), to determine total and virus-specific CD4 + cells [37, 38]; APC-conjugated rat anti-mouse
CD8a and a PE-conjugated tetramer specific for the CD8
immunodominant epitope present in the spike (S) glycoprotein, spanning amino acids 510–518 (S510-518), to
identify total and virus-specific CD8 + cells [37, 38]. Data
were collected using a Novocyte flow cytometer and analyzed with FlowJo software (Tree Star Inc.).

Slides were washed extensively in 1X TBS, 0.2% Tween20 and 0.2% Triton-X followed by antigen retrieval, an
incubation with 1mM sodium citrate at 85oC for 3 min.
Slides were stained with FluoroMyelin Green (Invitrogen,
Cat#F3465, 1:300) for 20 min and washed again with 1X
TBS. After blocking in 5% BSA (w/v) and normal goat
serum for 1 h, samples were incubated in primary antibody, Anti-SMI-32 (nonphosphorylated neurofilament
H; Biolegend, Cat# 801,701, 1:1000) overnight at 4 °C.
Slides were washed in 1X TBS and incubated in secondary antibody, Alexa 594 goat anti-mouse (Invitrogen,
Cat# A11005, 1:500) for 1 h at room temperature. Slides
were mounted with Fluoromount-G with DAPI (Invitrogen, Cat#00-4959-52). Confocal images were taken with a
40X oil objective and 63X oil objective (SP8; Leica). Mean
fluorescence intensity of axonal damage was measured
using FIJI [41] and was quantified based on the reported
integrated density of axonal damage normalized to thearea of the selected region.

Histology

Mice were euthanized at days 14 and 21 p.i. with isoflurane, and immediately cardiac perfused with 20mL of
1X PBS, and spinal columns were isolated and stored in
4% paraformaldehyde at 4 °C for 24–36 h. The length ofspinal cord extending from thoracic vertebrae 6–10 was
carefully removed and cryoprotected in 30% sucrose at
4 °C for a minimum of 3 days, then cut into 1-mm transverse blocks and processed to preserve the craniocaudal
orientation, and subsequently embedded in optimum
cutting temperature (OCT) compound (VWR, Radnor,
PA, USA). Spinal cord tissue was coronally cryosectioned
at eight microns (µm) thick. Sections were stained withhematoxylin and eosin (H&E) in combination with luxol
fast blue (LFB), with between 5 and 8 sections/mouse
analyzed. Areas of total white matter and demyelinatedwhite matter were determined with Image J Software anddemyelination was scored as a percentage of total whitematter from spinal cord sections analyzed [38–40].

Electron microscopy and analysis

Mice were euthanized at day 21 p.i. with isoflurane andimmediately underwent cardiac perfusion with 20mL
of 1X PBS followed by 20mL of 0.1 M cacodylate buffer
containing 2% paraformaldehyde/2% glutaraldehyde. The
length of spinal cord extending from thoracic vertebrae
6–10 was carefully removed and stored in 2% paraformaldehyde/2% glutaraldehyde overnight at 4 °C. Serial
ultrathin sections of spinal cord embedded in Epon epoxy
resin were stained with uranyl acetate-lead citrate andanalyzed as previously described [37]. Images were taken
at 1200X magnification in demyelinating regions within
the spinal cord ventral and lateral funiculus for Cst7-/mice (n = 6) and controls (n = 6). For each group, between
133 and 660 axons/mouse (7–20 images/mouse) wereanalyzed using Image J software. G-ratio was calculated
as a ratio of axon diameter to total outer fiber diameter[36, 37]. Axons were considered remyelinated if myelin
thickness was less than 0.5 μm and the g-ratio was greaterthan 0.65, and absence of myelin sheath was used as thecriterion for demyelinated axons [42].
10X Genomics chromium single cell 3’ single cell RNA
sequencing (scRNASeq)

Spinal cords were removed from uninfected Cst7-/(n = 5) and control mice (n = 5), and JHMV-infected Cst7/- (n = 6) and control mice (n = 6), at day 21 p.i. Cells wereisolated from spinal cords via homogenization and Percoll gradient as previously described, keeping cells in
RPMI with 10% FBS and on ice whenever possible. Isolated cells were incubated with anti-CD16/32 Fc block(BD Biosciences, San Jose, CA) at a 1:200 dilution. Cells
were stained with DAPI and APC-conjugated anti-CD45

Syage et al. Journal of Neuroinflammation(2024) 21:157(Biolegend, 103112) for 20 minutes on ice in 1X PBS containing 0.5% bovine serum albumin (BSA). Live CD45+
cells were enriched using both BD FACSAria II and BD
Fusion cell sorters (UC Irvine Stem Cell Research Center, Flow Cytometry Core), washed once with 0.04% BSA.
Individual samples were then labeled with 100 µl of aunique Cell Multiplexing Oligo (CMO), consisting of a
Feature Barcode oligonucleotide conjugated to a lipid(10X Genomics Chromium Single Cell 3’ CellPlex Kit),
for 5 min at room temperature. Samples were washedtwice with 1% BSA in PBS, counted, and samples werereconstituted to a concentration of 1,000 cells/µl andprocessed by the UC Irvine Genomics High-Throughput
Facility (https://ghtf.biochem.uci.edu) for single cell RNA
sequencing using the 10X Genomics Chromium Single
Cell 3’ platform. A NovaSeq 6000 sequencer was used
to perform RNA sequencing (200 cycles), and sequencing reads were processed using the 10X Genomics CellRanger pipeline. For JHMV infected Cst7-/- and control
groups, cells from 6 mice were multiplexed and pooled
for each individual group. For uninfected Cst7-/- andcontrol groups, cells from 5 mice per group were used.
To ensure adequate CD45 + cell numbers required to run
scRNA-sequencing were achieved, all 10 samples from
uninfected control and Cst7-/- groups were multiplexedand pooled together. Sequencing data from the Cst7/- and control groups were then separated out using the
10X Genomics’ Cell Ranger pipeline.
scRNAseq analysis

Using Seurat version 4.1.1, experimental groups were
filtered, integrated, normalized, and scaled. Genes thatwere found in less than 10 cells, as well as cells with lessthan 500 and greater than 3,500 detected genes, were
removed. Additionally, cells with more than 30,000
unique molecular identifiers (UMIs) and greater than
5% mitochondrial counts were regressed out. Genecounts per cell were normalized by total expression,
multiplied by a scale factor of 10,000, then transformed
to log scale. Resulting cells numbers per group were as
follows: control = 3,347 cells; Cst7-/- = 3,371 cells; control, JHMV = 3,840 cells; Cst7-/-, JHMV = 3,350 cells.
Downstream analysis was performed in Seurat, following
principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) dimensional
reduction [43, 44]. Gene expression signatures of CD45+
cells from spinal cords of uninfected and JHMV-infected
Cst7-/- and control mice at day 21 p.i. were scrutinized
and cells from the aggregated dataset were clusteredinto corresponding immune cell populations by a sharednearest neighbor (SNN) modularity optimization-based
clustering algorithm via the FindClusters function in the
Seurat analysis package in R. The resulting clusters were
defined by analysis of expression levels and distribution

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of population-specific immune cell markers. Once theclusters were established and identified, plots were generated using Seurat and ggpubr R packages [45].
Statistical analysis

GraphPad Prism was used to perform statistical analyses.
Data for each experiment is presented as mean ± standard error of mean (SEM) or standard deviation (SD).
Values from experimental groups were compared using
unpaired (two-tailed) t-test, Mann-Whitney U test, orWelch’s t-test. Wilcoxon test was used for analyzing
gene expression in scRNAseq clusters, and the resulting p values were corrected for multiple comparisons by
Holm-Sidak method with a p value of ≤ 0.05 considered
statistically significant.

Results
JHMV infection and Cst7 expression

To determine the mRNA expression profile of cystatin
F and its target, cathepsin C, we looked at expression
patterns in our previous scRNAseq dataset with adult
C57BL/6J mice, infected i.c. with JHMV at days 3, 7, and21 p.i., from both brains and spinal cords [29] (Fig. 1A).
At days 3 and 7 p.i., Cst7 expression within the brain wasalmost exclusively restricted to lymphocytes, including
CD4+, CD8 + T cells, NK cells, as well as microglia subpopulations (Fig. 1B, C). By day 21 p.i., expression of Cst7
within the spinal cords of infected mice was much higher
than at earlier time points and indicated that inflammatory lymphocytes and microglia, and to a lesser extent
macrophages, remained the primary source for Cst7
transcripts (Fig. 1D). Our finding that microglia were thedominant cellular source of cystatin F transcripts within
the brains and spinal cords of JHMV-infected mice isconsistent with reported expression patterns of cystatin F within the context of pre-clinical animal models
of demyelinating diseases [31–34]. Cystatin F can target
cathepsin C and L which regulates various immune cellfunctions including expression of cytokines. Expressionof transcripts specific for cathepsin C (Ctsc) and cathepsin L (Ctsl) in immune cell populations largely mirrored
expression of Cst7 with microglia, macrophages, andT cells being the prominent cellular sources expressingthese transcripts (Supplemental Fig. 1).
Ablation of Cst7 does not increase acute JHMV-induced
diseaseCst7-/- and control mice were infected i.c. with JHMV tomeasure viral burden within the CNS and evaluate clinical disease severity (Fig. 2A). Infected control and Cst7-/mice did not exhibit significant differences in viral titers
at day 7 p.i., indicating that control of viral replication
was not dramatically altered at this timepoint (Fig. 2B).
However, JHMV infection did result in more severeSyage et al. Journal of Neuroinflammation(2024) 21:157

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Fig. 1 Expression profile of Cst7 transcripts in CD45 + cells from JHMV-infected mice. (A) scRNAseq was conducted on CD45 + cells from brains or spinal
cords of JHMV-infected mice at defined times post-infection (p.i.) [29]. Violin plots showing expression levels of cystatin F (Cst7) within each cell cluster at(B) day 3 p.i. (brains), (C) day 7 p.i. (brains), and (D) day 21 p.i. (spinal cords). Dots shown in plots B-D represent individual cells

clinical disease in Cst7-/- mice compared to control mice
beginning at day 15 p.i., which remained more severe out
to day 21 p.i. (Fig. 2C). Cystatin F has been suggested
to play a role in suppressing target cells from secreting
immune cell chemoattractants [34, 46]. To determine ifloss of Cst7 function affected immune cell infiltration
into the CNS, we performed flow cytometric analysis on
brains of JHMV-infected mice at day 7 p.i. Representative
gating for microglia (CD11b+CD45lo) and macrophages/
myeloid cells (CD11b + CD45hi) is shown in Fig. 2D.
Using this approach, we found no significant differencein numbers of either microglia or macrophages/myeloid
cells between control and Cst7-/- mice (Fig. 2D). We next
looked at CNS inflammatory CD4 + and CD8 + T cells andvirus-specific CD4 + and CD8 + T cells via tetramer staining (Fig. 2E, F). We did not detect significant differences
in infiltration of any of these cell populations between
control and Cst7-/- mice (Fig. 2E, F). These results indicate that cystatin F is not crucial in host defense during
acute JHMV infection, as determined by control of viral
replication and immune cell infiltration into the CNS.
Cystatin F and neuroinflammation in mice persistently
infected with JHMV

JHMV-infected control mice and Cst7-/- mice were sacrificed at days 14 and 21 p.i. to assess effects of silencing
cystatin F expression on viral burden and neuroinflammation within the CNS (Fig. 3A, H). qPCR analysis ofviral RNA showed no differences in transcript levels between Cst7-/- mice and control mice in the spinal

cords at day 14 p.i. (Fig. 3B). Moreover, viral RNA levels
within the spinal cord were lower in the spinal cords ofboth Cst7-/- and control mice at day 21 p.i. and no differences in viral RNA levels detected (Fig. 3I). Thesefindings further support the notion that cystatin F is not
required for immune-mediated control of JHMV replication within the CNS. Flow cytometric analysis of spinal
cords at day 14 indicated increased numbers of microglia(p < 0.01, Fig. 3C) and total CD8 + T cells (p < 0.05, Fig. 3F)
in Cst7-/- mice compared to control mice yet there were
no differences in either macrophages (Fig. 3D), CD4 + T
cells (Fig. 3E), or virus-specific CD8 + T cells (Fig. 3G)
between Cst7-/- mice or control mice. By day 21 p.i.,
no differences were observed between Cst7-/- mice andcontrol mice with regards to microglia, macrophages or
inflammatory T cells (Fig. 3J-N).
Cystatin F influences the severity of demyelination andremyelination in JHMV-infected mice

Spinal cords were removed from uninfected and JHMVinfected Cst7-/- and control mice at days 14 and 21 p.i.
to evaluate axonal damage and the severity of demyelination (Fig. 4A). Axonal damage in spinal cords was evaluated using SMI32 staining [47, 48] and revealed increased(p < 0.05) levels of damage in Cst7-/- mice compared tocontrol mice at day 21 p.i. (Figs. 4B, C). LFB staining of
spinal cords from uninfected Cst7-/- mice showed no difference in myelin formation compared to uninfected control mice (data not shown). Compared to control mice,
JHMV infection of Cst7-/- mice resulted in an increase

Syage et al. Journal of Neuroinflammation(2024) 21:157

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Fig. 2 Cystatin F does not play a prominent role during acute disease following JHMV infection. (A) Cst7−/− mice and Cst7+/+ littermate controls were i.c.
infected with JHMV. Brains were isolated at day 7 p.i. for flow cytometric analysis and viral titers (n = 7–9), and clinical disease was recorded. (B) Viral titers
on brains at day 7 p.i. showed no significant difference between Cst7-/- (n = 7) and control mice (n = 9); level of sensitivity of plaque assay is ~ 100 PFU/g
tissue (C) Increased clinical disease from day 15 to 21 p.i. was marked by a more severe hind-limb paralysis in Cst7-/- mice (n = 26) compared to controls(n = 33). (D) Representative flow cytometric plots show microglia (CD45loCD11b+) and infiltrating macrophage/myeloid cells (CD45hiCD11b+) in the brains
of infected Cst7-/- (n = 8) or control mice (n = 6). Quantification of flow data indicates no difference in microglia or macrophage/myeloid cell numbers. (E)Representative staining for CD8 + and S510-518 tetramer revealed no difference in infiltrating CD8 + T cells and virus-specific CD8 + T cells between infected Cst7-/- and controls. (F) Staining for CD4 + and M133-147 tetramer showed similar numbers of infiltrating CD4 + T cells (p = 0.66) and virus-specificCD4 + T cells between infected groups. Data are presented as average ± SEM; ns - not significant; *p < 0.05, **p < 0.01

Syage et al. Journal of Neuroinflammation(2024) 21:157

Page 8 of 17

Fig. 3 Silencing of Cst7 enhances immune cell infiltration into the spinal cord during early chronic disease following JHMV infection. (A) Cst7-/- andcontrol mice were i.c. infected with JHMV, and spinal cords were isolated at day 14 p.i. to evaluate immune cell infiltration and viral burden. (B) There were
no differences in viral RNA between infected Cst7-/- mice (n = 3) and controls (n = 3) at day 14 p.i. Quantification of flow cytometric data from spinal cords
comparing (C) microglia (CD45loCD11b+), (D) macrophage/myeloid cells (CD45hiCD11b+), (E) CD4+ T cells, (F) CD8+ T cells, and (G) virus-specific CD8+ T
cells (CD8+S510-518 tetramer+) between control (n = 5) and Cst7-/- (n = 8) mice demonstrates amplified infiltration of CD8 + T cells (p < 0.05) and microglia(p < 0.01) in Cst7-/- mice at day 14 p.i. (H) Cst7-/- and control mice were i.c. infected with JHMV, and spinal cords were isolated at day 21 p.i. to evaluate
immune cell infiltration and viral burden. (I) No differences in viral RNA between infected Cst7-/- mice (n = 5) and controls (n = 5) were found at day 21
p.i. Quantification of flow cytometric data from spinal cords comparing (J) microglia, (K) macrophage/myeloid cells, (L) CD4+ T cells, (M) CD8+ T cells, and(N) virus-specific CD8+ T cells between control and Cst7-/- animals demonstrates no differences in immune cell populations in Cst7-/- mice at day 21p.i.,(n = 5). Data are presented as average ± SD; ns - not significant; *p < 0.05; **p < 0.01

in spinal cord demyelination at days 14 (p < 0.05) and 21(p < 0.001) (Fig. 4D-F). Electron microscopic (EM) analysis of spinal cord sections was performed to evaluate ifremyelination was impacted by the absence of cystatin
F. High magnification (1200X) images of the ventral

and lateral funiculus of spinal cords of JHMV-infected
Cst7-/- mice, compared to controls, at day 21 p.i. wereused to assess differences in remyelination. Measurement of g-ratio, the ratio of the inner axonal diameterto the total outer fiber diameter, is commonly employedSyage et al. Journal of Neuroinflammation(2024) 21:157

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Fig. 4 Cystatin F is necessary for remyelination following JHMV-induced demyelination. (A) Cst7−/− mice and littermate controls were i.c. infected with
JHMV, and spinal cords were isolated at days 14 and 21 p.i. (B) Representative images showing increased SMI32 staining in spinal cords of JHMV-infected
Cst7-/- mice compared to control mice; upper panels display DAPI (blue), fluoromyelin (green), and SMI32 (red); lower panels show only SMI32 staining at
day 21 p.i. (C) Quantification of SMI32 staining in Cst7-/- mice (n = 7) and control mice (n = 7). (D) Representative spinal cord tissue stained with H&E andLFB (stains myelin fibers blue) from infected Cst7-/- and control mice at day 21 p.i., revealing demyelination within the ventral funiculus and lateral whitematter columns (outlined by black line). Quantification of spinal cord demyelination at (E) day 14 p.i. (n = 4, control; n = 6, Cst7-/-) and (F) day 21 p.i. (n = 15,
control; n = 12, Cst7-/-) between control and Cst7-/- mice. (G) Electron micrographs showing ultrathin spinal cord sections from control and Cst7-/- miceat day 21 p.i. highlighting myelinated, demyelinated, remyelinated axons and degenerated axons. Histological comparisons in the ventral funiculus andlateral white matter columns between Cst7-/- mice (n = 6) and controls (n = 6) were made from electron micrographs, including (H) ratios of myelinated,demyelinated axons, and remyelinated axons calculations, and (I) g-ratios (axon diameter/total fiber diameter), Scale bar for F is 2 μm. For H and I, 133–660
axons were analyzed per mouse. For SMI-32 analysis, data represents mean ± SD. All other data represent the mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001

Syage et al. Journal of Neuroinflammation(2024) 21:157

as a structural index of remyelination, with lower ratios
indicating more extensive myelination [36, 37, 49]. Representative EM images revealed an overall increase indemyelinated axons, with accompanying decreased
remyelination in Cst7-/- demyelinated axons, withaccompanying decreased remyelination in Cst7-/- micecompared to control mice (Fig. 4G). Cst7-/- mice exhibited a significant (p < 0.05) decrease in the ratio of myelinated and remyelinated axons and an increase in the ratio(p < 0.001) of demyelinated axons compared to infected
control mice (Fig. 4H). These data correlated with theincreased (p < 0.01) g-ratios in spinal cord axons from
infected Cst7-/- mice compared to control mice, further
indicating that remyelination is impaired in the absenceof cystatin F (Fig. 4I).
scRNAseq reveals immune cell diversity between Cst7-/and control mice following JHMV infection

To better understand the molecular mechanisms bywhich cystatin F impacts neuroinflammation and demyelination, we performed scRNAseq on spinal cords of
JHMV-infected Cst7-/- and wildtype littermate control mice at day 21 p.i. CD45 + cells were flow sorted
and enriched, and subsequently Cell Multiplexing Oligo(CMO) labeled and pooled before undergoing scRNAseq. The total number of cells from uninfected control(3,347 cells) and Cst7-/- (3,371 cells) and JHMV-infected
control (3,840 cells) and Cst7-/- (3,350 cells) groups wereused for unbiased clustering analysis that produced 21
unique cell clusters in combined uninfected and infected
mice (Fig. 5A, B). To corroborate the algorithm-assisted
identification of clusters, we assessed expression of
known cellular markers in our dataset and found that
expression of these markers corresponded with respective cell cluster identities (Fig. 5C). Uninfected control
mice showed limited numbers of microglia subpopulations expressing Cst7 transcripts, while Cst7 expression
was undetectable in Cst7-/- mice, confirming theCst7em1Aduci allele as being null (Fig. 5D). Cst7 transcripts
were detected in microglia/macrophage and T cells populations in the spinal cords of JHMV-infected mice at day
21 p.i. but were again not detected in Cst7-/- mice, as
expected (Fig. 5D). These findings indicate that low levelsof Cst7 transcripts are present within the CNS of healthy
uninfected mice and are increased under neuroinflammatory conditions following JHMV infection.
Loss of Cst7 function produces altered expression of genes
related to phagocytosis and remyelination following JHMV
infection

We have previously reported increased transcription ofgenes associated with remyelination in the spinal cords of
JHMV-infected mice, including Cst7, Igf1 (insulin growth
factor 1), and Lpl (lipoprotein lipase). There was one

Page 10 of 17

microglia subpopulation (MG1) within the spinal cords
of JHMV-infected control mice that expressed all threetranscripts (Fig. 6A, C). Further examination indicateddecreased expression of Igf1 transcripts in MG1 cells
from Cst7-/- mice compared to control mice, while there
were no differences in expression of Igf1 and Lpl in MG1
between infected controls and Cst7-/- mice (Fig. 6A,
C). Expression of these transcripts within inflammatory
monocytes and macrophages revealed a broader distribution of expression in subpopulations of these cells;
though, neither population of cell was a prominent cellular source of these transcripts as compared to microglia (Fig. 6B, D). Macrophage subpopulations, Mac1 andMac2, trended to be a more prominent source for Cst7
and Igf1 transcripts, respectively, whereas inflammatory monocytes expressed higher levels of Lpl (Fig. 6D).
Expression of Lpl transcripts was reduced (p < 0.01) ininflammatory monocytes from infected Cst7-/- micecompared to controls (Fig. 6D). These findings indicatethat silencing of Cst7 did not dramatically affect expression of transcripts associated with remyelination in
microglia or monocytes/macrophages in JHMV-infected
mice, suggesting that alternative mechanisms were associated with increased white matter damage and reduced
repair. Expression of transcripts encoding anti-inflammatory mediators, including IL-10 (Il10), TGFβ (Tgfb1), andAnnexin A1 (Anxa1), were also evaluated in inflammatory myeloid and T cells. No significant changes in thesetranscripts were detected in spinal cord microglia (Supplemental Fig. 2A) and monocytes/ macrophages (Supplemental Fig. 2B). For inflammatory CD4 + T cells, nodifference in Anxa1 and Tgfb1 transcripts were detected
yet Il10 transcripts were increased (p < 0.05) in CD4 + T
cells in Cst7-/- mice compared to controls (Supplemental
Fig. 2C) while no differences in any of these transcripts
were detected in inflammatory CD8 + T cells (Supplemental Fig. 2D).
Impaired phagocytosis of myelin debris by myeloid
cells has been associated with impaired remyelination in
JHMV-infected mice [28, 36, 50]. Therefore, one potential pathway associated with inefficient remyelination in
JHMV-infected mice includes reduced phagocytosis ofmyelin debris by microglia. Within the spinal cords of
JHMV-infected Cst7-/- mice, there was increased expression of transcripts associated with phagocytosis within
spinal cord microglia, including Abca1 (ATP binding
cassette subfamily A member 1, p < 0.05) and Apoe (apolipoprotein E, p < 0.05), compared to infected controls at
day 21 p.i. (Fig. 6E), while there were no differences in
the number of Abcg1 transcripts (ATP binding cassette
subfamily G member 1) between infected Cst7-/- andcontrol mice (Fig. 6E). Within spinal cord monocytes/
macrophages, there were increased (p < 0.001) of Abca1,
Abcg1, and Apoe transcripts in infected Cst7-/- mice

Syage et al. Journal of Neuroinflammation(2024) 21:157

Page 11 of 17

Fig. 5 ScRNAseq reveals the immunological landscape within spinal cords of JHMV-infected Cst7-/- mice during chronic disease. Cst7−/− mice (n = 6) andcontrols (n = 6) were i.c. infected with JHMV, spinal cords from JHMV infected and uninfected mice were isolated day 21 p.i., and CD45 + cells were sorted
for scRNAseq. (A) UMAP plot of scRNAseq data revealing 21 distinct cell clusters (aggregate data from uninfected and JHMV-infected Cst7-/- mice andcontrols at day 21 p.i.). (B) UMAP plots showing the immune landscape in uninfected and JHMV-infected Cst7-/- and control mice at day21 p.i. (C) Dot
plot presenting expression of cell type specific markers within the 21 cell clusters. Dot size is representative of the percentage of cells from the clusterthat express the gene, while the degree of color intensity exhibits the average expression level of the gene. The dashed boxes highlight commonly anduniquely expressed genes of clusters within overarching cell types. (D) Expression pattern and levels of Cst7 transcripts across each sample

compared to infected control mice (Fig. 6F). These findings indicate that deletion of Cst7 increases expressionof genes associated with phagocytosis within myeloid
cells and that deficient phagocytosis of myelin debris in
JHMV-infected Cst7-/- mice may not be responsible for
deficient remyelination, but instead could be representative of a response to the elevated levels of demyelination
found in Cst7-/- mice.
Increased transcripts of monocyte/macrophage and T cellchemoattractant chemokines in spinal cords of JHMVinfected Cst7-/- mice

We have previously shown that chemokines associatedwith macrophage and T cell recruitment contributeto increased demyelination in JHMV-infected mice
[49, 51, 52]. Cystatin F has been linked with influencing genes associated with inflammation and regulating
T cell effector function [34, 53–56]. Therefore, we next
examined if increased demyelination in JHMV-infected
Cst7-/- mice correlated with dysregulated expression ofproinflammatory chemokines, leading to increased T
cell and myeloid cell accumulation in the spinal cord. We
examined expression of chemokine transcripts encoding monocyte/macrophage chemokines, Ccl2 and Ccl5;
T cell chemoattractant chemokines, Cxcl9 and Cxcl10;
and Tnf-alpha in monocytes, macrophages, and microglia within the spinal cords of infected Cst7-/- and control mice at day 21 p.i. The level of transcripts encoding

Syage et al. Journal of Neuroinflammation(2024) 21:157

Page 12 of 17

Fig. 6 Cst7 ablation produces altered expression of genes related to remyelination and phagocytosis following JHMV infection. UMAP plots highlighting (A) microglia and (B) monocyte/macrophage clusters. Expression of transcripts encoding remyelination markers cystatin F (Cst7), insulin-like growth
factor-1 (Igf1), and lipoprotein lipase (Lpl) in (C) microglia and (D) monocytes/macrophages. For C and D, top rows show comparisons between clusters,
while bottoms rows compare between uninfected and JHMV-infected Cst7-/-, and control mice, in specific clusters. Expression of phagocytic markers was
examined in uninfected and JHMV-infected Cst7-/- and control mice, revealing an overall increase in expression of Abca1, Abcg1, and Apoe (apolipoprotein-E) in infected Cst7-/- compared to controls in both (E) microglia and (F) monocyte/macrophage clusters. In C-F, normalized expression values wereused, and random noise was added. Box plots show interquartile range, median value (bold horizontal bar), and average expression per sample (red dot).
Wilcoxon test was used; ns (not significant) p > 0.05, *p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001

Ccl2 was significantly (p < 0.001) increased in Cst7-/mice compared to control mice in inflammatory monocytes but not macrophages or microglia (Fig. 7A). There
was no difference in expression of Ccl5 in monocytes,
macrophages, or microglia between controls and Cst7/- mice (Fig. 7A). Expression of Cxcl9 was significantly
increased in monocytes (p < 0.01), macrophages (p < 0.05)
and microglia (p < 0.001) in Cst7-/- mice compared tocontrols (Fig. 7A), while expression of Cxcl10 was only
significantly (p < 0.001) increased in monocytes of Cst7/- mice compared to controls (Fig. 7A). Tnf transcripts
were only elevated in macrophages (p < 0.05) and microglia (p < 0.001) in Cst7-/- mice compared to control mice
(Fig. 7A). We also determined that IFN-g response signatures in microglia (p < 0.006) (Fig. 7B) and inflammatory

monocytes/macrophages (p < 0.002) (Fig. 7C) from
infected Cst7-/- mice were increased in CD8 + T cells(p < 0.002) and CD4 + T cells (p < 0.005) from Cst7-/compared to control mice (Fig. 7D-F). Expression of
IFN-g transcripts was elevated in both effector (p < 0.05)
and cycling (p < 0.001) CD8 + T cells (Fig. 7G) but neither
CD4 + T cells or memory CD4 + T cells (Fig. 7H) from
infected Cst7-/- mice compared to controls. Examination of other markers of CD8 + T cell activation indicatedno differences between Cst7-/- and control mice in transcripts encoding granzyme B (Gzmb) in either effector orproliferating CD8 + T cells (Fig. 7). Expression of perforin
transcripts (Prf1) was increased (p < 0.01) in proliferating
CD8 + T cells from Cst7-/- mice compared to controls(Fig. 7G). For CD4 + T cells, there was only increased

Syage et al. Journal of Neuroinflammation(2024) 21:157

Page 13 of 17

Fig. 7 Cst7 ablation augments T cell activation and chemokine expression. (A) Levels of transcripts encoding macrophage chemoattractants, Ccl2 andCcl5, T cell chemoattractants, Cxcl9 and Cxcl10, and tumor necrosis factor-alpha (TNF-α, Tnf) in monocyte, macrophage, microglia populations. Gene set
enrichment analysis (GSEA) for IFN-γ responses in combined microglia (B) and monocyte/macrophage (C) clusters revealed elevated expression of genes
related to IFN-γ responses in JHMV-infected Cst7-/- mice compared to controls. (D) UMAP plot highlighting CD8+ T cell clusters (Eff. CD8+, Cyc. CD8+,
Mem. CD8+) and CD4+ T cell clusters (CD4+, Mem. CD4+). IFN-γ responses were also amplified in combined CD8+ T cells (E) and CD4+ T cells (F) in infected
Cst7-/- mice compared to controls. (G) Eff. and Cyc. CD8+ T cells from infected Cst7-/- mice have increased expression of CD8+ T cell activation markers,
granzyme b (Gzmb), perforin (Prf1), and IFN-γ (Ifng), compared with controls. Furthermore, expression levels of CD4+ T cell activation markers, Cd44 (CD44),
Icos (inducible T cell co-stimulator), and Ifng (IFN-γ), were augmented in CD4+ T cells and Mem. CD4+ T cells from infected Cst7-/- mice compared tocontrols (H). In A, G-H, normalized expression values were used, and random noise was added. Box plots shows interquartile range, median value (boldhorizontal bar), and average expression per sample (red dot). Wilcoxon test was used; ns - not significant; *p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001(p < 0.05) expression of transcripts encoding activation
marker CD44 (Cd44) in memory CD4 + T cells from
Cst7-/- mice, with no differences in transcripts encoding
for inducible T cell co-stimulator (ICOS, Icos) or IFN-g
in either CD4 + T cells or memory CD4 + T cells between
Cst7-/- and control mice (Fig. 7H). These findings indicate that in the absence of cystatin F, there is enhanced
expression of proinflammatory chemokines, associatedwith increased inflammatory monocytes/macrophages
and activated T cells leading to white matter damage.

Discussion
The ubiquitously expressed cystatin C has received more
attention in the realm of neurodegenerative diseases,
including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and MS, for its inhibitory effect on cathepsin
B [57–59]. However, the role of cystatins and their target

Syage et al. Journal of Neuroinflammation(2024) 21:157

cathepsins in CNS disease remains a relatively new fieldof research and not well characterized. Unlike cystatin
C, cystatin F is secreted as an inactive disulfide-linked
dimer and only becomes active upon proteolytic cleavage of 15 N-terminal residues when endocytosed by target cells, where it is trafficked to endosomal/lysosomal
compartments, suggesting that its primary function is tomodulate surrounding cells, in addition to acting in an
autocrine manner [60–62]. Cystatin F is more commonly
known for its use by cancer cells to immunosuppress
granule-mediated cytotoxicity of effector CD8 + T cells
and NK cells [55, 56, 63, 64].
Cystatin F has recently been found to play a role inmodulating disease severity in pre-clinical mouse models of demyelination, including experimental autoimmune encephalomyelitis (EAE), as well as toxin andgenetic models of disease [31–34]. In addition, cystatin F
has been found at the leading edges of plaques in postmortem tissue from MS patients, further supporting a
potential role in both demyelination and remyelination[31–34]. Expression of Cst7 transcripts is dramaticallyreduced in mice in which microglia have been ablated,
and this was associated with increased demyelination
and reduced remyelination in response to JHMV infection, arguing for a protective role for cystatin F in regulating chronic viral-induced neurologic disease [30]. The
current study was undertaken to better understand thefunctional role of cystatin F in regulating host immune
responses following JHMV infection of the CNS, as well
as further evaluate its role in influencing neuroinflammation, demyelination, and remyelination using mice lacking Cst7 function.
Previous studies have shown that microglia are important in aiding in host defense as well as in limiting demyelination and enhancing remyelination in response to
JHMV infection [36]. This likely involves a multitude of
specialized microglial functions, including by enhancing antigen presentation to CNS infiltrating T cells,
phagocytosis of myelin debris, which inhibits OPC differentiation and remyelination [65–69], recruiting andpromoting differentiation of OPCs [70, 71], and altering
the microenvironment to encourage remyelination andsurvival of OPCs via secretion of growth factors and signaling mediators [72–76]. Microglia depletion prior to
JHMV infection leads to an increase in mortality associated with impaired control of viral replication, decreased
expression of MHC class II and muted CD4 + T cell
responses highlighting an important role for microglia inhost defense following JHMV infection of the CNS [27,30]. Viral replication within the brains of JHMV-infected
Cst7-/- was not altered compared to infected control
mice and there were no differences in either clinical disease severity or mortality during acute disease arguingthat cystatin F does not play a critical role in generation

Page 14 of 17

of an effective anti-viral T cell response. Targeted depletion of microglia also leads to increased demyelinationand limited remyelination, associated with reduced levelof transcripts for Igf1, Lpl, and Cst7 [77–79]. Impaired
remyelination in JHMV-infected Cst7-/- mice was not
associated with a dramatic alteration in transcripts
encoding either Igf1 or Lpl, indicating that cystatin F
may not contribute to remyelination by modulation ofremyelination-associated gene expression. More recently,
remyelination in JHMV-infected mice has been shown tobe related to efficient phagocytosis of myelin debris, andmicroglia appear to be critical in this process [28, 36, 50].
In the absence of Cst7 expression in JHMV-infected mice,
there was an increase in expression of transcripts associated with myeloid cell phagocytosis/lipid metabolism,
including Apoe, Trem2, and Cd36, in the Cst7-/- micecompared with controls, which argues against impaired
myelin phagocytosis by either microglia and/or inflammatory monocytes/macrophages.
Cystatin F is known to act through inhibiting its target protease, cathepsin C, which cleaves pro-enzymes,
including granzymes in CD8 + T and NK cells, which
regulates cytotoxicity of these cells [46, 55, 56, 63, 64].
Furthermore, cathepsin C has also been linked withregulating microglia activation and expression of proinflammatory genes [46, 58, 80, 81]. Overexpression ofcathepsin C aggravates neuroinflammation in a model oftraumatic brain injury (TBI) via increased expression ofmacrophage chemoattractant chemokine, Ccl2, and neutrophil chemoattractant Cxcl2 [53], and genetic silencing
of cystatin F resulted in increased demyelination, associated with increased Cxcl2 gene expression in the cuprizone model of demyelination [34]. Consistent with thesefindings are our results indicating increased expressionof transcripts encoding proinflammatory chemokines by
inflammatory monocytes/macrophages and microglia.
Our findings support and extend these earlier studies anddemonstrate that, in the absence of cystatin F, there isincreased expression of transcripts encoding the monocyte/macrophage attractant chemokine, Ccl2, as well as
T cell chemoattractant chemokines, Cxcl9 and Cxcl10,
in inflammatory monocytes/macrophages and microglia
in infected Cst7-/- mice compared to controls. Whether
this is due to specific roles for cystatin F in influencing
expression of these chemokines in myeloid cells is notknown and warrants further investigation. We have previously demonstrated an important role for both CXCL9and CXCL10 in attracting T cells into the CNS of JHMVinfected mice which amplify spinal cord demyelination[8, 49, 82–84]. In addition, CCL2 has an important rolein attracting monocyte/macrophages into the CNS that
increases demyelination severity [51, 52]. The increase in
severity of spinal cord demyelination in JHMV-infected
Cst7-/- mice was associated with increased numbers ofSyage et al. Journal of Neuroinflammation(2024) 21:157

microglia and CD8 + T cells at day 14 p.i. when compared
to control mice. While demyelination worsened by day
21 p.i. in infected Cst7-/- mice, there were no significant
differences in immune cell infiltration within the spinal
cords between Cst7-/- mice and control mice. However,
we did identify increased expression of both IFN-g andperforin transcripts in CD8 + T cells present in the spinal
cords of infected Cst7-/- mice. With regards to inflammatory T cells, Ifng transcripts were increased in both effector and cycling CD8 + T cells in Cst7-/- mice yet were
not detected in CD4 + T cells or memory CD4 + T cells.
Nonetheless, increased expression of CD8 + T cell IFN-g
was associated with elevated IFN-g responses by myeloid
cells as well as CD8 + and CD4 + T cells, indicating that,in the absence of cystatin F, there is a heightened stateof immune cell activation, and this is associated withincreased myelin damage. In further support of this arestudies from our laboratory and others demonstrating
both IFN-g and CXCL10 increase apoptosis of oligodendrocyte progenitor cells, and this would further explain
the increased in demyelination and paucity in spinal cord
remyelination in JHMV-infected Cst7-/- mice [85, 86].
Increased expression of perforin transcripts in proliferating CD8 + T cells from Cst7-/- mice suggests enhanced
cytolytic activity by these cells. Cystatin F is an important
regulator of cytolytic activity in both NK cells and T cells[54, 55], and increased cystatin F levels correlate with
reduced CTL cytotoxicity [56]. Increased CTL activity/
perforin secretion may also be contributing to the extensive spinal cord demyelination observed in the Cst7-/mice [87, 88]. It is also important to mention that we did
not detect changes in transcripts encoding anti-inflammatory cytokines IL-10, TGFb, and Annexin 1. Nonetheless, it is inevitable that germline ablation of Cst7 affects
gene expression in other cells types specifically resident
cells of the CNS e.g. astrocytes, oligodendroglia and neurons that may have impact neuroinflammation as well as
demyelination. We are currently examining the impact ofgene expression in these cells following JHMV infectionof Cst7-/- mice. Collectively, these findings indicate that
influencing cystatin F expression within the CNS may
offer the opportunity to regulate inflammation and/or
pathogenic T cell activity, which may impact the severityof neuroinflammation and demyelination.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03153-0.
Supplementary Material 1
Acknowledgements
The authors acknowledge the Chao Family Comprehensive Cancer Center’s
support of the Transgenic Mouse Facility shared resource that is funded, inPage 15 of 17part, by the National Cancer Institute of the National Institutes of Health underaward number (P30CA062203) at the University of California, Irvine.
Author contributions
A.R.S., K.N.G. and T.E.L. designed and performed experiments, interpreted data,prepared figures, and wrote the main manuscript text. C.H., S.K., and G.R.M.
generated the Cst7-/- mice. C.P., K.M.M., K.F., A.D., Y.C. and G.O. performedexperiments and assisted in preparation of figures.
Funding
This work was supported by funding from the National Institutes of Health(NIH) grant R35NS116835 to TEL; NIH awards R01NS083801, RF1AG056768,
and RF1AG065329 to K.N.G. G.O. was supported by NIH Training Grant
T32AI007319-34. A.R.S. was supported by NIH Microbial Pathogenesis Training
Grant T32AI055434.
Data availability
All data and materials will be made available upon request to thecorresponding author. The Cst7-/- mice (C57BL/5J-Cst7em1Aduci/J, Strain#:037727) have been deposited at The Jackson Laboratory. The RNAseq data
will be deposited in NCBI’s Gene Expression Omnibus.

Declarations
Ethical approval
All animal work performed in this study was conducted following approval by
the UC Irvine (USA) Institutional Animal Care and Use Committee.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Neurobiology & Behavior, School of Biological Sciences,
University of California, Irvine 92697, USA
2
Department of Molecular Biology & Biochemistry, School of Biological
Sciences, University of California, Irvine 92697, USA
3
Transgenic Mouse Facility, ULAR, Office of Research, University ofCalifornia, Irvine 92697, USA
4
Department of Developmental & Cell Biology, University of California,
Irvine 92697, USA
5
Center for Virus Research, University of California, Irvine 92697, USA
Received: 14 September 2023 / Accepted: 12 June 2024

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