Van Roy and Kielian Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03174-9(2024) 21:179

Journal of Neuroinflammation

Open Access

RESEARCH

Tumor necrosis factor regulates leukocyte
recruitment but not bacterial persistence
during Staphylococcus aureus craniotomy
infection
Zachary Van Roy1 and Tammy Kielian1*
Abstract
Background Craniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the
14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus(S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infectionthat is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypicalproinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although
TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis
has not been explored.
Methods A mouse model of S. aureus craniotomy infection was used to investigate the functional importance of
TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates,
inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil
extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity.
Results TNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to thebrain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to
14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels
by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced
lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO
mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S.
aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF
KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity
in response to live S. aureus, whereas granulocytes were not affected.
Conclusion These findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection
via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action.

*Correspondence:
Tammy Kieliantkielian@unmc.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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Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

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However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signalsthat dictate S. aureus pathogenesis during craniotomy infection.
Keywords Craniotomy, Biofilm, S. aureus, Granulocytes, Macrophage, TNF, Lymphotoxin, TNFR1, TNFR2

Introduction
Access to the intracranial compartment is achieved
through craniotomy, wherein a piece of the skull is temporarily removed for numerous indications, including
brain tumor excision, hematoma evacuation, or implantation of a medical device [1]. In 3–5% of cases, surgical site infection complicates patient recovery and overhalf of craniotomy infections are caused by Staphylococcus aureus (S. aureus), which forms a biofilm on thesurface of the bone flap that evades immune and antibiotic clearance, progressing to chronic infection [1, 2].
Therefore, craniotomy infections require an additional
surgery to remove the biofilm nidus (i.e., bone flap) andlong-term antimicrobial therapy to eliminate residual
planktonic bacteria, underscoring the severity of these
infections [3–6]. Our previous work has demonstrated
compartmentalization of the immune response during
craniotomy infection, with adaptive immune cells andmonocytes preferentially homing to the infected brain
parenchyma. In contrast, the galea and bone flap are
dominated by granulocytic myeloid-derived suppressor
cells (G-MDSCs) and neutrophils (PMNs) that are alsopresent in the brain, albeit at a lower frequency [1, 7–10].
Both granulocyte populations acquire anti-inflammatory
activity within the biofilm milieu by their ability to inhibit
T cell proliferation and robust IL-10 production [7, 9].
However, proinflammatory responses are also elicitedduring S. aureus craniotomy infection, as demonstrated
by the importance of TLR2- and caspase-1-dependent
pathways for bacterial containment [11]. Therefore, it
is important to understand the relative contributions ofpro- vs. anti-inflammatory cytokines for dictating craniotomy infection outcome with the goal of leveraging thisinformation to augment the former, in combination with
antibiotics, to clear infection without the need for additional surgical procedures.
Tumor necrosis factor (TNF) was one of the firstcytokines to be described and is a prototypical proinflammatory mediator [12]. Although the cytokine waspreviously renamed TNF-α in 1985 when lymphotoxin-β
was identified and referred to as TNF-β, the discovery of
lymphotoxins-α and -β in 1993 demonstrated that there
is only one form of TNF [13]; therefore, we refer to thecytokine as TNF throughout the manuscript. TNF is
produced by a wide range of immune and parenchymal
cells within minutes following injury or infection [14, 15].
Consequently, TNF is tightly regulated at the transcriptional, translational, and post-translational levels [12,14] and signals through one of two non-redundant cellsurface receptors, namely TNFR1 and TNFR2. TNFR1
is constitutively and broadly expressed in most cell types[12, 14, 16] and is responsible for the majority of reported
effects of TNF in the literature [16], where receptor binding elicits mitogen-activated protein kinase (MAPK)
and NF-κB activation. TNFR2 expression is restricted toleukocytes and a limited number of additional cell types(i.e., endothelial cells) and is important for modulating neutrophil antimicrobial activity [17]. TNF signaling
through both receptors elicits pleiotropic effects, including NADPH oxidase and NLRP3 inflammasome priming in PMNs [1, 11], increased blood-brain barrier (BBB)
permeability during neuroinflammation [1, 18], generation and activation of myeloid-derived suppressor cells(MDSCs) [19–21], and promoting T cell activation [22].
In terms of S. aureus infection, TNF deficiency has beenreported to worsen a wide range of diseases, including
sepsis [23], brain abscesses [24, 25], arthroplasty [26], andskin abscesses [17]. The protective nature of TNF against
infection is also apparent in humans, as TNF polymorphisms [27] and the use of TNF-neutralizing biologics are associated with an increased risk of infection inpatients [28–30], including S. aureus [22].
Although TNF influences the function of all major
immune cell populations that respond to craniotomy
infection (i.e., PMNs, G-MDSCs, monocytes, macrophages, microglia, and CD4+ T cells), the importanceof TNF during CNS biofilm infection has not yet been
examined. Here, we show that TNF is critical for stimulating chemokine production and leukocyte recruitment
during acute craniotomy infection since both were significantly reduced in TNF KO mice. In addition, loss of
TNF in macrophages and TNFR1 or TNFR2 deficiency ingranulocytes affected phagocytosis, bacterial killing, andapoptosis in response to S. aureus. Despite these phenotypes, the loss of TNF or its receptors did not dramatically alter bacterial burden in vivo, suggesting either the
involvement of other inflammatory mediators and/or
redundancy in the TNF signaling pathway. The latter possibility was supported by the finding that lymphotoxin-α(Lta) expression was significantly elevated in granulocytes from TNF KO mice. Collectively, these findings suggest that TNF contributes to coordinating theimmune response to craniotomy infection but that otherinflammatory mediators are also involved.

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

Materials and methods
Mouse model of S. aureus craniotomy infection

Craniotomy infection was established in 8–10 week-old
male and female C57BL/6J wild type (WT; RRID: IMSR_
JAX:002365), Tnf−/− (TNF KO; RRID: IMSR_JAX:00300),
Tnfr1−/− (TNFR1 KO; RRID: IMSR_JAX:003242),
Tnfr2−/− (TNFR2 KO; RRID: IMSR_JAX:002620), or
Pad4−/− (PAD4 KO; RRID: IMSR_JAX:030315) mice aspreviously described [2, 7, 10, 11]. Briefly, a surgical planeof anesthesia was achieved by i.p. ketamine and xylazine
administration before exposing the cranium with a midline scalp incision. A pneumatic surgical drill (Stryker
Corporation) was used to produce a 3–5 mm bone flap on
the left side of the skull that was immediately incubatedwith 106 CFU of the methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 LAC13C [31] diluted
in 500 µL brain-heart infusion broth for 5 min at 37 °C,
which resulted in an infectious inoculum of 103 CFU.
Bone flaps were extensively rinsed in 1X PBS to eliminate bacterial broth, blotted to remove excess fluid, andreimplanted. The incision was sutured closed, and animals were monitored under external heat until regaining
ambulation and once per day thereafter until euthanized.
In some experiments, mice received systemic antibiotics.
Briefly, an antibiotic cocktail [25 mg/kg Rifampin (Mylan
Institutional; Morgantown, WV) + 5 mg/kg Daptomycin(Camber Pharmaceuticals; Piscataway, NJ) in 1X PBS]
was administered via i.p. injection (200–250 µL) starting on day 3 post-infection and every 24 h thereafter
until experiment conclusion. Control animals received
i.p. injections of an equivalent volume of sterile 1X PBS.
The craniotomy infection model is associated with a low
mortality rate (< 1%), which is primarily due to complications from anesthesia, and no overt differences in animal
appearance were observed between WT mice and any of
the KO strains examined.
Tissue harvesting and processing

Mice were euthanized by isoflurane overdose at days 7,
14, or 28 post-infection. Following perfusion with 1X
PBS to clear leukocytes from the vasculature, the bone
flap, subcutaneous galea tissue, and infected brain hemisphere were removed and placed in 10% fetal bovine
serum (FBS) in PBS. Tissues were then processed separately to obtain single-cell suspensions. Bone flaps were
initially vortexed for 30 s to dislodge adherent immune
cells, and an aliquot of this suspension was saved for flow
cytometry analysis. The remaining bone flap samples
were sonicated for 5 min to disperse biofilm, seriallydiluted, and plated on tryptic soy agar plates supplemented with 5% sheep blood for bacterial enumeration.
Galea samples were disrupted with the blunt end of a
5 mL syringe plunger, passed through a filter top FACS
tube, and saved for flow cytometry. Brain samples were

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homogenized through a 70 μm strainer and digested withcollagenase IV and DNase I for 15 min at 37 °C before
enzyme inactivation with the addition of FBS. Myelin
was removed using a 25% Percoll solution by centrifugation for 20 min at 520xg with no brake. The resulting
cell pellet was washed in PBS, passed through a filter top
FACS tube, and saved for flow cytometry analysis. Aliquots of galea and brain were taken immediately following homogenization for bacterial enumeration. Likewise,
where indicated, supernatant was collected from pelleted
homogenates for subsequent inflammatory mediatoranalysis or albumin ELISA.
Quantification of immune infiltrates and intracellular
cytokine staining

Single-cell suspensions from the bone flap, galea, andbrain were first incubated with TruStain FcX (Cat#101320; BioLegend) before cell surface marker staining with the following antibody panel: CD45-PE/Cyanine7 (RRID: AB_312979), Ly6G-Pacific Blue (RRID:
AB_2251161), Ly6C-PerCP-Cy5.5 (RRID: AB_1727558),
CD11b-AlexaFluor® 700 (RRID: AB_493705), F4/80Brilliant Violet 510™ (RRID: AB_2562622), CX3CR1Brilliant Violet 785™ (RRID: AB_2565938), CD3-BUV737(RRID: AB_2871136), CD4-Brilliant Violet 650™
(RRID: AB_2562529), CD8-Brilliant Violet 605™ (RRID:
AB_2562609), and NK1.1-APC/Cyanine7 (RRID:
AB_830871). For TNFR1 and TNFR2 KO experiments,
the following adjustments were made to the panel: MHCII (I-A/I-E)-Brilliant Violet 605™ (RRID: AB_2565894)
was included, CD8 was omitted, and Ly6C-FITC (RRID:
AB_1186135) was used instead of PerCP-Cy5.5. Dueto the lack of adaptive immune cells and microglia in
the galea and bone flap, CX3CR1, CD3, CD4, CD8, andNK1.1 antibodies were omitted from these samples.
Dead cells were identified and excluded from analysis using a Zombie UV Fixable viability kit (BioLegend;
Cat #423108) and Spherotech AccuCount beads (Cat#ACBP-100-10; 8.0–12.9 μm) were added to each sample
to enable reporting of absolute cell counts. All acquisition was completed on a BD LSR II-G cytometer withanalysis using FlowJo and the gating strategies depicted
in Additional File 1: Fig. S1.
When indicated, intracellular staining for TNF was
conducted using the Cyto-Fast™ Fix/Perm Buffer Set (BioLegend; Cat# 426803). Briefly, cells from the brain, galea,
and bone flap were incubated with 5 µg/mL brefeldin A
immediately ex vivo for 4 h. Samples were then surface
stained as described above, fixed, and incubated with
TNF-PerCP/Cy5.5 (RRID: AB_961434) using Cyto-Fast™
reagents according to the manufacturer’s protocol.

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

Quantification of cytokine and chemokine expression intissue homogenates and cell supernatants

Brain and galea supernatants were collected as describedabove and analyzed for inflammatory mediator expression using a MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Cat #HT17MG-14 K-PX25,
Millipore Sigma). Homogenates were first centrifuged at
14,000 rpm for 10 min at 4 °C to remove residual cellular material and assayed according to the manufacturer’s
instructions. A MAGPIX® xMAP instrument (Luminex)
was used for analysis with Belysa® Analyst software (Millipore Sigma), and values were corrected for total protein
content via BCA assay.
Murine IFN-ɣ, IL-6, IL-10, IL-12, CCL2, and TNF levels were quantified in cell-conditioned medium using
a Mouse Inflammation Cytometric Bead Array (Cat#552364, BD Biosciences). IL-12 production was minimal under the conditions tested in this study, and IFN-ɣ
is not produced by the innate immune cell populations
examined, so neither mediator was reported.
Generation of primary immune cells

All primary immune cells were prepared from WT, TNF
KO, TNFR1 KO, or TNFR2 KO mice. Macrophages and
G-MDSCs were derived from the bone marrow of 8- to
12-week-old animals as previously described [32, 33].
Briefly, bone marrow was extracted and filtered with a
70 μm cell strainer prior to red blood cell lysis in sterile
H2O, followed by rapid correction to isotonic conditions.
Macrophages were cultured in RPMI-1640 with 10% FBS,
penicillin/streptomycin/fungizone, 2 mM L-glutamine,and 10% conditioned medium from L929 fibroblasts
as a source of macrophage colony-stimulating factor(M-CSF) at 37 °C, 5% CO2. Medium was replaced ondays 3 and 5 with cells used on day 7 in vitro. G-MDSCs
were expanded from bone marrow in RPMI-1640 supplemented with 10% FBS, penicillin/streptomycin/fungizone, granulocyte-macrophage colony-stimulating factor(GM-CSF) and granulocyte colony-stimulating factor(G-CSF) (40 ng/mL each) for 3 days at 37 °C with 5%
CO2, whereupon IL-6 (40 ng/mL) was added. Cells wereisolated from the resulting mixed cell population on day
4 in vitro using Ly6G magnetic beads (Miltenyi Biotec).
Neutrophils were isolated on the day of use from bonemarrow with Ly6G magnetic beads following red blood
cell lysis.
Assessments of apoptosis, phagocytosis, bactericidal
activity, and neutrophil extracellular trap (NET) formation

Apoptosis was analyzed using a flow cytometry approach.
Leukocytes were added to a 96-well plate at a density of
2 × 105 cells per well and exposed to live S. aureus at a
10:1 multiplicity of infection (MOI; bacteria: leukocyte)
for 1 h prior to staining with 400 nM Apotracker Green

Page 4 of 15(Biolegend; Cat #427402) for 30 min at 37 °C. Dead cells
were identified using a Zombie UV Fixable viability kit(BioLegend). Leukocytes were distinguished from bacteria and cell debris by forward and side scatter gating.
Analysis was performed on a BD LSR-IIG cytometer andquantified by FlowJo.
Phagocytosis was assessed by incubating leukocyteswith either live S. aureus-GFP or S. aureus-dsRED strains[34] at a 10:1 MOI for 1 h. Dead cells were excluded, andleukocytes were distinguished from extracellular bacteria
and cell debris via forward and side scatter gating. Analysis was performed on a BD LSR-IIG cytometer and quantified by FlowJo.
Bactericidal activity was assessed by gentamicin protection assays as previously described [35]. Cells wereseeded in a 96-well plate (2 × 105 cells per well) prior tolive S. aureus exposure at a 10:1 MOI for 1 h at 37 °C. Following incubation, extracellular bacteria were eliminatedby high-dose gentamicin (100 µg/mL) treatment for
30 min at 37 °C. Cells were then washed and incubatedin RPMI-1640 medium supplemented with a gentamicin
maintenance dose (1 µg/mL) to prevent S. aureus outgrowth. At the indicated time points, cells were lysed in
H2O for 15 min, serially diluted, and plated on blood agar
to enumerate intracellular bacteria. Importantly, both
gentamicin protection assays and phagocytosis experiments utilized identical bacterial incubation periods(1 h) to permit the interpretation of bactericidal activity
along with any changes in bacterial uptake, which wereminimal.
For assessing NET formation, bone marrow PMNs
were plated in 8-well glass chamber slides at 4 × 105 cells/
well and exposed to live S. aureus-dsRed at a MOI of 10:1
for 1 h at 37 °C. Cells were then stained with 5 µM Sytox
Green (Invitrogen; Cat #S7020) to assess NET formationand imaged using a confocal laser scanning microscope(Zeiss 710) with a 40× oil objective. NETs were discriminated from dead PMNs based on differences in cell size,
shape, and transparency of Sytox Green staining characteristic of extracellular DNA release [7, 36, 37] and arereported as the percentage of total cells in each field ofview. Representative Z-stack images were taken (1 μm
slices) to illustrate NET formation.
Assessment of blood-brain barrier permeability

Blood-brain barrier (BBB) integrity was assessed using analbumin ELISA as described elsewhere [38–40]. Briefly,
supernatants collected from brain homogenates of micefollowing vascular perfusion were centrifuged to remove
residual cellular debris and assayed using a mouse Albumin ELISA Kit (Fortis Life Sciences; Cat# E99-134)
according to the manufacturer’s protocol. Data was normalized to total protein content via BCA assay.

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

RNA sequencing experiments

Single-cell suspensions were prepared from the galeaof WT and TNF KO mice at day 7 post-infection as
described above, whereupon live CD45+Ly6G+ granulocytes were purified by fluorescence-activated cell sorting(FACS). A total of 9 mice were infected for each group(WT and TNF KO) and galea tissues were pooled from
3 mice for a total of 3 biological replicates. RNA was isolated using a Quick-RNA Microprep Kit (Zymo Research;
Cat #R1050), and RNA sequencing libraries were prepared with a target of 50 ng RNA per sample. Libraries
were sequenced on a NextSeq500 instrument (Illumina)
with mid-output 150 cycles (75 paired-end reads) at asequencing depth of ~ 10 million paired reads per sample. All subsequent data processing and analysis wereconducted with Partek Flow Genomics Suite (RRID:
SCR_011860). Reads were quality checked and trimmed
before alignment to the mouse genome. Additional postalignment quality control steps were executed to remove
features with low read counts, and data was normalized
by counts per million plus 1 × 10− 4. A gene-specific analysis (GSA) approach was used to determine the optimal
differential expression model based on lowest Akaike
Information Criterion correction (AICc) and to control for false discovery rate, as described elsewhere [41,42]. Genes with p < 0.05 were considered differentiallyexpressed. Gene set enrichment analysis (GSEA) was
performed in Partek Flow to identify differentially regulated pathways with Ingenuity Pathway Analysis (IPA;
Qiagen) used as a secondary modality to explore differentially expressed pathways. The resulting dataset has been
deposited in the GEO database (GSE252481).
Statistics

Significant differences between groups for immune populations, inflammatory mediator expression, ELISA, orother assays were determined using either an unpaired
two-tailed Student t-test, multiple unpaired t-test, or
two-way ANOVA with Šidák multiple comparisonstest in GraphPad Prism (RRID: SCR_002798). Outliers
were removed only for Milliplex assays using the ROUT
method [43] (with Q = 1%; 0–2 observations per condition) in GraphPad Prism. A p-value of < 0.05 was used toreflect statistical significance.

Results
TNF mediates granulocyte recruitment during S. aureus
craniotomy infection

Our previous study identified a transcriptional footprintof TNF signaling during craniotomy infection, which wasparticularly elevated in granulocytes [8]. We have also
reported robust TNF production by G-MDSCs, PMNs,
macrophages, and microglia in vitro as well as in vivo
during craniotomy infection [7]. Given these reports

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and the documented importance of TNF in immunity toother S. aureus infections [17, 23–26], we first explored
how TNF influenced the immune response to S. aureus
craniotomy infection using our mouse model [1, 2, 7–11]
with TNF KO and WT animals. Dramatic reductions inleukocyte recruitment were observed in TNF KO micein a time-dependent manner, where granulocytes weresignificantly decreased uniformly in the brain, galea, andbone flap of TNF KO mice at day 14 post-infection, bothby relative abundance (Fig. 1D-F) and absolute count(Additional File 1: Fig. S2D-F). Interestingly, these deficiencies recovered to WT levels by day 28 post-infection(Fig. 1G-I; Additional File 1: Fig. S2G-I). At earlier intervals (i.e., day 7) no changes in relative immune cell abundance were observed between TNF KO and WT micein the brain (Fig. 1A); however, by absolute cell counts,
G-MDSCs, PMNs, monocytes, and NK cells were significantly decreased in TNF-deficient mice (Additional
File 1: Fig. S2A). G-MDSCs in the galea and bone flap
and PMNs in the galea were also modestly reduced in
TNF KO mice at day 7 post-infection (Fig. 1B-C; Additional File 1: Fig. S2B-C). Intracellular cytokine staining
confirmed the absence of TNF production in immune
populations from the brain, galea, and bone flap of TNF
KO animals (Fig. 2B-D), and sham craniotomy elicited
few immune infiltrates (Additional File 1: Fig. S3A-D),
directly implicating infection as the cause of leukocyte
recruitment, as opposed to danger-associated molecularpatterns released from damaged tissues. Despite the transient defects in leukocyte recruitment in TNF KO mice,bacterial burdens were similar between WT and TNF
KO animals across the 28-day infection period (Fig. 2A)
and antibiotic administration did not reveal additionalchanges in bacterial abundance between TNF KO andWT conditions (Additional File 1: Fig. S3E). These results
illustrate the temporal importance of TNF for leukocyte
recruitment but presumably not antimicrobial capabilities during craniotomy infection given the stability ofbacterial growth, suggesting the existence of mechanismsthat can compensate for TNF loss.
TNF-dependent leukocyte recruitment is reflected bydifferential chemokine production

Given that granulocytes are the predominant cellular
infiltrate in the galea and bone flap during craniotomy
infection [1] and are in close proximity to the biofilm [2],we next explored the potential mechanism responsible
for TNF-dependent granulocyte recruitment. We firstassessed whether TNF loss affected BBB integrity to regulate leukocyte influx into the CNS during craniotomy
infection. This was based on extensive literature showing that TNF production, particularly by microglia, regulates BBB properties to promote peripheral immune cell
influx [1, 18, 44–46] and BBB permeability is reduced inVan Roy and Kielian Journal of Neuroinflammation(2024) 21:179

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Fig. 1 TNF modulates granulocyte recruitment during craniotomy infection. TNF knockout (KO; n = 7–35) and wild type (WT; n = 11–35) mice were subjected to craniotomy infection and sacrificed at days 7 (A-C), 14 (D-F), and 28 (G-I) post-infection, whereupon immune infiltrates were quantified fromthe brain (A, D, G), galea (B, E, H), and bone flap (C, F, I). Data was pooled from 2–5 independent experiments (mean ± SEM) and analyzed by multiple
unpaired t-test. Mono, monocyte; NK, NK cell; *, p > 0.05; **, p > 0.01; ****, p > 0.0001

TNF-deficient mice [47]. Albumin is a large plasma protein that is excluded from the CNS by an intact BBB [48]but accumulates during neuroinflammatory or pathologic conditions when the BBB is compromised [40, 49].
Albumin content in brain homogenates was equivalent
between TNF KO and WT mice throughout the courseof craniotomy infection (Additional File 1: Fig. S4A),
indicating that differences in BBB permeability were notresponsible for the transient reductions in leukocyte
recruitment observed in TNF-deficient animals (Fig. 1).
Among its pleiotropic effects, TNF is known to stimulate the production of other inflammatory mediators[50], including IL-6 [51], CCL3, CCL4, and CXCL2 [52].
To determine whether TNF loss impaired chemokine
production to explain why leukocyte recruitment was
reduced in TNF-deficient animals, a panel of 25 cytokines and chemokines was quantified in the galea andbrain of TNF KO and WT mice at days 14 and 28 postinfection. TNF was detected in the galea of WT mice atboth time points, with significant reductions observedin TNF KO animals (Fig. 3A). Significant decreases in
CCL3, CCL4, and CXCL2 were observed in the galea of
TNF KO mice at day 14 post-infection (Fig. 3A), corresponding to the timepoint and anatomical location where
granulocyte recruitment was maximally reduced (Fig. 1E;
Additional File 1: Fig. S2E). Further, these chemokines
rebounded to WT levels at day 28 post-infection, mirroring the recovery of leukocyte infiltrates during craniotomy infection (Fig. 1H; Additional File 1: Fig. S2H).
Other cytokines (Additional File 1: Fig. S4B) and chemokines (Additional File 1: Fig. S4C) in the brain werelargely unaffected in TNF-deficient mice, likely due tothe low expression of TNF in the brain of WT animals
during craniotomy infection (Fig. 3B). IL-6 production
was significantly reduced in TNF KO macrophages in
response to live S. aureus (Additional File 1: Fig. S5A),
and TNF was undetectable as expected (Additional File 1:
Fig. S5D). In accordance with our in vivo findings (Additional File 1: Fig. S4B-C), TNF KO and WT macrophages
produced largely equivalent amounts of CCL2 and IL-10

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

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Fig. 2 TNF does not impact bacterial burden during S. aureus craniotomy infection. TNF knockout (KO; n = 7–35) and wild type (WT; n = 11–35) mice
were subjected to craniotomy infection and sacrificed at the indicated time points. (A) Bacterial burden was assessed at days 7, 14, and 28 post-infection(data was pooled from 2–5 independent experiments and analyzed by two-way ANOVA). (B-D) Intracellular staining for TNF was performed at day 7 postinfection in cells obtained from the brain (B), galea (C), and bone flap (D) (n = 5/group) to confirm the absence of TNF in KO cells (data are representativeof two experiments (mean ± SEM) and analyzed by non-parametric multiple Mann-Whitney tests). CFU, colony forming unit; Mono, monocyte; **, p > 0.01;****, p > 0.0001; ns, not significant(Additional File 1: Fig. S5B-C). Overall, these results suggest that TNF is crucial for inducing chemokine and IL-6
production during craniotomy infection, specifically in
the galeal compartment, aligning with previous reports[50–52].
TNF-deficient granulocytes upregulate lymphotoxin-α
during craniotomy infection

While TNF influenced chemokine production and leukocyte recruitment to the galea during craniotomy
infection (Figs. 1 and 3), these changes were not destabilizing enough to alter bacterial burden (Fig. 2A). This
makes craniotomy infection unique from other S. aureus
models where TNF is critical for bacterial containment[17, 23–26]. To explore why S. aureus titers were notaffected following TNF loss, we first examined whether
TNF influenced macrophage, PMN, and G-MDSC
responses to S. aureus- the major cellular infiltrates

during craniotomy infection [1]. Macrophage activity was
selectively regulated by TNF, since apoptosis (Fig. 4A),
phagocytosis (Fig. 4B), and bactericidal activity (Fig. 4C)
were significantly reduced in TNF KO macrophages in
response to live S. aureus, whereas granulocyte activation
was largely unaffected by TNF loss (Fig. 4A-C). Examination of PMN bactericidal activity at earlier time points(2 h post-stimulation; Additional File 1: Fig. S6E) as well
as apoptosis and cell death at later intervals (6 h and 24 hpost-infection; Additional File 1: Fig. S6A-D) revealed
only subtle differences in TNF KO vs. WT PMNs, whichwere unlikely to be biologically relevant based on theirlimited magnitude. Collectively, these experiments indicate that TNF is critical for macrophage antimicrobial
capabilities and survival, in agreement with previously
reported anti-apoptotic and proinflammatory effects of
TNF signaling [12, 16, 50, 53].

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Fig. 3 TNF influences the production of select chemokines during craniotomy infection. TNF knockout (KO; n = 8–10) and wild type (WT; n = 8–10) mice
were subjected to craniotomy infection and sacrificed at the indicated timepoints, whereupon galea (A) and brain (B) tissues were homogenized, andinflammatory mediators were quantified from resulting supernatants. Data was pooled from 2 independent experiments (mean ± SEM) and analyzed by
non-parametric multiple Mann-Whitney tests. *, p > 0.05; **, p > 0.01; ***, p > 0.001; ns, not significant

Since TNF production was most robust in the galea,
which is dominated by granulocyte infiltrates, one possibility to account for the lack of altered bacterial burden in
TNF KO mice may be due to granulocyte insensitivity to
TNF loss. However, TNF signaling has been reported topromote granulocyte function during S. aureus infection[17] and was transcriptionally enriched in granulocytes
during craniotomy infection in WT mice [8]. Therefore,bulk RNA-seq was used to compare the transcriptionalsignatures of TNF KO and WT granulocytes to identify
potential compensatory mechanisms in the context of
TNF loss. Granulocytes (CD45+Ly6G+) were FACS purified from the galea of TNF KO and WT mice at day 7
post-infection for bulk RNA-seq. A total of 294 and 85
genes were upregulated in TNF KO vs. WT granulocytes,
respectively (Fig. 5A). Gene set enrichment analysis confirmed that TNF signaling pathways were diminishedwith TNF loss (TNF Receptor Superfamily Binding,
GO:0032813; TNF receptor binding, GO:0005164) concomitant with decreases in reactive oxygen/nitrogenspecies and inflammatory pathways (Fig. 5B). Similarfindings were obtained using Ingenuity Pathway Analysis(IPA; Fig. 5F). In contrast, multiple pathways associatedwith protein misfolding/unfolded protein response (UPR)

were enriched in TNF KO granulocytes (Fig. 5C). A moredetailed examination of genes in the TNF receptor binding pathway confirmed decreased Tnf, Traf3, and Traf1
levels in TNF KO granulocytes; however, lymphotoxin-α(Lta) expression was dramatically increased (Fig. 5D).
In addition, IPA identified LTA (LTα) as the third mostupregulated molecule in TNF KO cells relative to WT(Fig. 5E). Importantly, LTα shares 50% homology to TNF[54] and can signal through both TNFR1 and TNFR2[55], possibly compensating for TNF deficiency in KOmice during craniotomy infection.
TNF receptors have unique effects on leukocyte function
but do not influence S. aureus craniotomy infection
outcomeSince TNF-deficient granulocytes augmented Lta expression, which can signal through both TNFR1 and TNFR2,
we next investigated how the loss of either receptor regulated leukocyte function. A recent report revealed a rolefor TNF and its receptors in neutrophil extracellular trap(NET) formation [17]; however, no significant differences in NET formation were observed in vitro in TNF,
TNFR1, or TNFR2 KO PMNs relative to WT cells (Additional File 1: Fig. S7A-I). Accordingly, in vivo experiments

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Fig. 4 TNF modulates apoptosis, phagocytosis, and bactericidal activity in macrophages but not granulocytes. Primary leukocytes were challenged withlive S. aureus at a MOI of 10:1 (bacteria: leukocyte), whereupon apoptosis (A) and phagocytosis (B) were quantified by flow cytometry (n = 5/group). Bacterial killing (C) was measured by gentamicin protection assays (n = 4–8/group). Data is representative of 2–3 independent experiments (mean ± SEM) andanalyzed by two-way ANOVA. CFU, colony forming unit; *, p > 0.05; ****, p > 0.0001

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

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Fig. 5 Lta expression is heightened in TNF knockout (KO) granulocytes during S. aureus craniotomy infection. TNF KO and WT mice were subjected tocraniotomy infection and sacrificed on day 7 post-infection, whereupon live CD45+Ly6G+ granulocytes were isolated from galea tissues by FACS (n = 3
biological replicates, each pooled from 3 mice) and RNA isolated for bulk RNA-seq. (A) Volcano plot depicting genes significantly upregulated in WT (blue;
85 genes) or TNF KO (red; 294 genes) mice. Gene set enrichment analysis of pathways significantly decreased (B) or increased (C) in TNF KO granulocytes
compared to WT. (D) Genes in the TNF receptor binding pathway (GO:0005164) were analyzed between TNF KO and WT granulocytes. (E-F) Ingenuity
Pathway Analysis was conducted to predict differentially regulated molecules (E) and pathways (F). All reported transcriptional comparisons are statistically significant (p < 0.05)

with mice incapable of forming NETs (PAD4 KO) [56]revealed no defects in bacterial containment (Additional File 1: Fig. S7J), suggesting that NET formation isdispensable during craniotomy infection. In contrast,
TNFR1 and TNFR2 were found to differentially regulate
leukocyte phagocytosis, bactericidal activity, and apoptosis in response to S. aureus. Both TNFR1 and TNFR2
deficiency decreased apoptosis in G-MDSCs (Fig. 6A-B),
whereas PMN apoptosis was only affected by TNFR2 loss
(Fig. 6B). Interestingly, macrophage apoptosis was unaffected by either TNFR1 or TNFR2 (Fig. 6A-B). Phagocytosis was modestly reduced in both TNFR1 and TNFR2
KO G-MDSCs, with all other cell types unaffected(Fig. 6C-D). Finally, TNFR1 was critical for promoting
macrophage bactericidal activity (Fig. 6E), suggesting thatsimilar observations in TNF KO macrophages (Fig. 4C)
were likely TNFR1-dependent. Conversely, TNFR2 was

important for bacterial containment by G-MDSCs andPMNs (Fig. 6F). However, since bactericidal activity was
not affected in either TNF KO granulocyte population(Fig. 4C), this suggests that LTα may compensate for theloss of TNF in these cells via TNFR2 signaling. Collectively, these findings illustrate additional contributions of
TNF signaling in granulocytes that only manifest in thecontext of receptor loss.
Given these results, we next investigated whether
TNFR1 and/or TNFR2 signaling influenced S. aureus
craniotomy infection. The phenotype of TNFR1 KO
mice was partially reminiscent of TNF KO animals, as
revealed by significant reductions in G-MDSC, PMN,
and macrophage infiltrates in the brains of TNFR1 KO
mice with no change in bacterial burden (Fig. 7A and
C). However, leukocyte influx was not altered in either
the galea or bone flap (Fig. 7D and E). TNFR2 loss led

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

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Fig. 6 TNFR1 and TNFR2 differentially modulate macrophage and granulocyte apoptosis, phagocytosis, and bactericidal activity. Primary leukocytes from
TNFR1 (A, C, E) or TNFR2 (B, D, F) KO mice were challenged with live S. aureus at a MOI of 10:1 (bacteria: leukocyte), whereupon apoptosis (A-B) and phagocytosis (C-D) were quantified by flow cytometry (n = 4–5/group). Bacterial killing (E-F) was measured by gentamicin protection assays (n = 4/group). Datais representative of 1–3 independent experiments (mean ± SEM) and analyzed by multiple unpaired t-test. CFU, colony forming unit; *, p > 0.05; **, p > 0.01;***, p > 0.001; ****, p > 0.0001

Fig. 7 TNFRs do not impact bacterial burden during S. aureus craniotomy infection. TNFR1 (A, C, D, E) or TNFR2 (B, F, G, H) knockout (KO; n = 4–5) andwild type (WT; n = 5) mice were subjected to craniotomy infection and sacrificed at day 7 post-infection, whereupon bacterial burden (A-B) and immune
populations (C-E, F-H) were assessed. Results were analyzed by (A-B) two-way ANOVA and (C-E, F-H) multiple unpaired t-test (mean ± SEM). CFU, colony
forming units; Macro, macrophage; Mono, monocyte; *, p > 0.05; **, p > 0.01; ***, p > 0.001

to significant decreases in MHCII+ activated microglia in the brain (Fig. 7F) and the galea displayed minor
alterations in leukocyte recruitment (Fig. 7G), although
this was not sufficient to influence bacterial burdens ineither compartment (Fig. 7B). In general, similar trends

were observed for absolute leukocyte counts in infected
TNFR1 and TNFR2 KO animals (Additional File 1: Fig.
S88). Collectively, while granulocytes augment Lta
expression in the context of TNF deficiency, the fact thatbacterial burdens were not dramatically altered in TNF,

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

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TNFR1, or TNFR2 KO mice indicates that TNF signaling
in isolation is not sufficient to alter the course of craniotomy infection.

to planktonic S. aureus, which diverged from our invivo results showing equivalent bacterial burden during TNF KO or WT craniotomy infection that is typified
by biofilm formation. Other in vivo data presented here
also supports this concept, where NET formation had
no impact on S. aureus craniotomy infection. However,
NET formation was found to be protective in a mousemodel of S. aureus skin and soft tissue infection, reflective of planktonic growth, by TNF signaling throughTNFR2 [17]. Therefore, the similarities in bacterial burden between WT and TNF, TNFR1, and TNFR2 KO
mice observed in this study support the ineffectivenessof TNF-induced NETs to combat biofilm infection. This
earlier report [17] identified that TNFR1 was responsible
for granulocyte recruitment, which agrees with our findings where the greatest reductions in PMN and G-MDSC
infiltrates were observed in the brains of TNFR1 compared to TNFR2 KO mice. Nevertheless, TNFR1 andTNFR2 deficiency did not affect bacterial burden in vivo,
despite phenotypic changes in granulocyte phagocytosis,
apoptosis, and bactericidal activity in vitro, reflecting the
involvement of additional factors in the biofilm inflammatory milieu that promote infection persistence, which
remain to be identified. Given our recent findings that
immune responses during human craniotomy infection
are highly conserved with the mouse model at the cellular and transcriptional levels (manuscript in revision) anddespite the reported association between anti-TNF biologic use and an increased risk of S. aureus infection [22],our findings suggest that the biofilm features of S. aureus
during craniotomy infection may circumvent a negative outcome in this regard, although this remains highly
speculative. However, it is important to note that this lineof reasoning may not extend to other causative pathogensof craniotomy infection [65], which could be exacerbatedby TNF depletion and remains to be assessed.
Another concept underscored by this work surrounds
the biological redundancy of TNF signaling, which is acomplex system deeply integrated with cellular function[50, 55]. We identified a novel increase in Lta expressionin TNF KO granulocytes, suggesting a potential compensatory response since LTα can signal through both
TNFR1 and TNFR2 [54]. This was supported by the finding that apoptosis, phagocytosis, and bactericidal activity were similar in PMNs from TNF KO and WT mice,
whereas significant deficits were observed for these samemeasures under TNFR2 and, to a lesser extent, TNFR1
deficiency. These findings demonstrate that TNF deletion
may cause unexpected transcriptional changes that could
mask or alter phenotypes. In addition to Lta, our analysis revealed a marked increase in pathways associatedwith misfolded proteins/UPR in TNF KO granulocytes.
This finding was surprising, as TNF has been reported toinduce the UPR [66–68] and, therefore, was expected to

Discussion
This study demonstrates the importance of TNF signaling for leukocyte recruitment during S. aureus craniotomy infection, where dramatic reductions in cellular
influx were observed in the brain, galea, and bone flap of
TNF KO mice during the first two weeks post-infection.
Interestingly, these changes largely returned to WT levels
by day 28, underscoring the temporal complexity of theimmune response during craniotomy infection. Fluctuations in leukocyte recruitment coincided with concurrent
decreases and recovery in the levels of several granulocyte-recruiting chemokines, including CCL3, CCL4, andCXCL2 in TNF KO mice, likely accounting for reduced
PMN and G-MDSC abundance at earlier time points.
Interestingly, despite impaired leukocyte recruitment
up to day 14 post-infection in TNF KO animals, this did
not translate to altered bacterial burden in any infection
compartment analyzed. This finding contrasts with priorreports of heightened bacterial growth or even lethality
with TNF deficiency in other models of S. aureus infection [17, 23–26] or alternative pathogens [52, 57–60].
Our RNA-seq studies revealed heightened Lta expression in TNF KO granulocytes, which led us to speculatethat LTα may compensate to protect TNF KO mice fromexaggerated pathology since it binds the same receptors
as TNF (i.e., TNFR1 and TNFR2) to exert downstream
effects. However, this possibility was not supported bysubsequent experiments utilizing TNFR1 and TNFR2
KO mice. Instead, this suggests that other factors uniqueto the craniotomy infection microenvironment influence
the TNF-independent phenotypes reported here. One
possibility relates to the nature of S. aureus growth during craniotomy infection, namely biofilm formation on
the infected skull bone [1, 2, 10, 11]. Biofilm infections
are chronic [61] and thus often exhibit divergent attributes from other bacterial infections that are typified byplanktonic growth (i.e., abscesses, sepsis) [1, 2, 10, 31,
34, 62–64]. To our knowledge, only one study reported
heightened infectious burden in TNF KO mice using a
biofilm model (orthopedic biofilm infection) [26], which
described only modest increases in bacterial growth. In
contrast, work with non-biofilm infection models often
revealed more profound increases in pathogen burden,sometimes resulting in lethality [17, 23–25, 52, 57–60].
Therefore, TNF may be less critical for an effective antibacterial response in the context of biofilm infection.
This dichotomy between the importance of TNF acrossplanktonic and biofilm infections is supported by ourin vitro data, where TNF deficiency decreased macrophage phagocytosis and bactericidal activity in response

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

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be reduced with TNF deficiency. However, the UPR has
also been shown to promote TNF production by immune
cells [69, 70], so this may reflect a compensatory effort by
granulocytes to increase TNF levels, although this was
futile in the context of Tnf deletion. Another footprint of
TNF action in vivo was decreased PMN and G-MDSC
recruitment in all three tissue compartments of TNF KO
animals, which peaked at day 14 and recovered by day 28
post-craniotomy infection. This phenotype was linked toreduced chemokine production, which paralleled trends
in cellular abundance. Interestingly, an earlier study
reported nearly identical changes in a model of tuberculosis (TB) infection with TNF KO mice [52], in agreementwith our findings. For example, leukocyte recruitment to
the liver and spleen was impaired in TB-infected TNF KO
mice up to day 14 post-infection, with recovery to (andsometimes exceeding) WT levels at day 28, identical to
the time frames presented in the current study. Additionally, CCL3, CCL4, and CXCL2 levels were low at day 14
and recovered by day 28 post-infection, also in agreementwith our findings [52]. The striking parallels between thereport from Roach et al. and our data, despite differing
pathogens, infection sites, models, and methodologies,
suggest that the relationship between TNF, chemokines,
and leukocyte recruitment is partially cell-intrinsic andnot mediated by microenvironmental factors. While
Roach et al. did not explore the mechanism responsible
for TNF effects during TB infection, our data may provide some insights. Namely, heightened LTα productionin the context of TNF deficiency may be sufficient overextended periods to stimulate CCL3, CCL4, and CXCL2
expression. However, because TNF had no impact onbacterial burden during craniotomy infection, this model
is suboptimal to pursue this idea further. Additional
work investigating the role of LTα in the presence andabsence of TNF deficiency in a TNF-dependent model(such as TB infection) would be required to explore thispossibility. Interestingly, progranulin (PGRN) and TNF
also share the same receptor, and PGRN has a stronger
affinity for TNFR than TNF [71]. In addition, PGRN is
an important neurotrophic factor and plays an importantrole in infection, inflammation, and immunity [72–75].
Although grn expression was low and not significantly
different between WT and TNF KO granulocytes during
craniotomy infection, it is possible that other cell types
are a source of PGRN, which could be pursued in futurestudies that are outside the scope of the current report.
Additionally, although G-MDSCs exhibited modest bactericidal activity in vitro, it is unclear whether this would
extend in vivo given the documented anti-inflammatory
characteristics of G-MDSCs within the complex craniotomy infection microenvironment [9, 10, 76].
In conclusion, we demonstrate a role for TNF in leukocyte recruitment to the site of craniotomy infection by its

ability to promote chemokine production. Additionally,
we present evidence for a potential compensatory mechanism for the lack of TNF via enhanced Lta transcription.
Despite these changes, infection was not worsened by
TNF loss at any time point examined, which may result
from S. aureus-derived factors unique to biofilm growth.
These findings underscore the pleiotropic effects of
TNF during bacterial infection and the heterogeneity in
immune responses that are likely tissue niche-dependent.
Abbreviations
BCA	Bicinchoninic acid assay
CCL3	Chemokine (C–C motif ) ligand 3 (MIP–1α)
CCL4	Chemokine (C–C motif ) ligand 4 (MIP–1β)CD3	cluster of differentiation 3
CD4	cluster of differentiation 4
CD45	cluster of differentiation 45
CD8	cluster of differentiation 8
CFU	Colony forming unit
CNS	Central nervous system
CO2	Carbon dioxide
CX3CR1	CX3C motif chemokine receptor 1
CXCL2	Chemokine (C–X–C motif ) ligand 2 (MIP–2)
ELISA	Enzyme–linked immunosorbent assay
FACS	Fluorescence–activated cell sorting
FBS	Fetal bovine serum
GEO	Gene expression omnibus
GFP	Green fluorescent protein
G-CSF	Granulocyte colony stimulating factor
GM-CSF	Granulocyte-macrophage colony stimulating factor
IL-6	Interleukin-6
IPA	Ingenuity pathway analysis
KO	Knockout
LTα	Lymphotoxin–α
MAPK	Mitogen–activated protein kinase
G-MDSC	Granulocytic myeloid-derived suppressor cell
MHCII	Major histocompatibility complex II
MOI	Multiplicity of infectionNET	Neutrophil extracellular trap
NF-κB	Nuclear factor-kappa B
PBS	Phosphate–buffered saline
PGRN	Progranulin
PMN	NeutrophilRNA	Ribonucleic acid
TB	Tuberculosis
TNF	Tumor necrosis factor
TNFR1	Tumor necrosis factor receptor 1
TNFR2	Tumor necrosis factor receptor 2
UPR	Unfolded protein response
WT	Wild type

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03174-9.
Supplementary Material 1
Acknowledgements
The authors thank Rachel Fallet for managing the mouse colony.
Author contributions
ZVR and TK designed, and ZVR conducted the experiments presented here.
ZVR wrote the manuscript with editing provided by TK.

Van Roy and Kielian Journal of Neuroinflammation(2024) 21:179

Funding
This work was supported by the National Institutes of Health/National Instituteof Neurological Disorders and Stroke R01 NS107369 and National Institute ofAllergy and Infectious Diseases R01 AI169788 (both to TK), T32 NS105594, and
UNMC Graduate Research Fellowship (to ZVR). The UNMC DNA Sequencing
Core receives partial support from the National Institute for General Medical
Science (NIGMS; INBRE - P20GM103427-14 and COBRE − 1P30GM11076801). Both the UNMC DNA Sequencing and Flow Cytometry Research Cores
receive support from The Fred & Pamela Buffett Cancer Center Support Grant(P30CA036727).
Data availability
Transcriptomic data from bulk RNA-seq studies has been deposited in the
GEO database (GSE252481). All other data in this manuscript are available
upon reasonable request.

Declarations
Ethics approval
This study was conducted according to the recommendations in the Guide
for the Care and Use of Laboratory Animals of the National Institutes of Healthand complies with the Animal Research: Reporting of In Vivo Experiments
guidelines. The protocol was approved by the University of Nebraska Medical
Center Institutional Animal Care and Use Committee (#16-123-10).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Pathology, Microbiology, and Immunology, University of
Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha,
NE 68198-5900, USA
Received: 12 March 2024 / Accepted: 14 July 2024

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