Bergersen et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03176-7(2024) 21:189

Journal of Neuroinflammation

Open Access

RESEARCH

Toxoplasma infection induces an aged
neutrophil population in the CNS that is
associated with neuronal protection
Kristina V. Bergersen1, Bill Kavvathas1, Byron D. Ford1,2 and Emma H. Wilson1*
Abstract
Background Infection with the protozoan parasite Toxoplasma gondii leads to the formation of lifelong cysts inneurons that can have devastating consequences in the immunocompromised. In the immunocompetent individual,
anti-parasitic effector mechanisms and a balanced immune response characterized by pro- and anti-inflammatory
cytokine production establishes an asymptomatic infection that rarely leads to neurological symptoms. Several
mechanisms are known to play a role in this successful immune response in the brain including T cell production of
IFNγ and IL-10 and the involvement of CNS resident cells. This limitation of clinical neuropathology during chronic
infection suggests a balance between immune response and neuroprotective mechanisms that collectively prevent
clinical manifestations of disease. However, how these two vital mechanisms of protection interact during chronic
Toxoplasma infection remains poorly understood.
Main text This study demonstrates a previously undescribed connection between innate neutrophils foundchronically in the brain, termed “chronic brain neutrophils” (CBNeuts), and neuroprotective mechanisms during
Toxoplasma infection. Lack of CBNeuts during chronic infection, accomplished via systemic neutrophil depletion,
led to enhanced infection and deleterious effects on neuronal regeneration and repair mechanisms in the brain.
Phenotypic and transcriptomic analysis of CBNeuts identified them as distinct from peripheral neutrophils andrevealed two main subsets of CBNeuts that display heterogeneity towards both classical effector and neuroprotectivefunctions in an age-dependent manner. Further phenotypic profiling defined expression of the neuroprotective
molecules NRG-1 andErbB4 by these cells, and the importance of this signaling pathway during chronic infection wasdemonstrated via NRG-1 treatment studies.
Conclusions In conclusion, this work identifies CBNeuts as a heterogenous population geared towards both classicalimmune responses and neuroprotection during chronic Toxoplasma infection and provides the foundation for future
mechanistic studies of these cells.
Keywords Neutrophils, Brain, Chronic infection, Toxoplasma gondii, Neuroprotection, Immune response*Correspondence:
Emma H. Wilson
emmaw@ucr.edu
1
Division of Biomedical Sciences, School of Medicine, University ofCalifornia, Riverside, Riverside, CA, USA
2
Present address: College of Medicine, Howard University, Washington,
D.C., USA
© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

Introduction
Ingestion of the protozoan parasite Toxoplasma gondii
leads to a lifelong infection characterized by the formation of cysts inside neurons of the brain. Prevalence rates
vary, but it is estimated that one third of the world’s population is currently infected with Toxoplasma [1]. While
infection is often asymptomatic due to constant pressureof the host immune response, an immunocompromised
state can result in Toxoplasmic encephalitis which is fatal
if left untreated [2]. The presence of the parasite in thebrain leads to recruitment of both innate and adaptive
immune cells which are required to control parasite replication while maintaining a balanced tissue environment[3–6].
Innate neutrophils and chemokine-dependent neutrophil recruitment have previously been identified as vital
sources of protection against acute Toxoplasma infection[7–10]. Two independent research groups have recently
identified a small but defined population of these cells inthe brain between 2- and 4-weeks post infection (wpi) [5,11]. The Dunay group has partially characterized thesecells and has also shown that depletion of neutrophils at
the start of chronic infection leads to increased parasiteburden and inhibited immune cell recruitment to thebrain [5]. The presence of neutrophils in the brain during
chronic infection suggests a more versatile role of thesecells than previously thought. Literature focusing on
potential alternative roles for neutrophils in non-Toxoplasma models describes two basic classes of these cells
based on their differential expression of markers such as
CD15 and CD49d among others [12–14]. More recent
works investigating neutrophils in homeostasis, tissue
injury, and non-Toxoplasma models of infection have
expanded upon this to include a much broader range ofpreviously undescribed neutrophil heterogeneity [15–18]. Therefore, the chronic presence of neutrophils in thebrain may point to an important yet undefined role forthese innate immune cells in maintaining brain homeostasis during chronic Toxoplasma infection.
Despite cyst-containing neurons and brain inflammation caused by peripheral immune cells like neutrophils,
Toxoplasma infection causes few clinical symptoms in the
immunocompetent host. However, underlying changes inneurochemistry and significant changes in the health ofneurons suggest a constant need to balance inflammationand protect against neuropathology [19, 20]. Recent workin our lab to determine Toxoplasma-induced changes in
immunological and neurological transcripts in the brain
supports these sub-clinical neurological changes, but also
reveals potential activated neuroprotective pathways thatremain poorly understood [21]. The concept of “neuroprotection” (prevention of neuronal death and maintenance of neuronal homeostasis during injury/infection)
has previously been investigated in models of central

Page 2 of 16

nervous system (CNS) injury, such as ischemic strokeand spinal cord injury, and during experimental cerebral
malaria (ECM) infection. In these models, neuroprotective functions are activated predominantly via signalingof the endogenous ligand Neuregulin 1 (NRG-1) throughits main receptor ErbB4 [22, 23]. Furthermore, resolution of neuropathology in a model of ischemic stroke
occurs through the clearance of damage signals, debris,and revascularization by macrophage scavenger receptor
1 (MSR1) [24]. MSR1 transcripts are upregulated during Toxoplasma infection and correlate with increasingchronicity [21]. While the sources of these three molecules and their contribution to neuroprotection havebeen investigated in other disease models, their cellularsources and roles in protecting against neuropathology
during chronic Toxoplasma infection remain unknown.
In this study, we present a previously undescribed connection between “chronic brain neutrophils” (CBNeuts)
and neuroprotective mechanisms during chronic Toxoplasma infection. Lack of CBNeuts after chronic infection was well-established led to a decrease in neuronal
regeneration suggesting a potential undefined role ofthese cells in neuroprotection. Phenotypic characterization of CBNeuts identified their expression of neuroprotective molecules including NRG-1, ErbB4, and MSR1
among others. CBNeuts had distinct phenotypic andtranscriptomic profiles compared to peripheral neutrophils (PNeuts) including CNS-specific signatures thatfurther distinguish two distinct CBNeut populations. The
combined data reveal that CBNeuts are capable of acting simultaneously as immune effector and neuroprotective cells and may play a vital flexible role in maintaining
brain homeostasis during chronic Toxoplasma infection.

Materials and methods
Animals, parasites, and mouse infections/kinetics studies

Animals. All research was conducted in accordance with
the Animal Welfare Act, and all efforts were made tominimize suffering. All protocols were approved by theInstitutional Animal Care and Use Committee (IACUC)
of the University of California, Riverside. Female 6–8
week old WT C57BL/6J mice were obtained from Jackson Laboratories and were maintained in a pathogen-freeenvironment in accordance with IACUC protocols at the
University of California Riverside.
Parasites and Infections. The ME49 strain of T. gondii
was maintained in cyst form by continuous passaging in
SW and CBA/J mice. Female 6–8 week old WT C57BL/6J
mice were infected with 10 ME49 cysts per mouse in
200 µl of sterile 1x PBS solution via intraperitoneal (IP)injection. Naïve controls received 200 µl of sterile 1x PBS
solution via IP injection.
Mice were infected as described above and sacrificed at
the following acute and chronic time points: 1week post

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

infection (wpi) (an acute time point where little/no cyst
formation is seen in the brain), 2wpi (an early chronic
time point where cysts are actively forming and immune
cells are recruited), 4wpi and 6wpi (2 mid-chronic time
points where cysts have been fully formed and a chronic
inflammatory state is well-established), and 11wpi (a latechronic time point where increased parasite reactivation
can sometimes be seen). At each time point, half brain
and whole spleen were harvested for flow cytometry,
and the other half brain and one lobe of liver were harvested for immunohistochemistry, Western blot, or parasite burden, respectively. For single cell RNA sequencing
experiments, whole brain was used.
Neutrophil depletion and NRG-1 treatment studies

Neutrophil Depletion. After 4 weeks of infection, whenchronic infection was well-established, a cohort ofinfected mice were injected with 500 µg/mouse of Ly6G
depleting mouse monoclonal antibody in vehicle (sterile
1x PBS) via 200 µl IP injection every other day for 14 days
according to previously published protocols [5]. A cohort
of infected control mice received 200 µl/mouse of vehicle(sterile 1x PBS) intraperitoneally every other day for 14
days to control for depletion injections.
NRG-1 Treatment. After 2 weeks of infection, whencysts were actively forming in the brain, a cohort ofinfected mice were injected with 5 µg/kg/day of NRG-1(R & D Systems) in vehicle (sterile 1x PBS, 0.1% BSA)
via 100 µl IP injection every day for 7 days according topreviously published protocols [23]. A cohort of infected
untreated control mice received 100 µl/mouse of vehicle(sterile 1x PBS, 0.1% BSA) intraperitoneally every day for
7 days to control for treatment injections.
Western Blot. Half brains were harvested 24 h after
NRG-1 treatment completion and placed in RIPA buffer.
Protein was extracted, normalized, and run on a Western
blot according to previously published protocols [19].
Blots were imaged separately for GLT-1 and β-Actin as acontrol and analyzed using ImageJ software.
Parasite burden and B1 gene analysis

Parasite burden in brain and peripheral liver was quantified as previously described [21, 25]. Briefly, DNA fromhalf brains and liver from naïve, infected control, andinfected neutrophil-depleted mice (n = 5/group) was
extracted and purified using a High Pure PCR Template
Prep Kit (Roche). DNA concentration of each sample wasdetermined via NanoDrop, and all DNA was normalized to 12.5 ng/µl before amplification. Parasite burden
was measured by amplifying the B1 gene of T. gondii by
RT-PCR.

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Cell processing for flow cytometry and cell sortingNaïve and infected female C57BL/6J mice were sacrificed and perfused intracardially with 20 mL of sterile 1x
PBS. Blood, spleens and half brains were harvested, andsplenocytes and brain mononuclear cells (BMNCs) were
processed according to previously published protocols[19, 25]. For neutrophil localization studies, whole brain
was harvested and dissected into 3 broad regions (cortex,
mid-brain region, and cerebellum) using brain morphology to define regions for each mouse. BMNCs were thenprocessed as above.
Flow cytometry

Processed cells were diluted with FACS buffer (4 g BSA,
50 mg EDTA, 1 L 1xPBS) to 1.0 × 106 cells/ml (or all cells
from respective brain regions) and transferred to FACS
tubes for staining. Cells were incubated with 1:10 FC
Block (BD) for 10 min on ice followed by fluorophoreconjugated or primary unconjugated antibodies for surface staining for 30 min protected from light. Cells were
washed with FACS buffer solution and incubated influorophore-conjugated secondary antibodies as neededfor 30 min protected from light. Fluorophore-conjugatedantibodies used during surface staining are as follows:
CD45 PE (Invitrogen), CD11b APCCy7 (Invitrogen),
Ly6G PerCPcy5.5 (Clone 1A8, BD), CD62-L APCCy7(BD), CD3 FITC (BD), CD4 APC (Invitrogen), CD4
APCCy7 (Invitrogen), CD8 PECy7 (Invitrogen), CD11b
PerCPCy5.5 (eBioscience), CD11b APC (eBioscience),
Ly6G BV510 Clone 1A8 (eBioscience), CXCR4 PECy5.5(ThermoFisher), and MMP9 AF647 (SantaCruz Biotech).
Primary unconjugated antibodies and their corresponding fluorophore-conjugated secondary antibodies are
as follows: NRG-1 primary antibody (Invitrogen) with
Alexafluor 647-conjugated secondary antibody (ThermoFisher); NRG-1 primary (Santa Cruz Biotech) with Alexafluor 568-conjugated secondary (ThermoFisher); MSR1
primary (Invitrogen) with Alexafluor 488-conjugated or
Q Dot 655-conjugated secondary (ThermoFisher); VEGF
primary (NovusBio) with Alexafluor 680-conjugated secondary (ThermoFisher); and biotinylated CD15 primary(Invitrogen) with PECy7-conjugated Streptavidin (eBioscience). Following surface staining, cells were washed,
fixed in 4% PFA, and resuspended in FACS buffer. For
intracellular staining, cells were spun at 1500 rpm in 0.3%
Saponin for 10 min to permeabilize cells and then incubated with FITC-conjugated ErbB4 (Santa Cruz Biotech)in Saponin to maintain permeabilization. After incubation, cells were washed and resuspended in FACS buffer.
Samples were acquired using either a BD FACS Canto
II flow cytometer or NovoCyte Quanteon machine,
NovoSampler Q, and NovoExpress Software at the UC
Riverside core facility. Analysis was conducted using
FlowJo software. For specific panels and gating strategy

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

used, see Supplementary Tables 1 and Supplementary
Fig. 1.
Cell sorting and single cell RNA sequencing

Cell Sorting. Neutrophils were sorted from brain andspleen based on CD11b/Ly6G positivity. BMNCs andsplenocytes were harvested as above at 4wpi. To obtaina large enough number of neutrophils for sequencing,
3 naïve and 3 infected mice were pooled for naïve andinfected neutrophil splenocyte samples, and 7 infected
mice were pooled for neutrophil BMNC sample. 3 separate infected mice were pooled for an unsorted BMNC
control sample. Cells were incubated in fluorescent-conjugated antibodies for CD11b APCCy7 (Invitrogen) andLy6G PE or PerCPCy5.5 (Clone 1A8; Invitrogen), rinsed,
and resuspended at 1.0 × 107 concentration in FACS buffer with 10% FBS to minimize cell death during sorting.
Neutrophils were sorted from pooled samples based on
gating strategy of Ly6G + CD11b + cells using a MoFlo
Astrios EQ Cell Sorter at 3–4% pressure to maximize cell
survival. Sorted cells were re-counted to determine viability, and a minimum of 1.0 × 104 sorted cells were used
for single cell sequencing.
10x
Sequencing
andAnalysis.
SortedLy6G + CD11b + cells and unsorted BMNC control
sample were processed for single cell RNA sequencing
according to 10x sample prep protocols for Steps 1–3(Manual: CG000204 RevD); instructions were followed
exactly. Time spent before loading sorted cells onto 10x
Chromium controller for Step 1 was < 1 h. Processed
samples were analyzed after Steps 2 and 3 via Bioanalyzer by the UCR Genomics Core facility for viability
and concentration before proceeding to next steps. Uponcompletion of the 10x protocol, samples were sent to the
UC San Diego IGM Genomics Center for sequencing(500 million reads/sample).
Immunohistochemistry and Immunofluorescence

A minimum of 4 biological replicates were used for eachgroup of mice (naïve, infected control, and infected neutrophil-depleted OR infected NRG-1-treated). Following
perfusion, half brains were harvested and post-fixed in
4% PFA for at least 24 h followed by 30% sucrose for 48 h.
Brains were frozen at -80 °C in optimal cutting temperature compound (OCT) and sectioned sagittally at 10 μm
thickness using a cryostat and charged slides.
H&E Staining. H&E staining protocol was as follows:
fixation of slides in 95% ethanol, incubation in hematoxylin followed by eosin stain for 30 s each (with fixation step in between), fixation in 95% and 100% ethanol,and final fixation using Citrisolv. Slides were sealed withcoverslips using Cytoseal (ThermoFisher) and dried overnight. Imaging was performed using ImageJ software.

Page 4 of 16

Cyst Quantification. For quantification of cysts from
H&E-stained brain sections, quantification was blinded.
Total numbers of cysts were quantified from each biological sample (n = 5 per group, 4 brain slices per sample).
Total counts per biological sample were calculated byadding numbers of cysts counted in each brain slice toaccount for biological variability in each group.
Immunofluorescence Staining. Slides were fixed andpermeabilized in 75% acetone/25% ethanol for 10 min atroom temperature. Slides were blocked with 5% Donkey
serum for 30 min at room temperature and incubatedwith primary antibodies at room temperature for 1 h protected from light. Primary antibodies and dilutions used
are listed: Goat T. gondii (1:300, Abcam); Rabbit SLPI
(1:200, Novus Biologicals); and Guinea pig GLT-1 (1:200,
Abcam). After primary incubation, slides were rinsed
three times with 1x PBS for 5 min each wash and incubated for 1 h protected from light at room temperature
with the following secondary antibodies (1:1000 dilution,
Thermofisher): Donkey anti-Rabbit Alexafluor 488, Donkey anti-Goat Alexafluor 568, and Chicken anti-Guinea
pig Alexafluor 488. For specific panels used, see Supplementary Table 2. Following secondary incubation, slides
were washed 3x in 1x PBS for 5 min each wash. Coverslips were mounted on slides using VectaShield Hardset
Mounting Medium with DAPI (Vector Labs), and slides
were dried overnight in the dark at room temperature.
Imaging was performed using a Leica inverted DMI6000
B microscope and Las-X software.
SLPI Quantification. For quantification of SLPI + cells,slides were blinded, and a minimum of 10 positively
stained cells were counted (from a minimum of 5 regionsof interest (ROIs) per section x 4 sections/ biologicalsample = minimum of 20 ROIs per biological sample)
from the cortex region of the brain (where the majority ofcyst formation occurs).
SLPI-positive staining of neuronal axons was quantified. Other cell types such as astrocytes can express SLPI,
but NeuN staining could not be used for colocalization
with SLPI expression on neuronal axons due to restriction of NeuN to the neuronal nucleus. Therefore, only
SLPI + neuronal axons were identified/quantified basedon accurate cortical neuron-axon morphology and different morphology/staining from SLPI + astrocytes seen inprevious studies [26, 27].
Statistical analyses

All experiments were repeated a minimum of 3 times
to confirm accuracy and consistency of results, and all
experiments were conducted with a minimum of n = 3 toaccount for biological variability. Statistical significancefor all experiments was determined by either 2- tailed
unpaired Student’s t-test or One-Way ANOVA with multiple comparisons, and a p-value < 0.05 was consideredBergersen et al. Journal of Neuroinflammation(2024) 21:189

statistically significant. The type of statistical test run foreach experimental result is indicated in the corresponding figure legends.
scRNAseq Analysis. Sequencing files were analyzed
using the following softwares: FileZilla, HPCC Cluster, Cell Ranger (version 5.0), and Loupe Browser (version 5.0). FastQC and MultiQC were used to evaluate
the quality of sequencing reads. Sequencing reads werealigned (fastq) to the mm10 mouse reference genome foranalysis, and the expression of transcripts was quantifiedin each cell. Low quality cells were excluded from analysis
using the Normalize function during analysis. Sequencing saturation was confirmed for each sample. After quality control checks, the following cells and average genereads per cell were obtained: Brain Neutrophils = 3,242
cells, 736 reads/cell; Infected Spleen Neutrophils = 6,804
cells, 782 reads/cell; Naïve Spleen Neutrophils = 4,813
cells, 702 reads/cell; BMNCs = 2,280 cells, 1,014 reads/
cell. For differentially expressed genes (DEGs), genes withp < 0.05 after comparison to control values were considered significantly enriched, and all genes discussed inthis study were identified as significantly altered. Selected
genes for heatmap analysis were confirmed as significantly altered (p < 0.05). The top 50 most significantly
enriched genes in each identified neutrophil subset wereused for Gene Ontology (GO) analyses, and the “Biological Processes” option was selected for the Mus musculus
host.

Results
Neutrophils persist in the brain throughout chronic
infection

As previously demonstrated [5], a small, well-defined
population of neutrophils (Ly6Ghi, CD11b+) was seen in
the brain during chronic infection (Supplemental Fig. 1)that were fully differentiated and mature based on theirexpression of CD11b, Ly6G, MHC I, and the lack of theproliferative marker Ki67 (Supp. Figure 1B). To determine the size and duration of this population over thecourse of infection, neutrophil percentages and absolute cell numbers were quantified from the brain tissue
at 1, 2, 4, 6, and 11wpi. Neutrophils were found within
the brain parenchyma as early as 1wpi with peak infiltration seen at 2wpi (Fig. 1A). These are time points when
the cell lytic tachyzoites are invading the brain prior tocyst formation [21]. A significant contraction of neutrophil numbers was observed at 4wpi, however, by latechronic stage, the number of neutrophils increased backto levels comparable with early chronic (2wpi) infiltration(Fig. 1A). Previous studies during chronic Toxoplasma
infection have found specific localization of infiltrating
inflammatory monocytes to the olfactory tubercle [11],and non-infection models have previously demonstrated
the presence of neutrophils in the olfactory bulb [28]. To

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determine the potential localization of neutrophils to aparticular part of the CNS, brains were perfused and the
cerebellum, frontal cortex and mid-brain were dissected,
anatomical areas that are easily identified and isolated.
The percentage of neutrophils was elevated in the cerebellum although absolute neutrophil numbers showed
little region specificity implying that neutrophils may
access the cerebellum more easily than other immune
cells (Fig. 1B and data not shown). These results indicate
a broad disbursement of neutrophils in the brain during
infection instead of localization to specific brain regions.
Following previous work by the Dunay group [5], neutrophils were depleted over the course of 2 weeks startingat 4wpi using a neutralizing Ly6G mAb (Fig. 1C; Supplemental Fig. 2) and parasite burden was measured. In theabsence of neutrophils, Toxoplasma B1 gene analysis andcyst burden was significantly elevated (Fig. 1C). These
results demonstrate an essential, persistent, and broadly
disbursed chronic neutrophil population in the brain,
which we termed “CBNeuts.”
CBNeuts are defined by distinct transcriptional and protein
signatures that confirm their tissue specificity

The landscape and immune requirements of neutrophils
during chronic infection are potentially very different
from the environment of acute peripheral inflammation
normally associated with neutrophil function [29]. This,in addition to our increasing understanding of trained
immunity and the role of neutrophils in the brain [30–34]
provoked us to investigate the transcriptional and phenotypic profiles of CBNeuts to determine if neutrophils thathave entered the brain have a unique signature. To do so,single cell RNA sequencing (scRNAseq) and flow cytometry were performed on sorted CBNeuts and peripheral neutrophils (PSNeuts) from infected and uninfected
spleens to assess tissue-specific gene and protein signatures. Cells were sorted from pooled brains and spleens
based on positive CD11b and Ly6G expression. Resulting 4-week-infected samples were aggregated following
scRNAseq, and transcriptomic profiles of CBNeuts vs.
PSNeuts were analyzed (Fig. 2A-B). Ly6G gene enrichment was evaluated in all sequenced samples (Supplemental Fig. 3A) to confirm neutrophil specificity. When
comparing all aggregated samples, CBNeuts clusteredseparately from splenic neutrophils and control unsorted
BMNC cells (Fig. 2A). When all sorted neutrophils were
aggregated and analyzed separately, a nearly complete
separation of CBNeuts (reds) was observed compared to
PSNeuts from infected (greens) and naïve (blues) mice
(Fig. 2B).
Based on the distinct separation of neutrophils fromthe brain compared to the spleen seen in our scRNAseq data, CBNeuts and PSNeuts were further analyzed at
4wpi for the expression of proteins known to play a role

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

Page 6 of 16

Fig. 1 Neutrophil persistence in the brain during chronic infection is vital for control of parasite replication. C57BL/6J mice were infected intraperitoneallywith 10 T. gondii cysts (n = 4 per time point) or injected with PBS as a control (n = 3) and analyzed for immune cells in the brain at both acute (1wpi) andchronic (2, 4, 6, and 11wpi) time points via flow cytometry. For location studies, mice (n = 5) were sacrificed at 4wpi, brains were dissected into 3 distinct
regions, and BMNCs were analyzed via flow cytometry to determine neutrophil location in the brain. For neutrophil depletion, a cohort of infected micereceived neutralizing Ly6G mAb treatment at 4wpi for 2 weeks to deplete neutrophils. (A) Frequencies (left) and numbers (right) of CD11b + Ly6G + neutrophils in brain at each time point. (B) Quantification of neutrophil percentages in each defined brain region. (C) Quantified parasite burden from wholebrain DNA via RT-PCR using T. gondii B1 gene (left) and cyst burden quantified via direct counting from H&E-stained slides (right). * = P < 0.05, ** = P < 0.01,“p-val < 0.05” indicates varying degrees of significance between indicated time points; significance determined via One-way ANOVA (A-B) or unpaired
student t-test (C), and error bars indicate SDin neutrophil function (Fig. 2C-E). Previously identified
proteins including MMP9, VEGF, CD62-L, CD15 andCXCR4 are found in peripheral neutrophils with a morealternative/protective function, including tissue repair
and angiogenesis [5, 15]. To determine the expressionof these molecules previously associated with protective neutrophil functions and general neuroprotection(NRG-1, ErbB4, and MSR-1) by our CBNeut population, BMNCs and peripheral splenocytes were incubatedwith antibodies to Ly6G, CD11b, and the proteins mentioned above, and multi-parameter flow cytometry was
conducted. When CBNeuts were compared to PSNeuts
as a whole, tSNE plots revealed mostly non-overlapping
populations (Fig. 2C). Striking visual differences between
CBNeuts and PSNeuts were observed in the expression of ErbB4, MSR1, VEGF, and CD62-L (Fig. 2D),
with CBNeuts uniformly expressing MSR1 and VEGF
compared to PSNeuts. tSNE clustering defined CBNeut

subsets based on noticeable differential expression of
ErbB4 and CD62-L. While populations of these defined
subsets were also seen in PSNeuts, they made up a muchsmaller proportion of the total population comparedto CBNeuts. NRG-1 expression did not further define
CBNeut subsets but was expressed uniformly (data notshown). Analysis of neutrophils that were migrating in
the blood served as another peripheral population anddemonstrated similar results to those seen in PSNeuts(data not shown).
Neutrophils can also be distinguished by age andchanges in migratory capability as reviewed by Peiseler
and Kubes [15]. Thus, it was hypothesized that differences in migration capability and age would be apparent
in CBNeuts compared to PSNeuts as chronic infection
progressed. To address this, the expression of CD15
(indicative of the ability to migrate [14]) and CXCR4(indicative of aged neutrophils [35]) was monitored

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

Page 7 of 16

Fig. 2 Transcriptomic and protein analysis of CBNeuts reveals a distinct tissue-specific profile. For transcriptomic profiling, CBNeuts (from n = 7 brains) and
PSNeuts (from n = 3 spleens) were sorted from infected mice at 4wpi via flow cytometry and prepped for scRNAseq along with chronically infected BMNCs(n = 3 mice) and naïve spleen neutrophil (n = 3) controls. Phenotypic profiles from CBNeuts and PSNeuts were also evaluated via flow cytometry at 4wpi.
For both, neutrophils were identified based on expression of CD11b and Ly6G. (A) UMAP plot of all aggregated scRNAseq samples: PSNeuts from infectedmouse (I, yellow), PSNeuts from naïve mouse (N, orange), CBNeuts (red), and BMNCs (purple). (B) UMAP plot of aggregated sorted CBNeuts, PSNeuts from
infected mouse spleen (I), and PSNeuts from naïve spleen (N). (C) tSNE plot of concatenated CBNeuts and PSNeuts at 4wpi. (D) tSNE plots of selected
alternative molecules from concatenated CBNeuts (top) and PSNeuts (bottom). tSNE plot scale shows populations with low expression (blue) to highexpression (red) of molecules. (E) Flow cytometry quantification of CD15 (left) and CXCR4 (right) expression by CBNeuts and PSNeuts at multiple chronic
infection time points (2, 4, and 11wpi). * = p < 0.05; significance determined via unpaired student t-test, and error bars indicate SD

over the course of chronic infection (Fig. 2E). The percent of CD15 + CBNeuts significantly increased from
2wpi (20%) to 4wpi (> 60%) and dropped back to baseline by 11wpi (20%) (Fig. 2E, left). In contrast, the proportion of CD15 + PSNeuts increased less from 2-4wpi
and remained constant (~ 30% of whole population) from

4wpi on. CXCR4 expression followed a similar trend in
CBNeuts, significantly increasing from 2- to 4wpi, however remained consistently high through 11wpi (Fig. 2E,
right). PSNeuts had the inverse relationship showing adecrease in the proportion of CXCR4 expression between
2-4wpi (Fig. 2E). These results confirm the hypothesizedBergersen et al. Journal of Neuroinflammation(2024) 21:189

differences in migration capability and age between
CBNeuts and PSNeuts over time.
Collectively, these results identify CBNeuts as transcriptionally and phenotypically distinct from PSNeuts
based on the expression of protection/repair-associatedproteins, migratory capability, and age.
CBNeuts are composed of two distinctly characterized
subsets

Having already identified distinct phenotypic characteristics of CBNeuts, it was also clear from our global
scRNAseq data that these neutrophils were not homogeneous. Indeed, from our scRNAseq analysis of sorted
CBNeuts, two distinct clusters (marked as “1” and “2”)Page 8 of 16

were confidently identified as neutrophils based on
their enrichment of Ly6G and Itgam (encodes CD11b)(Fig. 3A). Importantly, these clusters could also be identified in our original aggregated dataset (Fig. 2B). Remaining cell types not identified as “neutrophils” were noted
for the expression of lineage-specific markers (T cells,
macrophages/microglia, inflammatory monocytes, neurons, astrocytes, and oligodendrocytes; Supplemental
Fig. 3B) and were excluded from further analysis. An
additional potential neutrophil population (pink) waslater identified as red blood cells (Supp. Figure 3B) and
was also excluded from further analysis. The two remaining CBNeut clusters were further classified based onenrichment of Fut4 (encodes for CD15) and Cxcr4 [15].

Fig. 3 CBNeuts display age-dependent heterogeneity at the transcriptomic level. CBNeuts (from n = 7 brains) were sorted from infected mice at 4wpi
via flow cytometry and prepped for scRNAseq. A) UMAP plot of CBNeuts (red = neutrophil populations) identified as: (1) “Aged Extravasated” (AE) (Ly6Gint,
Fut4+, CXCR4+) or (2) “Recently Recruited” (RR) (Ly6Ghi, Fut4int, CXCR4-) subsets. Subsets also identified in aggregated dataset (Fig. 2B). Pink subset identified
as red blood cells. B-C) Heat maps of (B) neuroprotective genes (top panel) and genes relating to classical (NETosis) vs. alternative (angiogenesis) functions (bottom panel). (C) GO enrichment terms from top 50 most significantly upregulated genes in AE and RR CBNeuts

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

Cluster 1 (Fig. 3A, dark red) was enriched for Fut4 andCxcr4 and was termed “Aged Extravasated” (AE). Cluster
2 (Fig. 3A, light red) showed enrichment for Fut4 but not
Cxcr4 and was termed “Recently Recruited” (RR).
To determine the genes that define these two subsets of CBNeuts, analysis of differential gene expression
was conducted using RR CBNeuts as the control group.
“Neuroprotective Phenotyping” heatmap analysis demonstrated enrichment for Vegfa, Nrg1, Flt1 (encodes for
VEGF receptor), and Msr1 in AE cells, while RR cells
showed enrichment for Sell (encodes for CD62-L) and
Mmp9 and had increased ErbB4 compared to the AE
subset (Fig. 3B). In contrast, analysis of gene enrichment corresponding to classical NETosis vs. alternative
angiogenesis functions found enrichment of genes relating to both processes in each subtype (Fig. 3B). AE cells
demonstrated enrichment for Bmf (NETosis gene) andthe angiogenic genes Pdgfb, Vegfa, Kdr, and Hif1a; andRR cells were enriched for the NETosis-associated genes
Mapk3 and Padi4 and the angiogenic genes Mmp8 andMmp9. Specific indicators of apoptosis were also examined including Bcl2, Casp1, Akt1, and Akt2 and showedno significant enrichment in either subset indicating nocell death in either subset.
GO analysis of the top 50 most enriched genes in each
CBNeut subset revealed differential upregulation of GO
terms (Fig. 3C). AE cells were most enriched for GO
terms relating to negative immune regulation and neural development, while RR cells were enriched for terms
associated with classical antimicrobial and inflammatory neutrophil functions. Taken together, these results
demonstrate that CBNeuts are composed of two major
age-dependent subsets that can be distinguished by theirexpression of neuroprotective genes and transcriptionally-predicted functions.
CBNeuts are capable of limiting infection-induced
neuropathology, which may be partially dependent on
NRG1 signaling

Neutrophils in the CNS have been previously seen
encouraging neuronal regeneration [36, 37]. This occurs
partially via the production of secretory leukocyte protease inhibitor (SLPI) [26, 38]. To determine whether neutrophils perform similar functions during Toxoplasma
infection, we investigated SLPI expression in the brain
following depletion of neutrophils via immunofluorescence of mouse brain tissue (Fig. 4). SLPI expression
could be seen in the infected brain with characteristic
morphology of neuronal bodies and axons which wereclassified as “SLPI + cells”. SLPI + neuronal cell bodies and
SLPI + axons (shown by white arrows) were identified inboth infected control and neutrophil-depleted brains(Fig. 4A-B). When this data was blindly quantified, micelacking neutrophils had significantly fewer SLPI + cells

Page 9 of 16

when compared to infected control mice independent ofareas of parasite replication (Fig. 4A and C), supportinga neuroprotective phenotype for neutrophils in the CNS.
The persistence of CBNeuts and the results fromour neutrophil depletion studies suggested a previously unknown role of neutrophils in maintaining brain
homeostasis during chronic infection via neuroprotective
mechanisms. Previous work in our lab has also demonstrated the activation of potential neuroprotective andreparative pathways in the brain during chronic Toxoplasma infection [21], but the cellular sources of molecules involved in these pathways remain unknown.
One of the most striking differentiating moleculesin the Aged Extravasated CBNeut population was thehigh enrichment of Nrg1 (Fig. 3B) which encodes for the
NRG-1 protein that is responsible for a large majority ofneuroprotective functions in the brain [22, 23, 39, 40].
Neutrophils have not been previously documented as
being sources of NRG-1 therefore, to confirm this observation, we performed flow cytometry on BMNCs overthe course of infection and examined NRG-1 expression (Fig. 5). Analysis revealed that CBNeuts were consistent, significant, and almost uniform cellular sourcesof NRG-1 (> 60%) at all chronic infection time points(Fig. 5A) which corresponds with the results seen in
our initial tSNE analysis. As previously seen (Fig. 2D), a
percentage of CBNeuts also expressed the main NRG-1
receptor ErbB4 (20–60%), and the kinetics revealed astriking high percentage of neutrophils expressing ErbB4
at 2wpi that significantly dropped as infection progressed(Fig. 5B). Innate macrophages and CNS-resident microglia also expressed these molecules while adaptive T cells
did not express ErbB4 at any time point; however, a subpopulation of T cells (10–20%) did express NRG-1 which
increased over the course of infection (Fig. 5A).
Based on the consistent expression of these two neuroprotective molecules by the majority of CBNeuts, we
wanted to elucidate the relationship between neuroprotective molecule expression and the classical versus alternative phenotypes seen in our earlier transcriptional andphenotypic profiling. To do this, we further characterizedthese cells by looking at NRG-1, ErbB4, and CD15 protein expression together. At 4wpi, more than 80% of all
CBNeuts were NRG-1 + and were composed of two main
subtypes based on their expression of CD15 (Fig. 5C, leftand D, top). This integrin aids in the migration of neutrophils into tissues and has previously been used to identify broad “classical” (CD15+) and “alternative” (CD15-)
categories [14] with classical neutrophils primed as effector cells for pathogen killing and alternative neutrophils
associated with healing, tissue remodeling, and vascular
repair. Surprisingly, 51% of NRG-1 + cells expressed CD15
indicating their ability to migrate, with high expressionof NRG-1 correlating with expression of CD15. TheseBergersen et al. Journal of Neuroinflammation(2024) 21:189

Page 10 of 16

Fig. 4 Neuronal regeneration attempts during chronic infection are inhibited in the absence of neutrophils. C57BL/6J mice were infected intraperitoneally with 10 T. gondii cysts or injected with PBS as a control (n = 5 per group), and a cohort of infected mice received neutralizing Ly6G mAb treatment at
4wpi for 2 weeks to deplete neutrophils. (A) Immunofluorescence images of infected control and neutrophil-depleted brains, 40x images. Blue = DAPI,Green = SLPI, Red = T. gondii, Scale bar = 25 μm. White arrows indicate SLPI + cells. (B) Magnified images of selected regions (dashed white boxes) in (A)to show SLPI stained neuronal axons. (C) Blinded quantification of SPLI + cells. *=P < 0.05; significance determined via unpaired student t-test, and error
bars indicate SD

two subtypes were further distinguished by their differential expression of ErbB4. Alternative neutrophils that
did not express CD15 were more likely to express ErbB4(indicated by lower NRG-1 expression) and therefore
be responsive to NRG-1 (Fig. 5C, right and D, bottom).
Collectively, these results identify CBNeuts as consistentcellular sources of NRG-1 that are composed of 2 main
subtypes based on their differential expression of CD15
and ErbB4. The novel expression of NRG-1 and ErbB4 by
CBNeuts also suggested an important yet undescribedrole for NRG-1/ErbB4 signaling in limiting Toxoplasmainduced neuropathology.

A pathology previously documented in the brain during Toxoplasma infection is the downregulation of GLT1, a critical glutamate transporter expressed by astrocytes[19]. A build-up of glutamate caused by GLT-1 downregulation leads to neurotoxicity and cell death. To determine the importance of the NRG-1/ErbB4 signaling
pathway during chronic Toxoplasma infection, we treatedchronically infected mice with exogenous NRG-1 andlooked for the presence of GLT-1 downregulation in theinfected brain [19] via immunofluorescence and western blot. In naïve mice, GLT-1 was expressed relatively
uniformly throughout the brain, but as previously published, GLT-1 loss was seen in “patches” across the tissue

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

Page 11 of 16

Fig. 5 CBNeuts express neuroprotective molecules NRG-1 and ErbB4. C57BL/6J mice were infected intraperitoneally with 10 T. gondii cysts (n = 4 per time
point) or injected with PBS as a control (n = 3) and analyzed for immune cells in the brain and expression of neuroprotective molecules at both acute(1wpi) and chronic (2, 4, 6, and 11wpi) time points via flow cytometry. For NRG-1 treatment, a cohort of 2 week-infected mice were treated with 5 µg/kg/
day of NRG-1 daily for 7 days. (A-B) Time course quantification of expression frequencies (shown by percent of positive cells) of NRG1 (A)and ErbB4 (B) by
brain mononuclear cells (BMNCs). (C) Representative flow plots of NRG-1 and ErbB4 expression by CBNeuts at 4wpi. Numbers on flow plots represent the
average percentage of expression (n = 4). (D) Expression overlap of neuroprotective molecules distinguished by CD15+ (pink) and CD15- (blue) CBNeuts.
For graphs, ****=P < 0.0001 (remaining significance indicated as p < 0.05); significance determined via One-way ANOVA. For all graphs, error bars indicateSD

during chronic infection (Fig. 6A, white arrows). While it
did not return GLT-1 expression to baseline uninfectedlevels, administration of exogenous NRG-1 treatment
significantly rescued infection-induced downregulationof GLT-1, with fewer GLT-1 negative patches and more
uniform expression seen in the tissue following treatment (Fig. 6A). This was also quantified at the protein
level via western blot, and NRG-1 treatment significantly
enhanced the amount of GLT-1 in the brain compared to
infected controls (Fig. 6B, C). Collectively, these results
highlight the importance of NRG-1/ErbB4 signaling during chronic Toxoplasma infection and offer a potential
pathway of neuroprotection employed by CBNeuts andother NRG-1-expressing cells.

Discussion
The maintenance of a homeostatic brain environment is
critically important across a wide range of neurologicaland immunological diseases. However, the mechanisms
behind this are poorly understood during infection where
the need to control the pathogen through the immune
response must be balanced with maintenance of neuronal function. Under most circumstances, Toxoplasma is
an infection that does not lead to severe clinical pathology despite a continuous inflammatory response in the
CNS and the direct infection of neurons by the parasite.
It therefore provides a useful model to address mechanisms of neuro-immune balance. The work here collectively characterizes a population of chronic neutrophils
in the brain identified as “CBNeuts” during ToxoplasmaBergersen et al. Journal of Neuroinflammation(2024) 21:189

Page 12 of 16

Fig. 6 NRG-1 treatment rescues infection-induced GLT-1 levels associated with neurotoxicity. C57BL/6J mice were infected intraperitoneally with 10 T.
gondii cysts (n = 4 per time point) or injected with PBS as a control (n = 3). For NRG-1 treatment, a cohort of 2 week-infected mice were treated with 5 µg/
kg/day of NRG-1 daily for 7 days. (A) Immunofluorescence images of naïve, infected control, and infected NRG-1-treated mouse brain slices. DAPI (blue),
GLT-1 (green), and Toxoplasma (red); white arrows indicate areas of decreased GLT-1 expression. Images taken at 10x magnification, Scale bar = 100 μm.
(B) Western blot of GLT-1 from brain homogenate of naïve, infected control, and infected NRG-1-treated mice. Half brains were harvested and protein was
extracted, normalized, and run on a Western blot. (C) Quantification of Western blot results using GLT-1:β-Actin ratios. * = P < 0.05; significance determined
via unpaired student’s t-test., For all graphs, error bars indicate SD

infection that persists long after chronicity has been
established. Depletion studies confirm the importanceof neutrophils for the control of parasite replicationin the brain and demonstrate that these neutrophils
are significant sources of neuroprotective molecules
and aid in neuronal repair. CBNeuts are phenotypically distinct from peripheral neutrophils and display
CNS-specific heterogeneity at the transcriptional levelindicating potential balancing of classical infection control and alternative neuroprotective functions during
chronic Toxoplasma infection. While it is known thatneutrophils are important during acute and early chronic

infection, we now show that this is a highly persistent
population in the brain and is therefore unlikely to be afirst responder population either to parasite infection
or initial invasion of the CNS. This population remains
in the brain for the duration of chronic infection aftera brief drop in both percentage and total cell numbers
from 2- to 4wpi (Fig. 1). This drop is evidence of a lull
between acute and chronic neutrophils and specificallypoints to the development of a long-lasting chronic neutrophil population after initial die-off of acute infiltrating cells during early chronic infection once parasites
have formed chronic cysts inside neurons. Despite their

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

low expression of the proliferative marker Ki67 (Supplemental Fig. 1B), the increase seen in neutrophil percentage and number from 4- to 6wpi suggests that these cells
may be capable of either proliferation in the brain or are
replenished from the circulation once chronic infection isestablished. This is also supported by increased numbers
seen at 11wpi, when reactivation of the parasite can beseen prompting increased neutrophil infiltration to control parasite replication.
While there is evidence for a role of more “classical”
antimicrobial functions by CBNeuts to directly control parasite replication during chronic infection, thedata presented here introduce an alternative functionof CBNeuts to produce NRG-1 and assist in neuronal
repair mechanisms. NRG-1/ErbB4 signaling-dependentneuroprotection is vital for host survival in instances of
CNS damage such as ischemic stroke, spinal cord injury,
epilepsy, and experimental cerebral malaria infection[22, 23, 41–43]. Our study also demonstrates the abilityof NRG-1/ErbB4 signaling to rescue infection-induced
neuropathology in the form of GLT-1 loss via exogenous
NRG-1 treatment during Toxoplasma infection, a neuroprotective mechanism that has not been reported previously. NRG-1 production has been broadly characterized
predominantly in CNS-resident cells, specifically neurons
and microglia [44–46]. However, expression of NRG-1 bymigrating peripheral immune cells is less understood,and expression of NRG-1 and or NRG-1/ErbB4 signaling by neutrophils has not been previously documented.
The recruitment of NRG-1-expressing immune cells may
be an innate response to injury or an indication of thedegree of CNS insult and therefore a need for additional
peripheral support. CBNeuts were almost uniformly
positive for NRG-1 protein although scRNAseq suggeststhat AE neutrophils are higher expressers of NRG-1 transcripts. It is important to note that neutrophils were notthe only immunological source of NRG-1 as macrophages
and CNS-resident microglia also expressed NRG-1 andErbB4. In contrast, adaptive lymphocytes were rarely
sources of these molecules. There is increasing recognition of the connection between the nervous and immune
systems, however, this data points to a niche for adaptive
T cell immunity being primarily about controlling infection and immunomodulation and not neuroprotection atleast via these mechanisms. Innate neutrophil productionof neuroprotective molecules may indicate a shift in theneeds of the brain once adaptive lymphocyte responses
are established. This is supported by data demonstrating neutrophils pivoting to protective functions in the
CNS following spinal cord and optic nerve injury, ischemic stroke, and retinopathy [37, 38, 47–50]. In addition, expression of both NRG-1 and ErbB4 by CBNeuts(specifically CD15-) indicates the potential for these cells
to conduct autocrine and paracrine signaling throughPage 13 of 16

the NRG-1/ErbB4 pathway. These results also suggestthat this pathway could function as a feedback loop telling CBNeuts when to shift to a more neuroprotective vs.
antimicrobial population.
In other systems, neutrophils have been defined as“alternative” and have functions that aid in angiogenesis and tissue repair as reviewed by Peisler and Kubes[15]. The presence of different neutrophil subsets in thebrain during Toxoplasma infection supports the newly
acquired appreciation for neutrophil heterogeneity which
can be dependent on a multitude of factors including age
and tissue environment [16–18, 30, 31]. Our work suggests that despite the common urge to classify immune
cells as “N1” or “N2” in terms of function, these neutrophils are capable of acting as both anti-parasitic and tissue reparative in the brain microenvironment. There wasno enrichment of effector genes Mpo or Nox1 (encoding
for NADP oxidase) in either AE or RR neutrophils, likely
reflecting the significant post-translational regulation ofthese effector processes. However, CXCR4hi AE neutrophils have upregulated transcription of the effector genes
Bmf and Pdgfb and also Vegfa, and Kdr, noted for theirroles in tissue repair. The overlap of these seemingly contradictory functions is steadily increasing with the recognition that the production of NETs and peroxidases arealso required for tissue repair as well as effector mechanisms against pathogens [38, 47, 51, 52]. This underpins the concept of innate immune cells being as criticalto tissue homeostasis as they are in initiating immune
responses and may pinpoint neutrophil flexibility asimportant not only in Toxoplasma infection but also in
all models of CNS disease.
Our work not only identifies CBNeuts as previously
unknown sources of neuroprotective molecules but also
demonstrates distinct subsets that are specific to thebrain environment. Previous work has identified two
broad categories of neutrophils that differ in their functions. The first is a “classical” pro-inflammatory subsetthat is associated with classical macrophage activation,
increased granularity, NETosis, and the production ofTh1 cytokines IL-12 and TNFα [13, 14]. The second classis considered “alternative” based on anti-inflammatory
cytokine expression like IL-10 and IL-4, association withalternative macrophage activation, decreased granularity, and angiogenesis and vascular repair [12, 13]. Here,
we demonstrate the presence of two major CBNeut subsets that differ from peripheral neutrophils suggestingthat tissue-specific signals may drive the differentiation
or retention of neutrophil subsets at the site of infection. These CBNeuts can be differentiated into two main
populations, CXCR4hi AE cells and recently recruited(RR) CXCR4lo cells that have increased enrichment for
Fut4 indicating their increased migration potential [15].
It should be noted that corresponding CXCR4-enrichedBergersen et al. Journal of Neuroinflammation(2024) 21:189

AE cells show decreased Ly6G expression in heatmap
comparisons of gene expression between AE and RR
populations in Fig. 3B. This difference in Ly6G enrichment between these subsets stems from the use of RR
cells as the denominator group for these direct comparisons between populations. As the RR population has ahigher Ly6G enrichment, this results in the low expression seen in the AE group. This is supported by previous
work demonstrating that as neutrophils age, their expression of Ly6G decreases in preparation for recycling backto the bone marrow [15, 53]. Although both AE and RR
CBNeuts exhibit signs of functional heterogeneity, thelatter RR population has an enhanced effector signature indicated by increased expression of genes and GO
terms associated with canonical pro-inflammatory andanti-microbial functions including “chemokine production” and “neutrophil-mediated killing of gram-negative
bacterium.” This supports the hypothesis that as neutrophils enter the brain during infection, they are primarily
there as an anti-microbial responsive unit. However, as
infection becomes established with reduced parasite replication and cell lysis, neutrophils age and tissue-specificrepair signals dominate.
The evidence for heterogenous functions of neutrophils
in disease stems partially from their discovered roles inneuronal and angiogenic repair in both spinal cord andoptic nerve injury demonstrating direct and indirect
protection [37, 38, 48, 51, 54]. These functions discovered in non-infection instances may be applicable to thedemonstrated evidence of damaged neurons observed
during chronic Toxoplasma infection [19, 20, 55]. Additionally, the continuous latent reactivation of parasitic
cysts in the brain may not only require the continuouspresence of these protective neutrophils but may also
be a stimulus for them to remain in the brain and differentiate into reparative subsets. As such, their consistentpresence throughout chronic infection may ultimately bea signal of ongoing neuronal damage which is supported
by slowly worsening neuronal function and changes inbehavior over time [19, 56] and suggests increased plasticity of neutrophils in the brain and the dependence ofneutrophil function on brain microenvironment. The
infection-induced changes in neuronal circuitry indicate an increased need for neuronal repair attempts aswell as increased infection control as chronicity progresses. The recruitment of innate immune cells such as
neutrophils may be necessary for this process as specificneuronal circuitry is required to mobilize neutrophil
migration from the bone marrow [57, 58]. Previous work
has demonstrated the recruitment of innate inflammatory monocytes to the olfactory bulb of the brain perhaps
to remedy olfactory-dependent behavioral changes [11].
Neutrophil localization to this area has been observed
during homeostasis in zebrafish models [28] but was not

Page 14 of 16

observed in our murine model of Toxoplasma infection.
While our work demonstrated no evidence of neutrophil
localization in the brain, other changes in neuronal function may be dependent on neutrophil repair of previously
damaged and re-wired circuits.
The communication between the brain and peripherythat continues to draw in non-aged neutrophils (RR) evenafter 11wpi will be important to understand and may
require parasite recrudescence and continued CCL2dependent recruitment [59, 60]. In addition, a small
cluster of aged neutrophils is observed in the spleen.
This would either indicate differentiation of these cells
in the periphery prior to migration to tissues or trafficking of tissue-educated neutrophils back into circulationand secondary lymphoid compartments. Such a population could act as an indication of continued CNS inflammation and thereby be a stimulus for new neutrophil
recruitment. Whichever way these neutrophils differentiate, the question still remains as to what signals drive
the brain tissue specificity of these cells and what factors
encourage the differentiation into a protective phenotype. It is possible that neuronal antigens are somehow
travelling to peripheral sites to initiate this process, but
the mechanisms behind this subset differentiation remain
to be elucidated.
Collectively, this study demonstrates a persistent neutrophil population in the brain throughout the duration of chronic Toxoplasma infection termed “chronic
brain neutrophils” (CBNeuts). The depletion of thesecells during chronic infection leads to enhanced infection and infection-induced neuropathology indicating apreviously unknown role of neutrophils in maintenanceof brain homeostasis during infection. Expression of
NRG-1 by CBNeuts reveals a distinct brain-specific phenotypic and transcriptomic profile of these cells which
also display functional heterogeneity based on age andintegrin expression, and NRG-1/ErbB4 signaling playa role in limiting infection-induced neuropathology. In
conclusion, these chronic brain neutrophils may represent a vital flexible component of the balanced immune
response to chronic infection in the brain that Toxoplasma gondii is only one example of. Such capacity for
functional flexibility within these cells marks them as
potential therapeutic targets for enhanced neuroprotection during chronic Toxoplasma infection and presents aparadigm shift in our broader understanding of neutrophil functions in the brain as a whole.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03176-7.
Supplementary Material 1

Bergersen et al. Journal of Neuroinflammation(2024) 21:189

Acknowledgements
The authors would like to thank the members of the University of California
Riverside’s Center for Glial Neuronal Interactions (CGNI) and the Genomics
and School of Medicine Research COREs for all advice, use of equipment, andprocessing of samples where appropriate. We are also thankful to the UCSD
IGM Genomics Center for sequencing of RNA samples. Lastly, we sincerely
appreciate the work conducted by UCR’s IACUC and the animal husbandry
provided by Leslie Karpinski and Jennifer Johnson.
Author contributions
KVB and EHW designed and conducted experiments; BDF provided guidanceon project design; KVB, BK, and EHW analyzed data; KVB and EHW wrote themanuscript.
Funding
This work was supported in part by R01DA048815; R01AI158417; andR01AI124682 to EHW, R01NS125775 and R25GM119975 to BDF, and by UCR
Graduate Division to EHW.
Data availability
The data presented in this study are deposited in the Gene Expression
Omnibus (GEO) repository (link: https://www.ncbi.nlm.nih.gov/geo/) and areaccessible through GEO Series accession number GSE210883 (https://www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE210883).

Declarations
Ethical approval
The animal study was reviewed and approved by the Institutional Animal Careand Use Committee (IACUC) University of California Riverside.
Competing interests
BF holds patents related to the work being reported without direct corporate
involvement at the time. The remaining authors declare that the research was
conducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
Received: 22 May 2024 / Accepted: 15 July 2024

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