(2024) 21:207
Ohm et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03188-3

Journal of Neuroinflammation

Open Access

RESEARCH

The potential therapeutic role of itaconate
and mesaconate on the detrimental effectsof LPS‑induced neuroinflammation in the brain
Melanie Ohm1†, Shirin Hosseini1,2†, Niklas Lonnemann1, Wei He3, Tushar More3, Oliver Goldmann4, Eva Medina4,
Karsten Hiller3* and Martin Korte1,2*Abstract
Despite advances in antimicrobial and anti-inflammatory treatment, inflammation and its consequences remaina major challenge in the field of medicine. Inflammatory reactions can lead to life-threatening conditions suchas septic shock, while chronic inflammation has the potential to worsen the condition of body tissues and ultimately
lead to significant impairment of their functionality. Although the central nervous system has long been consideredimmune privileged to peripheral immune responses, recent research has shown that strong immune responses
in the periphery also affect the brain, leading to reactive microglia, which belong to the innate immune system
and reside in the brain, and neuroinflammation. The inflammatory response is primarily a protective mechanism
to defend against pathogens and tissue damage. However, excessive and chronic inflammation can have negative
effects on neuronal structure and function. Neuroinflammation underlies the pathogenesis of many neurologicaland neurodegenerative diseases and can accelerate their progression. Consequently, targeting inflammatory signaling pathways offers potential therapeutic strategies for various neuropathological conditions, particularly Parkinson’s and Alzheimer’s disease, by curbing inflammation. Here the blood–brain barrier is a major hurdle for potential
therapeutic strategies, therefore it would be highly advantageous to foster and utilize brain innate anti-inflammatory
mechanisms. The tricarboxylic acid cycle-derived metabolite itaconate is highly upregulated in activated macrophages and has been shown to act as an immunomodulator with anti-inflammatory and antimicrobial functions.
Mesaconate, an isomer of itaconate, similarly reduces the inflammatory response in macrophages. Nevertheless, moststudies have focused on its esterified forms and its peripheral effects, while its influence on the CNS remained largely
unexplored. Therefore, this study investigated the immunomodulatory and therapeutic potential of endogenously
synthesized itaconate and its isomer mesaconate in lipopolysaccharide (LPS)-induced neuroinflammatory processes.
Our results show that both itaconate and mesaconate reduce LPS-induced neuroinflammation, as evidenced by lower
levels of inflammatory mediators, reduced microglial reactivity and a rescue of synaptic plasticity, the cellular correlateof learning and memory processes in the brain. Overall, this study emphasizes that both itaconate and mesaconate
have therapeutic potential for neuroinflammatory processes in the brain and are of remarkable importance dueto their endogenous origin and production, which usually leads to high tolerance.
†

Melanie Ohm and Shirin Hosseini have contributed equally to this work.

*Correspondence:
Karsten Hiller
karsten.hiller@tu-braunschweig.de
Martin Korte
m.korte@tu-braunschweig.de
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 theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Ohm et al. Journal of Neuroinflammation(2024) 21:207

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Keywords Microglia, LPS, Neuroinflammation, Synaptic plasticity, Hippocampus

Introduction
The central nervous system (CNS), has only a limited
capacity for self-regeneration, and must therefore beparticularly well protected against potential damage. Fora long time, the brain was considered as an immuneprivileged organ, due to its separation from the periphery by the blood–brain barrier (BBB) and the absence oflymphatic vessels [1]. However, intensive research demonstrated that the CNS is neither isolated nor passive in
its interaction with the immune-system and even in theabsence of inflammation the immune system and CNS
communicate with each other.
Certain pathogens, including specific viruses and bacterial infections, whether located in the brain or even in
periphery, have the potential to impact the CNS. Among
those acute systemic inflammatory processes triggeredby lipopolysaccharide (LPS), the most common antigen
on the cell surface of most Gram-negative bacteria, leadto neuronal death and neurodegeneration, particularlyin the hippocampus, a brain area of high importance forprocesses of learning and memory [2–7]. Immune reactions in the brain are mainly characterized by the reactivity of brain resident immune cells, namely microglia.
Microglial reactivity results in production of various
cytokines and chemokines as well as the production ofcomplement component 1q (C1q), prostaglandin E2(PGE2) and free radicals (NO and ROS) [8–10]. In addition to microglia, astrocytes, cells with immunologicalproperties are involved in neuroinflammatory processes.
Inflammatory mediators released by microglia can initiate the transformation of astrocytes from a quiescentresting to a reactive phenotype [11], additionally they
produce inflammatory mediators actively contributing to
the inflammatory status of the brain [12, 13].
Although neuroinflammatory responses are part ofthe brain’s defense mechanism to eliminate cellulardebris and pathogens, as well as facilitate tissue repair,
prolonged chronic responses can result in various neuropathological manifestations [14, 15]. For example, neuroinflammation is associated with various neurologicaldisorders such as neurodegenerative diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple
sclerosis and even depression [16–18]. Recent studies
suggest that neuroinflammatory processes may not only
exacerbate but also trigger the onset of these diseases[15]. Therefore, inflammatory signaling pathways havebeen implicated as potential therapeutic targets for manyneurological diseases to counteract the extent of theinflammation.

Itaconate, a metabolite derived from the tricarboxylic
acid cycle (TCA), has attracted considerable interest as
an intriguing example of an immunomodulator with antiinflammatory and antimicrobial properties [19–21]. It
is predominantly produced in myeloid cells specificallyduring inflammation via the inducible aconitate decarboxylase 1 (ACOD1), also known as immune-responsive
gene 1 (IRG1) protein [19, 20, 22, 23]. The anti-inflammatory potential of itaconate was first described in Irg1deficient mice (Irg1−/−), unable to convert cis-aconitate
to itaconate and therefore have a lack in the itaconate
synthesis. Macrophages from these animals showed
increased release of inflammatory mediators induced by
LPS compared to wild-type (WT) [24]. However, moststudies have focused on investigating the effects of chemically esterified forms of itaconate, dimethyl itaconate(DMI) and 4-octyl itaconate (4-OI), which are known for
their higher cellular permeability [19]. Recently, it was
revealed that DMI and 4-OI possess much higher electrophilicity than the endogenously produced itaconate,
and such electrophilicity underlies the ability to activate
signaling pathways including nuclear factor erythroid2-related factor 2 (NRF2) [25]. Therefore, it is necessary
to focus on the biological effects of the endogenous formof itaconate rather than the chemical derivatives.
Recently described by He et al., the stimulation of macrophages with LPS results not only in the production ofendogenous itaconate but also mesaconate, an isomer of
itaconate. The only structural difference between thesemetabolites is the position of the double bond [19]. Bothmetabolites have been shown to reduce the inflammatory response in inflammatory peripheral macrophages[19]. In addition, intraperitoneal (i.p) injection of itaconate and mesaconate appears to reduce the extent of
LPS-induced systemic inflammation and prolonged survival following a lethal dose of LPS [19]. However, little
is known about the effect of these two metabolites within
the CNS.
Sun et al. demonstrated that the administration of itaconate has a neuroprotective effect against the extentof PD symptoms in the mouse model [26]. In addition,
treatment with itaconate was able to reduce the inflammatory symptoms caused by the reperfusion injury [27].
Therefore, we investigated here whether itaconate without chemical modification can alleviate the inflammatory processes in the rodent brain induced by peripheral
immune stimulation and to evaluate its therapeutic role.
In addition, the anti-inflammatory properties of mesaconate in various pathological conditions are still not clear.

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Since the use of endogenous molecules as therapeutics is of great importance due to their natural production and expected high tolerability, this study investigated
the therapeutic potential of itaconate and mesaconate inperipheral LPS-induced neuroinflammation in the particularly susceptible brain region, the hippocampus.
Our results show that pretreatment with exogenous itaconate or mesaconate alleviates LPS-triggered neuroinflammation. This effect is manifested by both the reduced
microglial activation and the prevention of LPS-induced
synaptic plasticity impairment, highlighting the ability ofboth metabolites to preserve synaptic functionality.

Material and methods
Animals

For the injection of itaconate and mesaconate followed
by LPS, 3-month-old C57BL/6J male mice were used.
Mice were bred and kept at the animal facility of the
TU Braunschweig under standard housing conditions
in a 12:12 light:dark cycle at 22 °C with food and water
available ad libitum. The performed mice experiments
were approved according to the animal welfare law inGermany.
To investigate the impact of Irg1-gene on LPS-induced
neuroinflammation, Irg1−/− and WT mice as controls
were used in the age of 6 months. Mice were bred andkept at the central mouse facility of the Helmholtz Centre
for Infection Research, Braunschweig, Germany.
All protocols used in this project have been reviewed
and approved by the local committees at TU Braunschweig and the authorities (LAVES, Oldenburg, Germany
33.19–42502-04–21/3734) according to the nationalguidelines of the animal welfare law in Germany (‘Tierschutzgesetz in der Fassung der Bekanntmachung vom
18. Mai 2006 (BGBl. I S. 1206, 1313).
Intraperitoneal injections

Mice were intraperitoneally (i.p.) injected with itaconate
and mesaconate on three consecutive days. The dosage of 250 mg/kg bodyweight was used because He et al.
demonstrated an immunoregulatory effect at this dosage [19]. Control mice received equal volumes of PBS,
the solvent of the metabolites. Prior to the injection mice
were checked for possible changes that might affect theexperiment. The bodyweight and other external factors
were monitored and documented daily. The bodyweight
was used to define the appropriate amount of the injection solutions and were injected i.p. accordingly. Subsequently, to induce a systemic immune stimulation 24 hafter the last metabolites injection mice received a duali.p. LPS (E. coli O127:B8, Sigma Aldrich L 3129, 0.5 mg/kg
bodyweight) stimulus or saline as control solution, at 24 hPage 3 of 30

intervals [7]. Three hours after the second LPS-injection,
mice were sacrificed and samples were collected.
To test the impact of the lack of the Irg1-gene in LPSinduced neuroinflammation Irg1−/− mice and respective
controls were injected two-times with LPS (0.5 mg/kg
bodyweight) or an appropriate amount of saline solution
as a control in a 24 h interval. Three hours after the second injection mice were sacrificed.
Primary microglial and astrocytic cultures stimulatedwith LPS

Primary glial cultures were prepared as described before[7]. Briefly, neonatal mouse brains (P2-P4) of mixed
genders, excluding the hippocampus, cerebellum andmeninges, were transferred into HBSS 1X on ice. Afterdissecting the brains mechanically, the tissue was centrifuged 5 min at 300 ×g at 4 °C and the pellet was strained(100 µm pores) after resuspension in HBSS followedby another centrifugation step (400 ×g, 5 min, 4 °C).
The resulting pellet was resuspended in 10 mL culture
medium (low DMEM + 10% FCS + 1% Penicilin/Streptomycin) and cultured in a poly-lysine coated T-75 flask
at 10% ­CO2 at 37 °C. The medium was initially replaced
after 3 days, followed by a weekly medium change After
2 weeks, once a confluent cell layer formed, microglia
were harvested by shaking the flasks at 180 rpm for 3 h
at 37 °C and plated in a 12-well plate (5×105 cells/well).
Depending on the experiment, following 24 h incubation microglia were either pre-incubated with itaconate
or mesaconate (10 mM) for 4 h before LPS (10 ng/mL)
was added for further 21 h, or they were only treated with
LPS for 24 h, until supernatant was collected and cells
were harvested.
Primary astrocytic cultures were obtained from neonatal mouse brains (P1-3) by decapitating, removing
the hippocampus, cerebellum and meninges, and processing in fresh HBSS 1X. Following a slide mechanically dissociation, HBSS was replaced by a dissociation
solution (9.1 mL DMEM low glucose medium, 400 µL
10 × Trypsin/EDTA, 200 µL 1M HEPES, 5 mg/mL in
0.15 mol/L NaCl DNAse, 100 µl 100 × Penicilin/Streptavidin). The mixture was incubated at 37 °C for 30 min,gently shaken every 4 min, and subsequently further dissociated by pipetting. After a centrifugation step (7 min
at 800 rpm), to obtain good dissociation, the glass micro
pipettes bore was fire polished to a smaller opening.
After discarding the supernatant and adding culture
medium (DMEM low glucose, 10% FCS, 1% Penicillin/
Streptavidin), the homogenate was passed through a 40
µm cell strainer and the cell suspension was plated inpoly-D-lysine-coated T75 flasks. Cultures were incubatedat 10% ­CO2, 37 °C for ~ 2 weeks, with a medium change
every 2–3 days. Once confluence was reached, overnightOhm et al. Journal of Neuroinflammation(2024) 21:207

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shaking at 220 rpm isolated astrocytes. Cells were passaged at a 1:2 ratio after two passages and overnightshaking. Harvested cells were plated in 12-well plates(5×105 cells/well) and, after 24 h, stimulated with 100 ng/
mL LPS for 24 h before supernatant collection and cell
harvest.

protein concentration within the test samples. To normalize the detected density of the ELISA the total protein
concentrations of the brain tissue samples were measured using a Bradford assay. Briefly, a 1:200 dilution of
the samples in 100 µL Bradford solution was added andmeasured at 595 nm using the Epoch microplate reader.

Stable isotope tracing of LPS‑stimulated primary microglia

Immunohistochemistry

The tracer medium was prepared using DMEM (GibcoTM) without glucose and glutamine, as well as phenol red. This medium was supplemented with either[U-13C6] glucose (at a final concentration of 5.5 mM)
along with unlabeled glutamine (at a final concentrationof 4 mM), or [U-13C5] glutamine (at a final concentration of 4 mM) combined with unlabeled glucose (at a final
concentration of 5.5 mM). Both tracers were purchasedfrom Cambridge Isotope Laboratories. Initially, cells were
cultured in standard low glucose DMEM medium, subsequently replaced with medium equipped with the tracers.
The cells were then further incubated for 24 h to achieve
isotopic equilibrium before stimulated with LPS.

Immunohistochemistry was performed for visualizationof different cell types and proteins. Mice were deeply
anesthetized by C
­ O2 and sacrificed via decapitation. The
fresh cerebral hemispheres were fixed in 4% paraformaldehyde (PFA) in PBS for 24 h, followed by cryoprotectionin 30% sucrose solution in PBS 1X for at least 24 h. On
the day of experiment, the hemispheres were frozen in
Tissue-Tek® (Hartenstein Laborversand) at − 70 °C. Frozen brain hemispheres were cut into 30 µm thick slices
using a Reichert Jung/Leica Frigocut 2800E cryostat
microtome. Five to six sections per mouse were used forfree-floating immunohistochemical experiments. The
sections were washed twice with PBS 1X for two minutes
each and three times with 0.1% Triton X-100 for five minutes. Subsequently, the sections were permeabilized forone hour in blocking solution (0.3% Triton X-100, 10%goat serum, 5% bovine serum albumin (BSA)) followed
by incubation of respective primary antibodies (IBA1,
1:1000, rabbit, WAKO 019–19741; CD68 clone FA-11,
rat, 1:500; Bio-Rad MCA 1957; GFAP, 1:1000, mouse,
Sigma Aldrich G 3893) diluted in blocking solution overnight at 4 °C. The next day sections were washed 3 times
with PBS 1X for 10 min each, followed by incubation in
respective secondary antibodies diluted 1:500 in PBS
1X for 2 h at RT (­Cy™3 AffiniPure goat anti-rabbit IgG
H + L, 111–165-144; ­Cy™5 AffiniPure goat anti-rat IgG
H + L, 112–175-167; C­ yTM2-conjugated AffiniPure Goat
Anti-Mouse IgG H+L, 111–165-144) Finally, the brainsslices were washed again three times with PBS 1X for
10 min each, stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min and mountedwith Fluoro-gel medium (Electron Microscopy Sciences,
Hatfield, PA) on glass slides.

Cytokine immunoassay of blood and brain tissue

Cytokine immunoassay of blood and brain tissue was
performed using Enzyme-linked immune sorbent assay(ELISA) as described before [28]. The mice were euthanized via ­CO2 asphyxiation, followed by decapitation.
The trunk was positioned over a collection tube to allow
for blood drainage. The blood was then allowed to clot
for 20 min at room temperature (RT), followed by centrifugation at 2000 ×g for 20 min. This protocol ensuresthe collection of a sufficient volume of blood for the
ELISA assay. The whole brain was carefully removed.
One hemisphere was frozen in liquid nitrogen and storedat − 80 ℃ until usage. The brain was homogenized by the
GentleMACS (Milteny Biotec, Protein_01 program) in
500 µL STKM lysis Buffer (containing: 250 mM sucrose,
50 mM Tris–HCl, 25 mM KCl, and 5 mM ­MgCl2) andprotease inhibitor mixture (Roche c­ Omplete™ Protease
Inhibitor Cocktail tablet). Subsequently, the samples were
centrifuged for 10 min at 13.000 × g at 4 °C. The supernatant was stored at − 70 °C until assayed. Mouse IL-1beta/
IL-1F2 DuoSet, Mouse IL-6 DuoSet, Mouse IL-10 DuoSet and mouse TNF-alpha DuoSet ELISA kits (R&D SYSTEMS) were used to determine cytokine levels according
to the manufacturer’s recommendations. For measuring
cytokines of cell culture supernatants cytokines werediluted 1:5 similar to blood samples. Brain homogenates
were diluted 1:2. The absorbance was measured using an
Epoch microplate reader at 450 nm connected to the Gen
5 software (BioTek). Lastly, the recorded optical densityof the reaction was compared with the optical density of
established standard samples in order to ascertain the

Golgi‑Cox staining

Golgi-Cox staining was performed using the FD Rapid­GolgiStainTM Kit (FD Neuro-technologies, Inc.) according to the manufacturer’s protocol. Briefly, mice weredeeply anesthetized using C­ O2 and sacrificed via decapitation. The brain was carefully dissected and one of thecerebral hemispheres was incubated in the Golgi solution
mixture according to the manufacturer protocol. Before
sectioning the brain in 150 µm coronal slices using a
Leica Vibratome (VT 1000S), the cerebral hemispheres
were embedded in 2% agar. The sections were dried on

Ohm et al. Journal of Neuroinflammation(2024) 21:207

gelatin-coated slides. In the following steps, the sections
were further processed for signal development according to the kit manufacturer’s guidelines. Finally, the sections were mounted using Permount (Thermo Fisher
Scientific).

Electrophysiological recordings
Hippocampi of mice from experimental groups were dissected and cooled in ice-cold carbonated (95% ­O2 and
5% ­CO2) artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 4.9 mM KCl, 1.2 mM ­KH2PO4, 2 mM
­MgSO4, 2 mM ­CaCl2, 24.6 mM ­NaHCO3 and 10 mM
D-glucose. Afterward, 400 µm thick slices were chopped
with a manual tissue slicer (Stoelting) and were kept at
32 °C with a constant flow rate (0.5 mL/min) of carbonated ACSF for 2 h until recording. Recordings were performed as previously described [29, 30]. Field excitatory
postsynaptic potentials (fEPSPs) were elicited throughelectrical stimulation along the CA3 to CA1 Schaffer
collateral pathway, using a lacquer-coated stainless-steel
electrode (5 MΩ; AM Systems). Measurements weretaken in the CA1 stratum radiatum, with the recording electrode positioned at least 20 μm from the stratum pyramidale in the apical dendritic layer. The signals
captured by the electrode were amplified and digitizedwith an AM Systems amplifier (model 1700) and a CED
1401 analog-to-digital converter (Cambridge Electronic
Design). Basal synaptic transmission was assessed byplotting an input–output curve relating afferent stimulus
intensity to the fEPSP slope, with the test stimulus set to
evoke fEPSPs at 40% of the maximal response. Short-term
plasticity was analyzed by administering two consecutive stimuli of equal intensity across various interpulseintervals (10, 20, 40, 60, 80 and 100 ms). For long-term
potentiation (LTP) studies, after establishing a stablebaseline, LTP was induced with theta-burst stimulation(TBS) comprising four 100 Hz bursts, repeated 10 times
at 200 ms intervals, and applied thrice every 10 s. fEPSP
slopes were monitored for an hour post-stimulation andnormalized to the pre-TBS baseline. Both acquisition andanalysis of the data were carried out using IntraCell software (version 1.5, LIN, Magdeburg, 2000).
RNA extraction was performed by using ­NucleoSpin®
RNA isolation kit (Macherey–Nagel, Düren, Germany).
Followed by cDNA synthesis with High Capacity cDNA
Reverse Transcription Kit (Thermo Fisher Scientific).
Quantitative RT-PCR was performed using T­aqMan®
using BlueProbe qPCR Mix (Biozym) and using thehouse keeping control GAPDH. Expression levels of target mRNA of IL-1β, IL-6 and BDNF was analyzed using
Total RNA and quantitative real time‑PCR

Page 5 of 30

the ∆∆Ct method and were normalized to the expression
level of the house keeping gene.
Primer sequences bought at EUROFINS:GAPDH sense (s): GCC​TTC​CGT​GTT​CCT​ACC​, antisense (a): CCT​CAG​TGT​AGC​CCA​AGA​TG probe (p):CGC​CTG​GAG​AAA​CCT​GCC​AAGTA;
IL-1β s: ACG​GAC​CCC​AAA​AGA​TGA​AG, a: TTC​TCC​
ACA​GCC​ACA​ATG​AG, p: AGA​GCA​TCC​AGC​TTC​
AAA​TCT​CGC​A;
IL-6: s: TGC​TAC​CAA​ACT​GGA​TAT​AAT​CAG​G, a:
AGG​ACT​CTG​GCT​TTG​TCT​TTC, p: CTT​CTG​GAG​
TAC​CAT​AGC​TAC​CTG​GAGT.
Flow cytometry analysis

Single cell suspension from fresh brain tissue was created using Adult Brain Dissociation Kit (Milteny Biotec,
130–107-677) and the GentleMACS Milteny Biotec), as
described before [7, 31] according to the manufacturer´sprotocol. Afterwards, the cells were resuspended in
FACS staining buffer (PBS 1X + 1% FCS + 0.1% Na-Azide)and stained for 30 min against mouse CD11b-PerCPVio700 Clone REA592 (1:50), CD45-APC (1:50), CD68PE Clone REA835 (1:50) (Milteny Biotec) in a 96-wellplate. The flow cytometry was conducted using B­ D® LSR
II Flow Cytometer and analyzed with FlowJo Software(version 10.8.1). Briefly, in the FACS analysis, forward
scatter (FSC) and side scatter (SSC) parameters wereused to select the region of interest (ROI), focusing on
single cells and excluding doublets to capture individual
microglia populations. Within these ROIs, cells wereanalyzed for CD11b and CD45 expression to distinguishmicroglia ­(CD11b+/CD45low) from other monocytes(minimum number of cells: 100.000). Microglia were further analyzed for CD68 expression, a marker for reactive
microglia.
Extraction of intracellular metabolites in microglial cell
cultureMetabolic extraction was performed as previously
described [19, 32, 33]. Briefly, 5 ×105 microglia on 12 wellplates were washed with 0.9% NaCl and quenched with
250 µL -20°C methanol. After adding an equal volumeof 4°C deionized water with 1 µg/mL D6-pentane-dioic
acid (C/D/N Isotopes) as internal standard plates weremaintained on an ice-cold metal plate. Cells were thoroughly scraped and flushed before transferring to tubes
containing 250 µL – 20 °C chloroform. Subsequently. The
extracts were vortexed at 1400 rpm for 20 min followedby 5 Min centrifugation at 17000 × g, both at 4 °C. 280 µLof the upper aqueous phase (polar phase) was transferred into gas chromatography compatible glass vials
containing a micro insert and the samples were lyophilized under vacuum at 4 °C in CentriVap Concentration

Ohm et al. Journal of Neuroinflammation(2024) 21:207

System (Labconco). The dried samples were capped andstored at 4 °C until measurement. The interphase wascollected for RNA isolation.

Extraction of metabolites from blood and brain
tissueMetabolic extraction from blood serum was performed
as previously described [34]. Briefly, 11 µL blood serum
combined with 100 µL of an ice-cold extraction solvent(consisting of methanol and water in an 8:1 ratio, maintained at − 20 °C) and 2 µg/mL of D6-glutaric acid asinternal standard. The mixture was vortexed (1400 rpm)
at 4 °C for 10 min a, then centrifuged (13000 × g, 4 °C)
for another 10 min to precipitate proteins and extract
metabolites. The supernatants (90 µL) were transferred
to glass vials, dried under cold conditions using a speedvac, and stored at − 20 °C until gas chromatographymass spectrometry (GC–MS) analysis.
Tissue metabolic extraction was performed as previously described [35]. Briefly, brain tissue samples(30–80 mg) were promptly placed into 2 mL reaction
tubes and snap-frozen in liquid nitrogen. The tubes werestored at – 70 °C until further processing. Upon processing, samples were homogenized in a 2 mL tube containing ceramic beads (1.4 mm, QIAGEN) and an extraction
fluid containing an internal standard (4 + 1 MeOH:H2O
and 64 µM 13C-Ribitol) in a volume ratio of 1000 µl
per 100 mg of tissue. Subsequently, the brain samples
were homogenized in a Retsch MM400 homogenizer.
Throughout the remainder of the experiment, the tubes
were diligently kept on ice. A second extraction fluid supplemented with a second internal standard (0.1 M HClcontaining 2 µg/mL D6 glutaric acid) was added, with thevolume ratio being 500 µl per 100 mg of tissue and tubes
were vortexed. Finally, chloroform (800 µL per 100 mg
of tissue) was added, followed by a 30-s vortexing stepand samples were shaken at 1400 rpm at 4 °C for 15 min.
After a centrifugation for 5 min at 17000 × g at 4 °C, thesupernatants (60 µL) were transferred to glass vials anddried under using a speed-vac (4 °C). The dried samples
were stored at − 20°C until GC–MS.
GC–MS measurement

GC–MS measurement of relative metabolite levels and isotope enrichment was performed as previously described [32]. Dried samples were derivatizedwith equal amounts of methoxylamine (20 mg/mL
in pyridine) and N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) or N-Methyl-N(trimethylsilyl)-trifluoracetamide (MSTFA) using aderivatization robot (Gerstel MPS). A sample volume of
1µL was injected in a SSL injector in splitless mode at
270 °C. GC–MS analysis was performed by an Agilent

Page 6 of 30

7890B gas chromatogram system coupled to an Agilent 5977B GC/MSD (MSD, Agilent Technologies),
equipped with a 30 m DB-35MS + 5m Duraguard capillary column (0.25 mm inner diameter, 0.25 µm film
thickness). Helium was used as the carrier gas witha constant flow rate of 1 mL/min. Metabolites weredetected in either full scan or selected ion mode. Processing of chromatograms and calculation of massisotopomer distributions and relative quantificationof metabolites were performed using the Metabolite
Detector software [36].
Imaging and image analysis
Imaging and quantification of microglial cell density

Z-Stack images of immunohistochemically stained
sections (five per animal) were acquired with a ZEISSImaging system (Imager.M2 AXIO) equipped with an
ApoTome and a 20 × objective (0.8 NA) at 1µm increments, focusing on the CA1 and DG regions of the
hippocampus to assess microglial density and the fluorescence intensity of IBA1, CD68, or GFAP. Imaging
settings, including light intensity and exposure time,
were uniform across all groups. Slides were coded to
conduct the analysis blindly using Fiji software (BioVoxxel). Eight slices were selected and converted toa 2D image by using “Z-Projection” tool with “maximum intensity” setting. Microglial density was analyzed by merging IBA1 and DAPI images, counting
all IBA1 + cells with nuclei manually with the “Multi
Point” tool, and calculating cell density (cells/mm2) inExcel. Fluorescence intensity analysis involved measuring the integrated density of selected areas and subtracting background readings from unstained regions
to obtain the fluorescence signal. All data were normalized within each staining to the mean of the control.
Statistical analysis

Data were presented as mean ± SEM, analyzed and visualized with GraphPad Prism 8 (GraphPad Software,
Inc, United States). Differences between groups wereassessed using two-way ANOVA (for LPS-injection
and metabolite treatment effects), with Fisher’s LSD
for post-hoc analysis. Exceptions included specific conditions, such as the final 5 min of electrophysiological measurements, where differences between saline
and LPS treatments were evaluated using an unpaired
t-test. The significance levels are denoted in the figurelegends, the significance value described as */#p < 0.05,**/##p < 0.01, ***/###p < 0.001, ****/####p < 0.0001, as
well as in extended tables. The “N/n” of the different
experimental groups, as well as the statistical test isindicated in the respective Figure legends. All analysis
were conducted blindly by the experimenter.

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Results
Itaconate and mesaconate pretreatment attenuates
LPS‑induced neuroinflammation

To investigate the possible immunomodulatory andpreventive effects of itaconate and mesaconate on LPSinduced neuroinflammation, 3-month-old male C57BL/6
mice received three injections of these metabolites(250 mg/kg) or PBS as the solvent of the metabolites as acontrol solution in a twenty-four-hour interval. Twentyfour hours after the last injection, mice received two
injections of either LPS (0.5 mg/kg) or saline, administered twenty-four hours apart. Three hours after thesecond LPS injection, the mice were sacrificed and thesamples were collected (Fig. 1A).
Daily bodyweight monitoring revealed that administration of itaconate and mesaconate did not prevent LPSinduced bodyweight loss, which indicates that the initial
immunological response is not compromised (Fig. 1B, C,
PBS-Saline vs. PBS-LPS, p < 0.0001; Ita-Saline vs. Ita-LPS
p < 0.0001; Mesa-Saline vs. Mesa-LPS p < 0.0001, Table 1).
Of note, twenty-four hours after LPS injection, all groups
showed significant body weight loss compared to thesaline-injected mice. However, the mice pretreated withitaconate showed a significant decrease in bodyweightcompared to the mesaconate pretreated mice receiving
LPS (Fig. 1C, Ita-LPS vs. Mesa-LPS, p = 0.0474, Table 1).
In view of the biologically short half-life of itaconate
and mesaconate, the concentrations of these metabolites in the blood serum and brain at the time of sacrificing (3 h after the last LPS or saline injection) were
determined using gas chromatography–mass spectrometry (GC–MS) measurements (Fig. 1D–F). In serum,
LPS injection resulted in elevated itaconate blood levels
across all LPS-injected groups compared to their respective controls, regardless of whether they were pretreatedwith the metabolites (Fig. 1: D, PBS-Saline vs. PBS-LPS,
p < 0.0001; Ita-Saline vs. Ita-LPS p = 0.0145; Mesa-Saline
vs. Mesa-LPS p = 0.0079, Table 1). Notably, mesaconate
pretreatment led to a reduction in serum itaconate levels in LPS-injected mice compared to those receiving LPS
without metabolite pretreatment (Fig. 1D, PBS-LPS vs.
Mesa-LPS p = 0.0455, Table 1). Moreover, comparison ofsaline injected mice with or without metabolite treatment
revealed that itaconate pretreated saline controls showeda non-significant trend of higher itaconate levels in the
blood compared to the other two saline treated groups(Fig. 1D). However, the mesaconate content in the blood
serum was too low to be reliably quantified by GC–MS
and no significant differences were found between thegroups (Fig. 1E).
In order to investigate whether parts of the injected
metabolites penetrate into the brain or if LPS-induced

Page 7 of 30

itaconate level in the brain, we conducted metabolite
extraction from brain tissue. Notably, mice pretreatedwith PBS or mesaconate showed a significant increaseof itaconate brain levels after LPS injection comparedto the respective controls (Fig. 1F, PBS-Saline vs. PBSLPS, p = 0.0499; Mesa-Saline vs. Mesa-LPS p = 0.0309,
Table 1). However, itaconate concentration in the brainof mice pretreated with itaconate showed no significant
changes after LPS injection compared to the corresponding mice injected with saline, indicating that LPS injection per se did not increase itaconate concentration in
the brain of these mice (Fig. 1F). Remarkably, mice pretreated with itaconate followed by saline injection had
higher itaconate concentrations in the brain than mice
pretreated with PBS or mesaconate followed by saline
injection. This could be the reason why a further significant increase of itaconate in the brain of mice pretreatedwith itaconate and injected with LPS was not observed(Fig. 1F, PBS-Saline vs. Ita-Saline p = 0.0390, Table 1).
Mesaconate was not detectable in the brain. Overall,these results indicate that LPS injection increases itaconate levels in both blood and brain. Intriguingly, mice
pretreated with itaconate showed higher levels of itaconate in the brain, suggesting that itaconate may reach thebrain and be taken up by resident cells and persist evenmore than fifty hours after the last injection.
Bacterial LPS injections induce a robust and transient
immune response in both the periphery as well as in the
CNS, characterized by an increased release of inflammatory mediators [6, 7, 37]. To evaluate the potential
benefits of itaconate and mesaconate in this context,
we quantified the levels of secreted specific cytokines
in blood serum and brain homogenates using enzymelinked immunosorbent assay (ELISA) (Fig. 2).
In the absence of itaconate and mesaconate, LPS
injection significantly increased the levels of all tested
inflammatory mediators in blood serum compared tosaline-injected controls (Fig. 2, PBS-Saline vs. PBSLPS, A: IL-1β p < 0.0001, B: IL-6 p = 0.0022, C: TNF-α
p = 0.0031, D: IL-10 p = 0.0211, Table 2). Conversely, inmesaconate pretreated mice, only IL-6 and TNF-α levels
increased significantly post-LPS, whereas mice pretreatedwith itaconate showed only a significant increase in IL-1β
upon LPS-challenge (Fig. 2, Mesa-Saline vs. Mesa-LPS,
B: IL-6 p = 0.0066, C: TNF-α p = 0.0066; Ita-Saline vs. ItaLPS, A: IL-1β p = 0.0305, Table 2). Remarkably, itaconate
pretreatment significantly reduced LPS-induced releaseof IL-6 and TNF-α compared to mice injected with LPS
without metabolite pretreatment (Fig. 2, PBS-LPS vs. ItaLPS, A: IL-1β p = 0.0002, B: IL-6 p = 0.0297, C: TNF-α
p = 0.0460, PBS-LPS vs. Mesa-LPS, A: IL-1β p = 0.0007,
Table 2).

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 8 of 30

Fig. 1 Peripheral LPS injection increases endogenous itaconate production in the blood and brain. A Illustration of the injection protocol usedin subsequent in vivo studies: Itaconate and mesaconate were administered intraperitoneally (250 mg/kg bodyweight) for three consecutive
days, succeeded by two intraperitoneal LPS injections separated by twenty-four hours (0.5 mg/kg bodyweight). Mice were euthanized threehours after the second LPS injection for additional analysis (Created with BioRender.com). B Daily body weight monitoring. C The bodyweightof the experimental groups was recorded twenty-four hours after the first LPS injection, with the graph showing the percentage change
in weight compared to the baseline. D, E Detected level of itaconate (D) and mesaconate (E) in the blood across the experimental groups, as wellas the detected itaconate level of itaconate in the brain (F). Data are presented as mean ± SEM and were analyzed with the with a two-way ANOVA(C) followed by Fisher’s LSD test; */#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, C: [N (number of mice per group) = 11–12; D–F: N = 3–5]

To assess the neuroinflammatory response to peripheral LPS exposure, inflammatory mediators in the wholebrain homogenates were measured (Fig. 2E–H). LPSinjection in PBS-pretreated mice significantly increased
levels of IL-1β, IL-6 and TNF-α in the brain comparedto saline controls (Fig. 2; PBS-Saline vs. PBS-LPS: E:

IL-1β p < 0.0001, F: IL-6 p = 0.0035, G: TNF-α p = 0.0013,
Table 2). We did not observe an increase in inflammatory mediators in mice pretreated with itaconate andmesaconate compared to the respective saline controls.
Metabolite pretreatment resulted in significantly lower
levels of IL-1β, IL-6 and TNF-α in brain compared toOhm et al. Journal of Neuroinflammation(2024) 21:207

Page 9 of 30

Table 1 Significances Fig. 1
Figure 1C

Bodyweight loss 24h after LPS

FLPS-injection (1, 61) = 104.9, p < 0.0001
Ftreatment (2, 61) = 0.1027, p = 0.9025

Figure 1D

Itaconate blood

FLPS-injection (1, 19) = 39.32, p < 0.0001

Figure 1E

Mesaconate blood

FLPS-injection (1, 19) = 3.3111, p = 0.0846

Figure 1F

Itaconate Brain

FLPS-injection (1, 18) = 4.213, p = 0.0550

Ftreatment (2, 19) = 2.232, p = 0.1346
Ftreatment (2, 19) = 0.9566, p = 0.4019
Ftreatment (2, 18) = 0.8434 p = 0.4466

Fig. 2 Itaconate and mesaconate ameliorate the release of cytokines in blood and brain after LPS stimulation. Levels of IL-1β, IL-6, TNF-α
and IL-10 were assessed in the blood (A–D) and brain (E–H) by ELISA. Gene expression levels of IL-1β (I), IL-6 (J) and BDNF (K) were quantified
in the hippocampus of all experimental groups using qPCR. Data are presented as mean ± SEM and were analyzed with the two-way ANOVA
followed by Fisher’s LSD test; */#p < 0.05, **/##p < 0.01, ***p < 0.001, ####p < 0.0001, [N (number of mice per group) = 4–5]

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Table 2 Significances Fig. 2
Figure 2A

IL-1β—Serum

FLPS-injection (1, 24) = 37.85, p < 0.0001

Figure 2B

IL-6—Serum

FLPS-injection (1, 24) = 18.90, p = 0.0002

Figure 2C

TNF-α—Serum

FLPS-injection (1, 24) = 18.61, p = 0.0002

Figure 2D

IL-10—Serum

FLPS-injection (1, 24) = 6.44, p = 0.0181;

Figure 2E

IL-1β—Brain

FLPS-injection (1, 24) = 27.62, p < 0.0001

Figure 2F

IL-6—Brain

FLPS-injection(1, 24) = 5.371, p = 0.0293

Figure 2G

TNF-α—Brain

FLPS-injection (1, 24) = 6.100

Figure 2H

IL-10—Brain

FLPS-injection (1, 24) = 0.03026,
p = 0.8634

Figure 2I

IL-1β—Hippocampus

FLPS-injection (1, 19) = 10.33, p = 0.0026

Figure 2J

IL-6—Hippocampus

FLPS-injection (1, 19) = 6.342, p = 0.0209

Ftreatment (2, 24) = 5.774, p = 0.0090
Ftreatment (2, 24) = 1.503, p = 0.2427
Ftreatment (2, 24) = 1.528, p = 0.2374
Ftreatment (2, 24) = 0.8772
Ftreatment (2, 24) = 5.749 p = 0.0091
Ftreatment (2, 24) = 1.781 p = 0.1900
Ftreatment (2, 24) = 1.560 p = 0.2306

Ftreatment (2, 24) = 1.219 p = 0.3131
Ftreatment (2, 19) = 10.33, p = 0.0009
Ftreatment (2, 19) = 6.037, p = 0.0093
Figure 2K

BDNF—Hippocampus

FLPS-injection (1, 19) = 18.24, p = 0.0004
Ftreatment (2, 19) = 0.04105, p = 0.9599

PBS-pretreated LPS-challenged mice (Fig. 2, PBS-LPS vs.
Ita-LPS: E: IL-1β p = 0.0002, F: IL-6 p = 0.0086, G: TNF-α
p = 0.0049; PBS-LPS vs. Mesa-LPS: E: IL-1β p = 0.0007,
F: IL-6 p = 0.0329, G: TNF-α p = 0.0304, Table 2). These
results were further supported by gene expression analyses for IL-1β and IL-6 in the hippocampus, demonstrating a significant reduction in itaconate- and mesaconate
pretreated mice compared to LPS injected mice without metabolite administration (Fig. 2, PBS-LPS vs. ItaLPS, I: IL-1β p < 0.0001, J, IL-6 p = 0.0003; PBS-LPS vs.
Mesa-LPS, I: IL-1β p < 0.0001, J: IL-6 p = 0.0005, Table 2).
These findings suggest that itaconate and mesaconate
pretreatment can attenuate LPS-induced inflammatory
responses, particularly in the brain, highlighting theirpotential to ameliorate neuroinflammatory processes.
Regarding the anti-inflammatory cytokine IL-10, LPS
only caused a significant increase in the blood serum inthe absence of itaconate and mesaconate, which we did
not observe in the other test groups. The IL-10 level inthe brain showed no significant differences between thegroups, which may indicate that both metabolites alleviate inflammation via modulation of pro-inflammatory
mediators rather than anti-inflammatory mediators.
Further, as the brain-derived neurotrophic factor(BDNF) can also attenuate neuroinflammation [38], thepossibility of itaconate and mesaconate to regulate BDNF

Page 10 of 30

levels in the brain was evaluated. For this purpose, we
analyzed BDNF gene expression levels in the hippocampus after LPS injection in all experimental groups. LPS
injection significantly lowered BDNF levels in mice pretreated with either PBS or mesaconate, in contrast to
saline-injected controls (Fig. 2K, PBS-Saline vs. PBSLPS, p = 0.0129; Mesa-Saline vs. Mesa-LPS, p = 0.0158,
Table 2). However, in itaconate-pretreated mice, thereduction in BDNF expression following LPS injection
was not statistically significant compared to saline controls (Fig. 2K, Ita-Saline vs. Ita-LPS, p = 0.0622, Table 2),
indicating no significant difference when comparing LPSinjected mice pretreated with itaconate to those treatedwith saline. These findings suggest that while itaconate
and mesaconate mitigated the LPS-induced upregulation of inflammatory mediators indicating their protective potential, itaconate only shows a trend towards aprotective effect against LPS-induced reduction in BDNF
expression, which may be of interest to investigate indetail in future studies.
Itaconate and mesaconate pretreatment attenuates
LPS‑induced microglial reactivity

Microglia, the brain’s resident immune cells, exhibit highsensitivity to environmental alterations, with an elevationin pro-inflammatory mediators prompting a transition
into a reactive phenotype [39, 40]. Having shown thatthe pretreatment with itaconate and mesaconate reduced
the extent of inflammatory mediators in both periphery
as well as in the brain following LPS exposure, in the following step we investigated whether the metabolite pretreatment is also sufficient in mitigating LPS-induced
microglial reactivity. Elevated microglial density andincreased IBA1 expression are recognized biomarkers
for neuroinflammation and increased microglial reactivity [41, 42]. Therefore, microglial density was quantified
by labeling microglia against ionized calcium-binding
adapter protein (IBA1), a cytoplasmic marker expressedin both microglia and macrophages, across the CA1 anddentate gyrus (DG) subregions of the hippocampus (representative images for CA1 and DG are shown in Fig. 3A,
B, respectively). LPS injection increased microglia density in the CA1 and DG of the hippocampus of PBS-pretreated mice compared to saline-injected controls, which
was also present in LPS-injected mice pretreated withmesaconate (Fig. 3C, D; PBS-Saline vs. PBS-LPS: CA1
p = 0.0024, DG p < 0.0001; Mesa-Saline vs. Mesa-LPS:
CA1 p = 0.0064, DG p = 0.0076, Table 3).
However, pretreatment with itaconate prevented thisLPS-induced increase in microglia density. Remarkably, acomparison of the LPS-injected groups with or without
metabolite pretreatment showed a significant reduced

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 11 of 30

Fig. 3 Itaconate and mesaconate dampen the local hippocampal inflammatory effects in microglia after LPS stimulation. Representative
images showing immunohistochemical staining for IBA1 and DAPI in the CA1 (A) and dentate gyrus (DG) (B) region of the hippocampus
for all experimental groups (magnification 20x, scale bar is 100 µm). The density of microglia was evaluated in the CA1 (C) and DG (D)of the hippocampus. Measurement of IBA1 fluorescence intensity in the CA1 (E) and DG (F). Data are presented as mean ± SEM and were analyzed
with the two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, **/##p < 0.01, ***/###p < 0.001, ####p < 0.0001, [N (number of mice per group) = 4
per group, Microglial density CA1 n (number of analyzed imaged per group) = 16–18; DG n = 18–20; ­IBA1+ fluorescence CA1 n = 17–18; DG
n = 18–20]. N = 4 per group, CA1, n (number of analyzed imaged per group) = 17–18; DG, n = 17–20; E: N = 6–7,]

number of ­IBA1+ cells in the DG subregion upon itaconate pretreatment, while mesaconate pretreatment
displayed only a slight decrease in LPS-induced microglia density (Fig. 3D, PBS-LPS vs. Ita-LPS, p = 0.0008,
Table 3).
In a next step we aimed to evaluate whether itaconate
and mesaconate pretreatment mitigates LPS-induced
upregulation of IBA1, potentially indicating diminished microglial reactivity (Fig. 3E, F). PBS-pretreatedmice injected with LPS showed significant increase in

IBA1-fluorescent intensity relative to their saline controls (Fig. 3, PBS-Saline vs. PBS-LPS, E: CA1 p = 0.0001,
F: DG: p = 0.0025, Table 3). Interestingly, in mice pretreated with itaconate or mesaconate, we could notobserve a significant elevation in IBA1 expression
compared to their respective controls. Pretreatment
with mesaconate significantly attenuated the LPSinduced increase in IBA1 fluorescence intensity acrossboth hippocampal regions, while itaconate specificallyreduced it in the DG compared to PBS-pretreated miceOhm et al. Journal of Neuroinflammation(2024) 21:207

Page 12 of 30

Table 3 Significances Figs. 3 and 4
Figure 3C

IBA1 cell density CA1

FLPS-injection (1, 99) = 16.77, p < 0.0001

Figure 3D

IBA1 cell density DG

FLPS-injection(1, 107) = 18.32, p < 0.0001

Figure 3E

IBA1 fluorescence intensity CA1

FLPS-injection(1, 101) = 12.81, p < 0.0005

Figure 3F

IBA1 fluorescence intensity DG

FLPS-injection(1, 108) = 7.583, p < 0.0069

Figure 4C

CD68 fluorescence intensity CA1

FLPS-injection(1, 101) = 1.042, p = 0.3098

Figure 4D

CD68 fluorescence intensity DG

FLPS-injection(1, 105) = 4.208, p = 0.0427

Figure 4F

FACS CD68

FLPS-injection(1, 35) = 7.988, p = 0.0077

Ftreatment (2, 99) = 0.5576, p = 0.5744
Ftreatment (2, 107) = 2.158, p = 0.1205
Ftreatment(2, 101) = 3.873, p = 0.0240
Ftreatment(2, 108) = 6.300., p = 0.0026
Ftreatment(2, 101) = 1.643, p = 0.1986
Ftreatment(2, 105) = 3.293., p = 0.0410
Ftreatment (2, 35) = 0.7647, p = 0.4731(Fig. 3E, F, PBS-LPS vs. Ita-LPS, DG p = 0.012, PBS-LPS
vs. Mesa-LPS, CA1 p = 0.009, DG p = 0.0004, Table 3).
Collectively, these results suggest the efficacy of itaconate and mesaconate in preventing the LPS-induced
increase microglial reactivity, with itaconate additionally showing the capability to reduce microglial density.
To elucidate the impact of itaconate and mesaconate on microglial reactivity, we examined their effects
on the activation marker Cluster of Differentiation
68 (CD68). CD68, a lysosomal protein upregulated
in mononuclear cells during inflammation, is a usefulmicroglial activation marker [43]. First, immunohistochemical staining for CD68 was performed (representative images for CA1 and DG are shown in Fig. 4A,
B, respectively). CD68 fluorescence intensity analysis indicated that LPS injection did not significantly
affect the CA1 subregion, irrespective of itaconate ormesaconate pretreatment (Fig. 4C). However, itaconate pretreatment injected with LPS significantly lowered CD68 intensity in the DG subregion comparedto LPS-injected mice pretreated with PBS (Fig. 4D,
PBS-LPS vs. Ita-LPS, p = 0.0358, Table 3), while mesaconate showed only a slight decrease. Further, fluorescence-activated cell sorting (FACS) analysis quantified
CD68 in whole brain hemisphere-derived single cellsuspensions, stained for CD11b, CD45 and CD68. Dueto the relatively small sample mass when only the hippocampi are used and a relatively small effect visible
in the hippocampus by immunohistochemistry, FACS
analysis was performed on whole brain homogenates.
Microglia were identified by intermediate CD45 andpositive CD11b expression, classified into ­C D68+ or­C D68− cells (Fig. 4E). LPS injection increased the number of C
­ D68+ microglia compared to saline controls,

but not in mice pretreated with itaconate or mesaconate (Fig. 4F). Mesaconate pretreatment even slightly
reduced the percentage of ­C D68+ microglia compared
to PBS pretreatment, though without statistical significance (Fig. 4F).
While microglia are recognized as the brain’s primary
immune cells, research has also highlighted astrocytes’significant role in immune responses and regulation,and both glial cells seem to influence each other [11, 44].
In response to neuroinflammation, astrocytes undergo
astrogliosis, marked by an increase in glial fibrillary
acidic protein (GFAP) expression [45]. To investigate
whether pretreatment with itaconate and mesaconate canmitigate LPS-triggered astrogliosis, we conducted immunohistochemical staining for GFAP—a protein foundin the cytoplasm of astrocytes—within the hippocampus (representative images for CA1 and DG are shownin Suppl 1A and Suppl. 1B, respectively). LPS-injection
resulted only in a slight statistically insignificant increasein ­GFAP+ fluorescence intensity in the DG (Suppl. 1D)
which was even less evident in the CA1 region (Suppl.
1C) of PBS-pretreated mice compared to saline-injected
controls. This was also the case for itaconate pretreatedmice which showed a slight, non-significant elevation in
the GFAP fluorescence intensity in comparison to saline
injected controls. However, LPS injection did not leadto a similar slight increase of GFAP in mesaconate pretreated mice which was even slightly decreased in the
CA1 region (Suppl. 1C). Furthermore, comparing all
LPS-injected groups, with or without metabolite pretreatment, revealed that prior exposure to mesaconate
resulted in a significant reduction in GFAP fluorescenceintensity in the DG compared to LPS-injected mice pretreated with PBS (Suppl. 1D, PBS-LPS vs. Mesa-LPS,

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 13 of 30

Fig. 4 Itaconate and mesaconate slightly dampen the local hippocampal inflammatory effects in microglia after LPS stimulation. Representative
images showing immunohistochemical staining for CD68 and DAPI in the CA1 (A) and dentate gyrus (DG) (B) region of the hippocampus for all
experimental groups (magnification 20x, scale bar is 100µm). CD68 fluorescence intensity in the hippocampal CA1 (C) and DG (D) regions for all
experimental groups. Plots representing gated cell population identified as microglia and histogram illustration of CD68 expression determinedby FACS. Microglia were characterized by intermediate expressions of ­CD45low and ­CD11b+. Cells showing CD68 expression above a threshold wereclassified as ­CD68+-expressing microglia (E). Analysis of CD68 expressing microglia cells (F). Data are presented as mean ± SEM and were analyzed
with the two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, ##p < 0.01, [C, D: N (number of mice per group) = 4 per group, CA1, n(numberof analyzed imaged per group) = 17–18; DG, n = 17–20; F: N = 6–7]

p = 0.0495, Table 4). These results indicate that pretreatment with mesaconate was even able to reduce the slightincrease in GFAP fluorescence intensity induced by LPS
injection in the DG.

Overall, these results suggest that pretreatment ofmice with itaconate or mesaconate is able to attenuate
the increased microglial reactivity induced by LPS, thus
supporting their anti-inflammatory properties. This isOhm et al. Journal of Neuroinflammation(2024) 21:207

Table 4 Significances Suppl. Figure 1
Suppl. 1C

GFAP fluorescence intensity
CA1

FLPS-injection(1, 114) = 0.01229, p = 0.9119

Suppl. 1D

GFAP fluorescence intensity
DG

FLPS-injection(1, 106) = 2.649, p = 0.1066

Ftreatment(1, 114) = 0.3035, p = 0.7388

Ftreatment(2, 106) = 2.683, p = 0.0730

associated with a less pronounced effect on astrocytic
reactivity.
Itaconate and mesaconate pretreatment rescued
the LPS‑induced impairment of synaptic plasticity

Previously it was shown that long-term potentiation(LTP), a key form of activity-dependent synaptic plasticity is impaired following a peripheral immune stimulation by LPS-injection in mice [6, 7, 46]. Therefore, after
demonstrating an anti-inflammatory potential of itaconate and mesaconate in the brain, we aimed to determine
whether pretreatment with itaconate and mesaconate
could mitigate the LPS-induced impairment on hippocampal network function. Therefore, electrophysiological recordings were performed as previously described[7, 29, 30] at the Schaffer collateral pathway connecting
the CA3 and CA1. Initially, we analyzed the relationship between the fEPSP slope and stimulation intensity
using input–output curves. (Fig. 5A, E, I, Table 5). The
LPS injection had no significant effect on the basal synaptic transmission compared to saline injected mice, irrespective of prior metabolite treatment. Nonetheless, at a

Page 14 of 30

stimulus intensity of 100 μA, mice pretreated with mesaconate and subsequently injected with LPS exhibited amarginally decreased fEPSP slope relative to control mice
(Fig. 5I, Mesa-Saline vs. Mesa-LPS, 100 μA stimulus,
p = 0.0452, Table 5).
Subsequent, the potential effects of LPS-injection on
short-term synaptic plasticity of CA1 neurons measured by paired pulse facilitation (PPF) was investigated.
No significant differences in PPF were detected between
the LPS- and saline-injected mice, regardless if theyreceived a metabolite pretreatment or not (Fig. 5B, F, J).
These findings suggest that LPS injection does not significantly affect basal synaptic transmission or presynaptic
function, and that pretreatment with itaconate or mesaconate does not elicit substantial modifications in theseparameters.
Furthermore, long-term synaptic plasticity was examined in the Schaffer collateral CA3 to CA1 pathway
induced by TBS, following 20 min of baseline recordings (Fig. 5C, G, K). Consistent with previous studies [6,7, 46], LTP induction was significantly impaired in the
hippocampus of LPS-injected mice compared to salineinjected controls, when pretreated with PBS (Fig. 5C),a phenomenon extending into the LTP maintenancephase (Fig. 5D, p = 0.0101, Table 5). Remarkably, the LPSinduced LTP deficit was not evident in acute hippocampal slices from mice pretreated with either itaconate ormesaconate (Fig. 4G, K), Also, no significant impairment of LTP in the maintenance phase was observedin mice pretreated with metabolites (Fig. 5H, L). Collectively, these findings indicate that pretreatment withitaconate and mesaconate effectively prevent the LPSinduced impairments in LTP, highlighting their potential

Table 5 Significances Fig. 5
Figure 5A

Basal synaptic transmission PBS-Groups

FLPS-injection(1, 32) = 0.4627, p = 0.5013

Figure 5B

Paired Pulse Facilitation PBS-Groups

FLPS-injection(1, 18) = 0.01634, p = 0.8997

Figure 5E

Basal synaptic transmission Ita-Groups

FLPS-injection(1, 37) = 0.4155, p = 0.5232

Figure 5F

Paired Pulse Facilitation Ita-Groups

FLPS-injection(1, 30) = 1.712, p = 0.2007

Figure 5I

Basal synaptic transmission Mesa-Groups

FLPS-injection(1,33) = 0.6316, p = 0.4324

Figure 5J

Paired Pulse Facilitation Mesa-Groups

FLPS-injection(1,29) = 0.008853, p = 0.9257

Figure 5N

Dendritic spine density CA1

FLPS-injection(1, 215) = 0.0006828, p = 0.9792

Figure 5O

Dendritic spine density DG

FLPS-injection(1, 223) = 3.514, p = 0.0622

Fstimulus(1.592, 50.93) = 224.5, p < 0.0001
Fstimulus(2.413, 43.43) = 6.576, p = 0.0019
Ftreatment(2,036, 75,32) = 348,4, p < 0,0001
FtreatmentF (1,718, 51,53) = 18,78, p < 0.0001
Ftreatment(1,973, 65,12) = 262,8, p < 0.0001
Ftreatment(2,167, 62,84) = 14,15, p < 0.0001
Ftreatment (2, 215) = 8.416, p = 0.0003
Ftreatment (1, 223) = 5.588, p = 0.0043

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 15 of 30

Fig. 5 Pretreatment with itaconate and mesaconate prevents LPS-induced impairment of LTP. A, E, I Input–output curves of fEPSP slopes in acutehippocampal slices. B, F, J. Paired-pulse facilitation (PPF) of the fEPSP slopes plotted as a response to the second stimulation over the first one
at different interpulse intervals (10, 20, 40, 60, 80, and 100 ms. C, G, K LTP was measured in hippocampal acute slices of all experimental groups. D,
H, L LTP maintenance phase (T 75–80 min). M Representative images of the Golgi-Cox staining in hippocampal apical dendrites of CA1 subregionof all experimental groups (scale bar is 5 µm). Hippocampal dendritic spine density in CA1 (N) and DG (O) across the experimental groups. Data
are presented as mean ± SEM and were analyzed with a repeated measure two-way ANOVA followed by Fisher’s LSD test (A, B, C, E, F, G, I, J, K)
or two-way ANOVA followed by Fisher’s LSD test (N, O) or unpaired t-test (D, H, L); */#p < 0.05, **p < 0.01, ****p < 0.0001, [A–L: N (number of mice
per group) = 4–5, n (number of analyzed hippocampal slices per group) = 16–20; N + O: N = 3–4, n = 27–41]

in preserving the cellular foundations of learning andmemory.
In addition to assess neuronal functions, we next
evaluated the effect of itaconate and mesaconate on the

LPS-induced changes in the neuronal structure. For thispurpose, we analyzed the hippocampal dendritic spine
density by Golgi-Cox staining (representative images
shown in Fig. 5M). The results demonstrated that a dual

Ohm et al. Journal of Neuroinflammation(2024) 21:207

LPS-stimulus did not lead to a significant decrease indendritic spine density neither in CA1 (Fig. 5N) nor in
DG (Fig. 5O). However, neurons of LPS-injected mice
pretreated with PBS revealed a slightly lower dendritic
spine density in CA1 neurons compared to its respective
control. Conversely, itaconate pretreated mice injectedwith LPS showed a significant higher dendritic spine
density in CA1 neurons than their saline-injected mice
(Fig. 5N, Ita-Saline vs. Ita-PBS, p = 0.0310, Table 5). Additionally, in mice pretreated with mesaconate, LPS injections did not lead to a decrease in dendritic spine density
in CA1 neurons and actually resulted in an increase inspine density in DG neurons, when compared to control mice injected with saline (Fig. 5O, Saline-Mesa vs.
LPS-Mesa p = 0.0508, Table 5). Comparing mice injectedwith saline, with or without itaconate or mesaconate
pretreatment, the results demonstrated that pretreatment with itaconate alone led to a decrease in dendritic
spine density in CA1 neurons, in contrast to both PBSpretreated and mesaconate-pretreated mice (Fig. 5N,
PBS-Saline vs Ita-Saline, p = 0.0076; Ita-Saline vs. MesaSaline, p < 0.0001, Table 5). Additionally, mice treatedwith Mesa-LPS showed an increased dendritic spine density in the DG region compared to those treated with ItaLPS (Fig. 5O, LPS-Ita vs. LPS-Mesa, p = 0.0023, Table 5).
These findings indicate that although LPS-induce impairments in LTP, the dual LPS-stimulus do not induce astrong dendritic spine loss.
Itaconate and mesaconate reduce the LPS‑induced
inflammation in microglia but not in astrocytes

After demonstrating a dampened LPS-induced neuroinflammation in mice pretreated with itaconate or mesaconate, we aimed to identify the neuroinflammatory
modulation of the two metabolites. Therefore, primary
microglia- and primary astrocytes were prepared fromneonatal mice and pretreated with itaconate or mesaconate before stimulation with LPS (Fig. 6A).
A four hour pretreatment of primary microglia withitaconate or mesaconate demonstrated an efficient uptakeof the metabolites from the medium (Fig. 6, B: itaconate:
untreated vs. Ita p < 0.0001, Mesa vs. Ita p < 0.0001, LPS
vs. Ita-LPS p < 0.0001, Ita-LPS vs. Mesa-LPS p < 0.0001;
C: mesaconate: untreated vs. Mesa p < 0.0001, Ita vs.
Mesa p < 0.0001, LPS vs. Mesa-LPS p < 0.0001, Ita-LPS
vs. Mesa-LPS p < 0.0001, Table 6). Moreover, whenmicroglia were additionally treated with LPS (10 ng/mL), the intracellular metabolite levels of itaconate andmesaconate increased compared to control cells only
treated with the metabolites without LPS stimulation(Fig. 6, B: Ita vs. Ita-LPS, p = 0.0137; C: Mesa vs. MesaLPS, p = 0.0131, Table 6). Notably, microglia pretreatedwith itaconate exhibited an increase in intracellular

Page 16 of 30

mesaconate levels, significantly less than that observed in
microglia cells treated directly with mesaconate (Fig. 6C,
Ita vs. Mesa p < 0.0001, Table 6). However, compared tocells that received no metabolite treatment, there was aslight increase in the level, although this increase did notachieve statistical significance. The increase in intracellular mesaconate level was only visible when cells werepretreated with itaconate. Microglia pretreated withmesaconate did not show an increase in the intracellularlevel of itaconate (Fig. 6B), which indicates a transformation of itaconate and mesaconate in a single-directional
matter, as reported in macrophages [19]. Considering theevidence suggesting that itaconate might transform into
mesaconate within microglia and based on the findingsof He et al. that mesaconate is produced from itaconate
in peripheral macrophages, we next aimed to know if thisconversion also takes place in primary microglia (Suppl.
2) [19]. To study metabolic fluxes in LPS-stimulated primary microglia, we treated these cells with a [U-13C]
glucose tracer, followed with analysis of the mass isotopomer distribution of downstream metabolites. If mesaconate originates from itaconate, its labeling patternshould mirror that of itaconate, albeit to a lesser degree,
as demonstrated by He and colleagues in their study on
peripheral macrophages [19]. Upon a twenty-four hour
LPS stimulation, the highest fraction found were M1
mass isotopomers of itaconate, which represents molecules directly resulting from synthesized cis-aconitate,while the second highest fraction were the M3 isotopologues, which were derived from cis-aconitate synthesized in the second round of the TCA cycle (Suppl. 2A).
A similar enrichment pattern was observed for mesaconate, whereas the isotopomer abundances of mesaconate
were all slightly lower than those of itaconate (Suppl. 2A),in line with the results from He et al. [19].
He et al. further demonstrated that in peripheral macrophages mesaconate synthesis could also be attributable from glutamine via the TCA cycle and itaconate. To
verify this in microglia, a [U-13C] glutamine tracer wasapplied to LPS-stimulated microglia (Suppl. 2B). Similar
to peripheral macrophages [19], LPS-treated microglia
showed a high abundance of M4 isotopomer of mesaconate and itaconate. Again, the mass isotopomer distribution of mesaconate was very similar to that of itaconate,
but in lower abundance (Suppl. 2B). These findings suggest that mesaconate and itaconate are generated fromthe same pathway while mesaconate is in a later position than itaconate, as previously described in peripheral
macrophages [19].
To verify that the observed decrease in microglial reactivity following in vivo LPS injection is due to a direct
influence of itaconate or mesaconate on microglia, weproceeded to measure the release of pro-inflammatory

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 17 of 30

Fig. 6 Pretreatment with itaconate or mesaconate dampen microglial but not astrocytic inflammatory response to LPS. A Experimental procedureof primary microglia and astrocytes pre-treated with itaconate and mesaconate followed by LPS stimulation (Created with BioRender.com). (B, C)
Signal intensity of intracellular itaconate (B) and mesaconate (C) in primary microglia after either treated with itaconate or mesaconate for 24 hor additionally stimulated with LPS (10 ng/mL) after 4 h pretreatment. Cytokine secretion of (D) IL-6 and (E) TNF-α, as well as cytokine gene
expression of (F) IL-6 and (G) IL-1β of primary microglia. Data are presented as mean ± SEM and were analyzed with a two-way ANOVA; */#p < 0.05,**/##p < 0.01, ***/###p < 0.001, ****/####p < 0.0001, [B, C: n = 11–12 biological replicates of 4 independent experiments. cytokine secretion: D:
N = 2–5 per experimental group, n = 6–19, E: N = 2–4, n = 6–21; gene expression F: N = 2–4, n = 6–21, G: N = 2–4, n = 6–21. Astrocytes: cytokine
secretion: H: N = 4 per experimental group, n = 8–11, I: N = 4, n = 8–11; gene expression J: N = 3, n = 6–9, K: N = 3, n = 6–9]

cytokines in primary microglia stimulated with LPS,
both with and without itaconate or mesaconate pretreatment. The LPS-stimulation led to an increased releaseof the pro-inflammatory cytokines IL-6 and TNF-α
into the supernatant of primary microglial cultures, incomparison to untreated control groups, regardless if
microglia were pretreated with itaconate or mesaconate(Fig. 6, untreated vs. LPS, D: IL-6 p < 0.0001, E: TNF-α
p < 0.0001; Ita-untreated vs Ita-LPS, D: IL-6 p < 0.0001,
E: TNF-α p < 0.0001; Mesa-untreated vs. Mesa-LPS, D:

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 18 of 30

Table 6 Significances Fig. 6
Figure 6B

Itaconate

Ftreatment(2,65) = 3–319, p = 0.0731
Ftreatment(2,65) = 85.00, p < 0.0001

Figure 6C

Mesaconate

Ftreatment(2,65) = 164.5, p < 0.0001
Ftreatment(2,65) = 2.166, p = 0.1459

Figure 6D

ELISA IL-6 Microglia

FLPS-stimulation (1, 65) = 82.47, p < 0.0001
Ftreatment(2, 65) = 1.330, p = 0.2716

Figure 6E

ELISA TNF-α Microglia

FLPS-stimulation (1, 73) = 248.2, p < 0.0001
Ftreatment (2, 73) = 3.367, p < 0.0399

Figure 6F

Gene expression IL-6 Microglia

FLPS-stimulation (1, 55) = 59.66, p < 0.0001
Ftreatment(2, 55) = 59.66, p = 0.0055

Figure 6G

Gene expression IL-1β Microglia

FLPS-stimulation (1, 69) = 107.2, p < 0.0001
Ftreatment(2, 69) = 2.251, p = 0.1130

Figure 6H

ELISA IL-6 Astrocytes

FLPS-stimulation(1, 52) = 299.8, p < 0.0001
Ftreatment(2, 52) = 2.693, p = 0.0771

Figure 6I

ELISA TNF-α Astrocytes

FLPS-stimulation(1, 52) = 87.77, p < 0.0001
Ftreatment (2, 52) = 0.6445, p = 0.5291

Figure 6J

Gene expression IL-6 Astrocytes

FLPS-stimulation (1, 37) = 51.45, p < 0.0001
Ftreatment(2, 37) = 1.494, p = 0.2376

Figure 6K

Gene expression IL-1β Astrocytes

FLPS-stimulation (1, 37) = 383.1, p < 0.0001
Ftreatment(2, 37) = 0.2987, p = 0.7436

IL-6 p < 0.0001; E: TNF-α p < 0.0001, Table 6). Remarkably, the pretreatment with both metabolites diminishedthe release of pro-inflammatory cytokines. Specifically,
the cells pretreated with mesaconate exhibited a significant decrease in both IL-6 and TNF-α while itaconate
only significantly decreased the IL-6 level (Fig. 6, LPS
vs. Mesa-LPS, D: IL-6 p = 0.0250; E: TNF-α p < 0.0001;
LPS vs Ita-LPS, D: IL-6 p = 0.0164; E: TNF-α p = 0.0555,
Table 6). These results are supported by the gene expression analysis of IL-1β and IL-6. The LPS-induced expression of IL-1β and IL-6 was reduced by both metabolites
in a highly significant manner (Fig. 6, untreated vs. LPS,
F: IL-6: p < 0.0001; G: IL-1β p < 0.0001, Ita-untreated vs.
Ita-LPS, F: IL-6 p = 0.0011, G: IL-1β p < 0.0001, Mesauntreated vs. Mesa-LPS, F: IL-6 p = 0.0245, G: IL-1β
p = 0.0002, LPS vs Ita-LPS C: IL-6 p = 0.0024, D: IL-1β
p = 0.0198; LPS vs Mesa-LPS, C: IL-6 p = 0.0004, D: IL-1β
p = 0.0006, Table 6).
Considering the findings by Cordes et al., showingthat itaconate treatment affects the metabolic programof astrocytes [27], we next investigated whether the pretreatment with itaconate or mesaconate also diminishes
the inflammatory response in astrocytes. To this aim,
we pretreated primary astrocytes, similarly to primary
microglia, with itaconate or mesaconate, followed by
LPS-exposure (Fig. 6H–K). Astrocytes stimulated with
LPS showed a significant release of IL-6 and TNF-α into
the supernatant compared to non-stimulated controls,
regardless of pretreatment with itaconate and mesaconate(Fig. 6, untreated vs. LPS, H: IL-6 p < 0.0001, I: TNF-α
p < 0.0001; Ita-untreated vs Ita-LPS, H: IL-6 p < 0.0001,
I: TNF-α p < 0.0001; Mesa-untreated vs. Mesa-LPS, H:
IL-6 p < 0.0001, I: TNF-α p < 0.0001, Table 6). Notably,
following LPS stimulation, the levels of these cytokines
were not decreased in the supernatant of astrocyte cultures pretreated with both itaconate and mesaconate, andeven IL-6 was increased by both itaconate and mesaconate (Fig. 6H, LPS vs. Ita-LPS p = 0.0076, LPS vs. MesaLPS p = 0.0342, Table 6). Similarly, the gene-expression
analysis revealed no reduction in LPS-stimulated IL-1β
and IL-6 expression by the pretreatment of both metabolites (Fig. 6, untreated vs. LPS, J: IL-6 p < 0.0001, K: IL-1β
p < 0.0001, Ita-untreated vs. Ita-LPS, J IL-6 p = 0.0001, K:
IL-1β p < 0.0001; Mesa-untreated vs. Mesa-LPS, J: IL-6
p < 0.0001, K: IL-1β p < 0.0001, LPS vs. Mesa-LPS, J: IL-6
p = 0.0177, Table 6).
Altogether, both itaconate and mesaconate mitigated
the in vitro inflammatory response in LPS-stimulated
microglia but not in astrocytes, therefore suggesting theirbeneficial effects observed in vivo attributable to thedirect immuno-modulation in and by microglia.
Impacts of Irg1‑deficiency and the lack of endogenous
itaconate synthesis on LPS‑induced neuroinflammation

Considering the fundamental roles of itaconate andmesaconate in the immune responses, we aimed toinvestigate how the absence of endogenous itaconate,resulted from Irg1-deficiency in Irg1−/− mice, impacts

Ohm et al. Journal of Neuroinflammation(2024) 21:207

LPS-induced neuroinflammatory response within
the brain. Previous research indicates that endogenous itaconate plays a crucial role to restrict inflammatory responses, Irg1−/− mice exhibit exacerbateddisease outcomes compared WT mice capable ofproducing itaconate [47–51]. In a LPS-induced septic shock model, Irg1−/− mice were subjected to more
severe symptoms than the WT mice [52]. Kuo et al.

Page 19 of 30

discovered that Irg1−/− mice, when subjected to stroke
paradigms, suffered from significantly worse brain
injuries [53]. These injuries were marked by larger cerebral infarcts, greater disruption of the blood–brain
barrier, and heightened microglial activation, in comparison to their WT counterparts [53]. However, theimpact of Irg1-deficiency on brain immunity remains
poorly understood. To evaluate the function of IRG1

Fig. 7 LPS triggers a strong inflammatory response in both WT- and Irg1−/−-mice. A Illustration of the injection protocol used in subsequent in vivo
studies to compare the inflammatory respond between WT- and Irg1−/−-mice (Created with BioRender.com). B Bodyweight of the experimentalgroups twenty-four hours after the first LPS-injection. Cytokines in the blood (C–F) and brain (G–J) using ELISA. Data are presented as mean ± SEM
and were analyzed with the two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, [B: N (number of mice
per group) = 8–9; C–J: N = 3–4]

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 20 of 30

and its associated endogenous production of itaconate
and mesaconate in LPS-induced neuroinflammation,
both WT and Irg1−/− mice received two i.p. LPS injections (0.5 mg/kg in a twenty-four-hour time interval) or
saline solution as control (Fig. 7A). Subsequently, theinflammatory responses within the brains were analyzed three hours after the second injection of LPS andcompared between the two genotypes. Twenty-fourhours post-injection, the bodyweight of LPS-injected
mice decreased in both Irg1−/− and WT mice, in contrast to saline injected mice, with no significant influence of the genotype (Fig. 7B, WT-Saline vs. WT-LPS
p < 0.0001; Irg1−/−-Saline vs. Irg1−/−-LPS p < 0.0001,
Table 7). To investigate whether the absence of IRG1
influences the LPS-induced inflammatory responses,
cytokine levels were measured in both blood serum aswell as in brain homogenates by ELISA (Fig. 7C–F). As
expected, the dual LPS injection induced secretion of
IL-1β, TNF-α, IL-6, and IL-10 in WT mice, which was
further strengthened in Irg1−/− mice (Fig. 7, Irg1−/−-Saline vs. Irg1−/−-LPS, C: IL-1β p = 0.0078, E: IL-6
p = 0.0019, D: TNF-α p = 0.0002; F: IL-10 p = 0.0005,
Table 7). It is noteworthy that although Irg1−/− showedslightly higher levels of pro-inflammatory mediators inthe blood serum following LPS-stimulation, these differences were not statistically significant when compared to LPS-injected WT mice.
Analysis of cytokine levels in whole brain homogenates
demonstrated that LPS-injection significantly elevatedcytokine levels in both genotypes when compared to theirrespective saline controls (Fig. 7, WT-Saline vs. WT-LPS,

G: IL-1β p = 0.0026, I: IL-6 p = 0.0410, J: IL-10 p = 0.0445;
Irg1−/− -Saline vs. Irg1−/− -LPS, G: IL-1β p < 0.0001, I: IL-6
p = 0.0024, H: TNF-α p = 0.0046, Table 7).
Notably, LPS-induced secretion of IL-1β, IL-6 andTNF-α were more significantly increased in Irg1−/− micecompared to WT mice. Taken together, our data indicate that the lack of endogenous itaconate production
only had a minor effect on the inflammatory cytokine
response triggered by LPS.
Alterations in the endogenous synthesis of itaconate
has no significant effect on LPS‑induced microglial
reactivity

In vitro experiments, earlier described in this study, using
primary microglia cultures of WT pretreated with itaconate before stimulated with LPS, demonstrated a reduced
inflammatory phenotype (Fig. 6). Consequently, weinvestigated whether the absence of IRG1 and its respective lack of itaconate production affects the LPS-induced
microglial reactivity in the hippocampus (Fig. 8A, B).
LPS-injection resulted in a significantly increased number of I­BA1+-cells within the hippocampal regions CA1
and DG of Irg1−/− compared to those Irg1−/− mice receiving saline (Fig. 8, Irg1−/−-saline vs. Irg1−/−-LPS, C: CA1
p = 0.0051; D: DG p = 0.0334, Table 8). Contrary, WT
mice only showed modest increase microglial density
in response to LPS when compared to saline-injected
WT mice. Notably, a comparison of LPS-injected mice
across both genotypes revealed a significant elevationin microglial density within the CA1 in Irg1−/− mice.
Moreover, post LPS-injection a significant increase in

Table 7 Significances Fig. 7
Figure 7B

Bodyweight loss 24h after LPS

FLPS-injection (1, 30) = 74.77 p < 0.0001

Figure 7C

IL-1β Serum

FLPS-stimulation (1, 10) = 12.76, p = 0.0051

Figure 7D

TNF-α Serum

FLPS-stimulation (1, 10) = 52.16, p < 0.0001

Figure 7E

IL-6 Serum

FLPS-stimulation (1, 10) = 15.40, p = 0.0051

Figure 7F

IL-10 Serum

FLPS-stimulation (1, 10) = 32.38, p = 0.0003

Figure 7G

IL-1β Brain

FLPS-injection(1, 10) = 51.53, p < 0.0001

Figure 7H

TNF-α Brain

FLPS-injection (1, 10) = 10.69, p = 0.084

Figure 7I

IL-6 Brain

FLPS-injection(1, 10) = 19.45, p = 0.0013

Figure 7J

IL-10 Brain

FLPS-injection (1, 10) = 9.122, p = 0.0129

Fgenotype (1, 30) = 0.3624, p = 0.5517
Fgenotype (1, 10) = 0.5936, p = 0.4588
Fgenotype (1, 10) = 0.1907, p = 0.6716
Fgenotype (1, 10) = 2.351, p = 0.1562
Fgenotype (1, 10) = 2.273, p = 0.1659
Fgenotype (1, 10) = 1.500, p = 0.2487
Fgenotype (1, 10) = 2.252, p = 0.1643
Fgenotype (1, 10) = 0.7014, p = 0.4219
Fgenotype (1, 10) = 0.1843, p = 0.6768

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 21 of 30

Fig. 8 Silencing Irg1 has no significant effect on IBA1 microglia reactivity in LPS-treated mice compared to WT. A Representative images
from immunohistochemical staining for IBA1 and DAPI within the CA1 (A) and DG (B) across the experimental groups (magnification 20x, scalebar is 100 µm). Microglial density in the hippocampal CA1 (C) and DG (D). Fluorescence intensity of IBA1 in the CA1 (E) and DG (F). Data arepresented as mean ± SEM and were analyzed with the two-way ANOVA followed by Fisher’s LSD test; */#p < 0.05, **/##p < 0.01, ***p < 0.001, [C, D:
N (number of mice per group) = 4–5 per group, CA1 n (number of analyzed imaged per group) = 16–20, DG n = 16–20; E, F: N = 4–5 per group, CA1
n = 16–20, DG n = 16–20]

Table 8 Significances Fig. 8
Figure 8C

IBA1 cell density CA1

FLPS-injection (1, 70) = 8.224, p = 0.0055
Fgenotype (1, 70) = 3.662, p = 0.0597

Figure 8D

IBA1 cell density DG

FLPS-injection (1, 70) = 6.132, p = 0.0157
Fgenotype (1, 70) = 0.02670, p = 0.8707

Figure 8E

IBA1 fluorescence intensity CA1

FLPS-injection (1, 71) = 21.09, p < 0.0001
Fgenotype (1, 71) = 2.156, p = 0.1465

Figure 8F

IBA1 fluorescence intensity DG

FLPS-injection (1, 69) = 12.17, p = 0.0008
FLPS-injection (1, 69) = 0.01242, p = 0.9116

IBA1 fluorescence intensity was observed in the CA1
and DG of WT mice (Fig. 8, WT-Saline vs. WT-LPS, E:
CA1 p = 0.0001, F: DG p = 0.0038, Table 8). In contrast,
Irg1−/− mice exhibited a significant enhancement in IBA1

fluorescence in the CA1 region and a notable, albeit nonsignificant, increase in the DG region (Fig. 8, Irg1−/−saline vs. Irg1−/−-LPS, E: CA1 p = 0.0218, Table 8). WhileLPS administration induced an inflammatory microglial

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Page 22 of 30

Table 9 Supplementary 3 and 4
Suppl. 3B

CD68 fluorescence intensity CA1

FLPS-injection (1, 71) = 4.305, p = 0.0416

Suppl. 3D

CD68 fluorescence intensity DG

FLPS-injection (1, 72) = 0.06944, p = 0.7929

Suppl. 4B

GFAP fluorescence CA1

FLPS-injection (1, 71) = 2.213, p = 0.1413

Suppl. 4D

GFAP fluorescence DG

FLPS-injection (1, 68) = 0.3305, p = 0.5672

Fgenotype (1, 71) = 3.407, p = 0.0691
Fgenotype (1, 72) = 0.1633, p = 0.6874
Fgenotype (1, 71) = 4.828, p = 0.0313
Fgenotype (1, 68) = 1.031, p = 0.3135

phenotype in mice, the absence of endogenous itaconate production, due to the deletion of Irg1, did not significantly affect this response. However, analysis of the
microglial activation marker CD68 via immunofluorescence showed no significant elevation in CD68 fluorescence in the hippocampal subregions after LPS injection,compared to mice injected with saline neither in WT nor
in Irg1−/− mice (Suppl. 3A-D). Only in the CA1 subregion of WT mice a slight trend towards increased CD68
levels after LPS-injection was observed (Suppl. 3B, WTSaline vs. WT-LPS p = 0.0675, Table 9). Additionally, thequantification of immunolabelled astrocytes via a GFAP

staining revealed no significant elevation in GFAP fluorescence intensity between LPS- and saline-injected miceof either genotype (Suppl. 4A-D). Only a slightly higher
GFAP expression was observed in both the CA1 andDG subregions of the hippocampus of WT mice after
LPS-injection, and in the CA1 subregion of Irg1−/− mice
(Suppl. 4B,D). Remarkably, although LPS injection leads
to heightened density and reactivity of microglial cells
within the hippocampus, the absence of IRG1 and theconsequent lack of endogenous itaconate production
did not significantly influence the observed increases in
microglial density and reactivity triggered by LPS.

Fig. 9 Irg1−/−-mice do not show an effect on LTP induced by LPS compared to WT-mice. A Input–output curves of field excitatory postsynaptic
potential (fEPSP) slopes in acute hippocampal slices of WT- (A) and Irg1−/−-mice (E). Paired-pulse facilitation (PPF) of the fEPSP slopes plotted
as a response to the second stimulation over the first one at different interpulse intervals (10, 20, 40, 60, 80, and 100 ms) in hippocampal slices
of WT- (B) and Irg1−/−-mice (F). LTP was measured in hippocampal acute slices of WT- (C) and Irg1−/−-mice. (G) LTP maintenance (T 75–80 min)
was assessed in hippocampal acute slices of WT- (D) and Irg1−/−-mice (H). Data are presented as mean ± SEM and were analyzed with the repeatedmeasures two-way ANOVA followed by Fisher’s LSD test (A, B, C, E, F, G) or (D, H) unpaired t-test.; #p < 0.05, [N (number of mice per group) = 3–4, n(number of analyzed hippocampal slices per group) = 15–20]

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Both WT and Irg1−/− mice exhibited LPS‑induced
impairments in synaptic transmission and plasticity

To investigate the role of Irg1-deletion in LPS-induced
neuroinflammation in greater detail, we examined theeffect of Irg1-deletion and its respective loss of endogenous itaconate production, on activity-dependent synaptic plasticity. Specifically, we assessed whether the
degree of impairment in synaptic plasticity induced
by LPS varies between WT and Irg1−/− mice. To thisend, electrophysiological assessments was conducted(Fig. 9). Analyzing basal synaptic transmission in WT
mice, no significant differences were observed between
those injected with LPS and those with saline (Fig. 9A,
E). However, in Irg1−/− mice LPS injection resulted in a
reduced fEPSP slope at stimulus intensities of 400, 500,
600, and 700 μA compared to Irg1−/− mice injectedwith saline, indicating a notable impairment in synaptic transmission (Fig. 9E, Irg1−/−-saline vs. Irg1−/−-LPS:
400 μA p = 0.0146, 500 μA p = 0.0132, 600 μA p = 0.0146,
700 μA p = 0.0248, Table 10). Subsequently, we assessedshort-term synaptic plasticity at the Schaffer collateralCA1 synapse PPF (Fig. 9B, F). No notable differences in
PPF were observed between treatments in either WTor Irg1−/− mice, indicating that presynaptic function
remained largely unaffected by LPS injection in both genotypes. Lastly, the long-term activity-dependent synaptic plasticity was examined (Fig. 9C, D, G, H). As before,
LTP was induced at the Schaffer collateral CA3 to CA1
pathway using TBS after a 20-min period of stable baseline recording (Fig. 9C, G). Our findings showed that LTP
was significantly impaired in the hippocampus of micefollowing LPS injection compared to saline-injected controls, regardless of genotype. This impairment extended
to both genotypes during the maintenance phase of LTP
post-LPS injection (Fig. 9, WT, D: p = 0.0119; Irg1−/− H:
p = 0.0228, Table 10).
Overall, our findings indicate that the deletion of theIrg1 gene and the respective absence of endogenous itaconate do not significantly influence the LPS-induced
impairment in activity-dependent synaptic plasticity
within the hippocampus.

Table 10 Fig. 9
Figure 9A

fEPSP WT

Ftreatment (1, 28) = 0.7518, p = 0.3933

Figure 9B

PPF WT

Ftreatment (1, 28) = 0.3322, p = 0.5690

Figure 9E

fEPSP IRG1-KO

Ftreatment (1, 38) = 5.572, p = 0.0235

Figure 9F

PPF IRG1-KO

Ftreatment (1, 38) = 0.009346, p = 0.9235

Fstimulus (1.856, 51.95) = 238.3, p < 0.0001
Fstimulus (2.825, 79.10) = 17.65, p < 0.0001
Fstimulus (2.217, 84.26) = 288.5, p < 0.0001
Fstimulus (2.106, 80.01) = 17.86, p < 0.0001

Page 23 of 30

Discussion
This work aimed to identify novel therapeutic agents that
could mitigate neuroinflammatory processes, thereby
reducing their profound long-term effects on neuronalhealth. We focused particularly on endogenous compounds, which are naturally synthesized by macrophages
and thus have higher biocompatibility and tolerability.
Itaconate and mesaconate, two prominent immunomodulatory agents, may not only be therapeutic candidates
for infection-induced inflammation in the periphery [19],but were also presented in this work for their potential
to attenuate neuroinflammatory processes triggered bybacterial endotoxins. The results of this study now clearly
indicate that the TCA metabolites itaconate and mesaconate can ameliorate bacterial endotoxin effects on therelease of pro-inflammatory cytokines, microglial reactivity and can rescue impaired synaptic plasticity in thebrain.
This is important to elucidate, since neuroinflammatory responses are known to have long-lasting deleterious
effects on brain, in particular on the hippocampus, anespecially vulnerable brain region [54–58]. Neuroinflammation can eventually impair cognitive functions suchas learning and memory processes well beyond the initial phase of sepsis or viral infection [6, 29]. Additionally,
neuroinflammation has been linked to the progression ofvarious neuropathological conditions, including neurodegenerative diseases like Alzheimer’s disease (AD) [14,15, 59–61]. Despite extensive research, an effective drug
treatment remains elusive.
The evidence obtained here suggests that the endogenous production of itaconate and mesaconate may not
be sufficient to attenuate neuroinflammation, as shownby the comparison of WT and Irg1−/− mice exposed to
LPS, while additional exogenous administration of these
immunomodulatory metabolites may be more effective
via cumulative effects.
The dosage of exogenous itaconate and mesaconate(250 mg/kg body weight) used in this study was chosen
based on findings by He et al., which showed that micetreated with these metabolites prior to administrationsof a lethal dose of LPS experienced extended survival
rate compared to untreated mice [19]. In addition, mice
pretreated with itaconate or mesaconate and injectedwith LPS showed less severe symptoms and a lower drop
in body temperature [19]. The LPS dose for our study,
0.5 mg/kg body weight, was chosen in line with our previous study, identifying the dose effective in triggering
neuroinflammatory symptoms through a dual LPS injection [7, 37].
The release of pro-inflammatory cytokines, which is a
crucial element in the signaling and initiation of inflammatory responses [62–64], showed a significant decreaseOhm et al. Journal of Neuroinflammation(2024) 21:207

in the release and production of IL-1β, IL-6 and TNF-α
in the brain when mice were pretreated with itaconate ormesaconate prior to LPS injection. This finding suggeststhat the two metabolites may attenuate a critical aspectin the initiation of a neuroinflammatory responses. Additionally, itaconate and mesaconate selectively decreased
IL-1β levels in blood serum, which may underscore theattenuated general disease-like symptoms in the LPSexposed mice. Furthermore, IL-1β injections into rodent
brains activates astrocytes and microglia cells accordingly as earlier studies have shown [65], highlighting
the role of IL-1β in contributing to neuroinflammatory
processes and emphasizing its suitability as a target forneuroinflammation therapy. These findings are along to
the previous investigations that showed ester of itaconate by promoting the expression of cyclic AMP-dependent transcription factor (ATF3) in mouse macrophages
and human blood monocytes can prevent LPS-induced
expression of IκBζ protein and IL-6 [66] which may also
be the case in microglia in this scenario. In addition,
dimethyl itaconate was shown to protect against LPSinduced pro-inflammatory mediators release in mice by
activating MAPKs and Nrf2 and inhibiting NF-kB signaling pathways [67]. Future studies can identify the effectsof metabolites in modulating NF-kB and IκBζ as underlying mechanistic signaling pathways. In another study, itaconate and its two isomers, mesaconate and citraconate,
were found to decrease the phosphorylation levels of Signal Transducer and Activator of Transcription 1 (STAT1)
and attenuate canonical type I IFN signaling [68]. Therefore, these signaling pathways may be involved in the
immunomodulatory effects of the metabolites on microglial cells but need to be investigated in future studies toreveal itaconates and mesaconates signaling pathways
in microglia. Conversely, we did not observe changes inlevels of the anti-inflammatory cytokine IL-10, which
actually slightly increased in mice pretreated with mesaconate prior to LPS injection compared to those injectedwith LPS alone. IL-10 is known for its strong anti-inflammatory properties [69]. It modulates tissue repair after an
inflammatory insult. LPS can upregulate microglial IL-10
release both in vitro and in vivo, probably due to thestimulation of endogenous repair mechanisms in preparation for the clearance of the acute insult [70, 71]. Ourfinding in this respect is remarkable, since it suggests thatthe anti-inflammatory function of itaconate and mesaconate in the brain may be mediated via the downregulationof pro-inflammatory cytokines, and not via an increased
production of anti-inflammatory cytokines.
Peritoneal injections of LPS have been shown toenhance microglial reactivity in the hippocampus [7,42], therefore we intended here to elucidate the potential
positive role of itaconate and mesaconate on microglial

Page 24 of 30

reactivity using as readouts cell density, IBA1 fluorescence intensity and the levels of the microglial activation marker CD68 in subregions of the hippocampus.
Only itaconate prevented the LPS-induced increase in
microglia density. Moreover, previous studies have demonstrated that LPS elevates IBA1 expression [41, 72].
Of note, pretreatment with both itaconate and mesaconate significantly reduced the fluorescence intensity of
IBA1 immunostaining in the hippocampus following
LPS injection compared to mice pretreated with PBS.
Elevated microglial density and IBA1 expression levels
are indicative of both increased microglial reactivity andconsequently neuroinflammation triggered by LPS injection [42]. Considering the different effects of itaconate
and mesaconate on microglia density and IBA1 expression in the hippocampus, it is possible that pretreatment
with these metabolites modulates microglial reactivity
differently and in co-supporting way. Mesaconate could
attenuate aspects of the pro-inflammatory response, as
shown by decreased IBA1 expression, while itaconate
could prevent microglial proliferation in addition toattenuating pro-inflammatory responses. Previously, itaconate was shown to play a complex role in influencing
macrophage polarization, suggesting a possible influenceon macrophage phenotype [73, 74]. It is reasonable toassume that both itaconate and mesaconate regulate theinflammatory state of microglia, the macrophages residing in the brain, even though they appear to function
differently. However, this hypothesis needs to be investigated by further studies analyzing the effect of itaconate
and mesaconate on microglial polarization possibly leading to different functions and properties. In this respect
it is noteworthy, that He et al. have shown that the two
metabolites have different effects on cellular respirationand metabolic changes in macrophages [19]. Althoughboth itaconate and mesaconate possess immunomodulatory potential, they seem to interact with cellular metabolic pathways in distinct ways [19], which might leadto the different regulatory states of the microglia. It wasdemonstrated that itaconate, but not mesaconate, inhibited Succinate dehydrogenase (SDH) in macrophages,
while mesaconate treatment did not [19]. Furthermore,
although mesaconate attenuated glycolytic activity similarly to itaconate, only itaconate was able to suppress
TCA activity and cellular respiration [19]. Therefore,
there are some differences in the mechanisms of actionof these two metabolites on cells. It is therefore plausiblethat they also have different effects on the metabolic status of microglia, which may ultimately affect their functions and reactivity, a phenomenon that requires further
investigation in future studies. This is even more remarkable considering that there are only minor differences
between itaconate and mesaconate, both just differ due

Ohm et al. Journal of Neuroinflammation(2024) 21:207

to the different position of a single proton and the placement of the double bond.
Recent research suggests that the categorization of theactivation states of microglia, as opposed to peripheral
macrophages, is more complex than previously assumed:
The traditional binary categorization of microglia into
“M1—classically activated, pro-inflammatory” and“M2—alternatively activated, anti-inflammatory/tissue
repairing” is now considered simplistic [75]. It is more
likely that microglia exhibit a spectrum of functionaland morphological phenotypes that cannot be reduced
to two categories (M1/M2 –reactive/not reactive). It
may be that microglia from itaconate- and mesaconatepretreated mice injected with LPS have adopted different
stages of reactivity that differentially modulate microglial
proliferation and reactivity. To gain a more comprehensive understanding of how pretreatment with itaconate
and mesaconate may modulate LPS-induced microglial
reactivity, further research involving morphological studies, gene expression profiling, and functional assessments
may be beneficial.
LPS injection resulted in increased expression of CD68,
a microglial activation marker, throughout the brain ofmice pretreated with control PBS, indicating microglial
reactivity in response to inflammation [76, 77]. Conversely, mice pretreated with itaconate and mesaconate
exhibited a non-significant increase in CD68 levels as
determined by FACS analysis. However, immunohistochemical examination of CD68 expression in hippocampal microglial cells did not reveal this effect very clearly.
This finding emphasizes a modulation of microglial reactivity in mice pretreated with itaconate and mesaconate,
which could be even more pronounced in other brain
regions besides the hippocampus.
Part of the brains immune response in addition to
microglia are astrocytes. However, when we quantified
astrogliosis by analyzing GFAP fluorescence intensity our
results indicate that the dose of LPS used in this study
was not sufficient to induce severe astrogliosis, as LPS
did not result in a significant increase in fluorescenceintensity compared to corresponding saline injected
controls. These results are consistent with other studies, e.g. Norden et al. came to similar conclusions andobserved an increase in IBA1 expression twenty-fourhours after LPS injection (0.33 mg/kg i.p. LPS), but nochange in GFAP fluorescence at such a low dose of LPS,
despite an increase in astrocytic cytokine profiles [42].
Further evidence along this line was presented by Wendeln et al., which observed that the number of G
­ FAP+
astrocytes increased only after three low-dose LPS injections (0.5 mg/kg) [37]. Furthermore, Kang et al. reported
increased GFAP fluorescence intensity following five LPS
injections (250 µg/kg) [78]. However, it is noteworthy

Page 25 of 30

that in our study LPS-injected mice without pretreatment exhibited a more pronounced tendency for elevated GFAP expression compared to the corresponding
controls.
Beyond the in vivo studies, in vitro investigations using
isolated microglia and astrocyte cultures were carriedout to identify the specific cell type through which theobserved anti-inflammatory effects of metabolite treatment are mediated. Microglia pretreated with itaconate
or mesaconate, but not astrocytes, showed a reduction in
pro-inflammatory cytokines following LPS stimulation.
This suggest that the anti-inflammatory effects observedin vivo are primarily mediated by the modulation of
microglial function, whereas astrocytes may play a lessimportant role in initiating the immunoregulatory effects
of itaconate and mesaconate within the brain. Considering that activated microglial cells have the potential toinduce astrocytic reactivity [11], the slight increase in
GFAP immunostaining in the LPS-injected mice pretreated with itaconate and mesaconate compared to themice receiving LPS alone could be attributed to reduced
microglial activation induced by the metabolites.
Intraperitoneal injection of LPS has been shown to
induce neuroinflammation leading to structural andfunctional changes in neuronal cells, particularly impairing cognitive function [6, 7, 79, 80]. In line with previous
findings, the results here further supported the notionthat mice injected with LPS exhibited impaired longterm potentiation (LTP) at CA3-CA1 synapses in the hippocampus. This impairment in LTP was rescued whenmice were administered itaconate or mesaconate prior to
LPS injection, supporting the potential of the two metabolites to prevent the consequences of neuroinflammation
on synaptic plasticity. Previous reports suggested that
inflammatory mediators play a role in impairing neuronal
synaptic transmission and plasticity [81–83]. The detrimental effect of LPS on synaptic plasticity is attributedin part to the activation of pro-inflammatory cytokines
such as IL-1β [80, 84–86]—a cytokine that in this study
showed the most significant reduction in brain levels in
LPS-injected mice when pretreated with itaconate ormesaconate compared to mice injected with LPS alone.
While previous studies indicate that a low basal level ofIL-1β is essential for the proper regulation of synaptic
plasticity, excessively elevated levels of this cytokine cannegatively affect synaptic transmission [81]. In addition,
IL-1β has been found to impair signaling from N-methyld-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors[87, 88], although the exact regulatory mechanism of themetabolites in this scenario remains unclear [81].
In a next step of the study to determine the importance of endogenous itaconate levels in attenuating

Ohm et al. Journal of Neuroinflammation(2024) 21:207

neuroinflammatory processes and adverse sequelae
in vivo, we injected Irg1-deficient mice (Irg1−/−) with
LPS. The Irg1 gene encodes the enzyme IRG1, whichcatalyzes the synthesis of itaconate [20]. Previous studies have shown that Irg1−/−mice exhibit an enhanced
inflammatory response and a more severe disease phenotype compared to WT mice with endogenous itaconate
production [47–51]. In a model of septic shock, miceadministered a single injection of LPS exhibited stronger
symptoms of sepsis compared to their WT counterparts [52]. Although the effects of Irg1 deficiency on
the peripheral immune response are well documented,
there are few studies looking at the effects on the brain.
Nevertheless, Kuo et al. et a discovered that Irg1−/− mice
suffered more severe brain injury after stroke [53]. In
addition, Daniels et al. showed that mice lacking Irg1 weremore susceptible to infection by neurotropic flaviviruses,
including Zika virus, than WT mice, with Irg1−/− micehaving a higher viral load [89]. Despite these findings,
the role of Irg1 deficiency in brain-specific inflammation is less explored. Here, Irg1−/− mice showed only
slightly higher susceptibility to LPS injections compared
to WT mice, with both genotypes experiencing similar
body weight loss after peripheral LPS administration. Of
note, pro-inflammatory mediators in the blood and brainof Irg1−/− mice were slightly elevated compared to WT
mice, although not significantly. Contrary to expectations based on previous studies, such as Yang et al., which
reported significant differences in body weight loss andhigher cytokine levels in Irg1−/− mice, these results could
not be replicated in this study, possibly due to variations
in LPS dosage, timing of blood analysis or number of LPS
injections [52]. In addition, the number of LPS injections,using a double injection strategy in the current study, hasbeen shown to induce a strong stimulation of cytokines
in the brain, but could induce immune tolerance in the
periphery, reflected by altered cytokine levels in the
blood, possibly explaining the observed results [37].
Analysis of microglial cells also revealed only minorchanges between the two genotypes, with microglial density being higher in LPS-injected Irg1−/− mice than in
WT mice injected with LPS. However, there were no significant changes in the fluorescence intensity of IBA1 andCD68 immunostaining. Together with the slight increase
in inflammatory mediators in Irg1−/− mice compared to
WT, this suggests that the absence of IRG1 has a limited
effect on LPS-induced neuroinflammation. Furthermore,no changes were observed in astrocytes with respect tothe intensity of GFAP immunofluorescence. When activity-dependent synaptic plasticity was examined in the
hippocampus of both WT and Irg1−/− mice, LTP wasfound to be impaired after two peripheral LPS injections.
However, the extent of this LPS-induced impairment

Page 26 of 30

was not significantly different between Irg1−/− and WT
mice, suggesting that the impairment was not enhancedby the absence of IRG1 or, by extension, by the absenceof endogenous itaconate. Overall, our results suggest that
deletion of Irg1 does not significantly affect the neuroinflammatory response to a double injection of a low doseof LPS. This contradicts the original hypothesis that theabsence of endogenous itaconate production in Irg1−/−
mice would exacerbate neuroinflammation. Previous
research has emphasized the role of IRG1 in itaconate
production due to its immunomodulatory effects via
multiple pathways. However, the functions of IRG1 go
beyond immunomodulation [78, 79]. Li et al. pointed out
the involvement of IRG1 in oxidative stress and foundincreased ROS levels in immunostimulated macrophages[79]. IRG1 is also associated with antigen processing byenhancing MHC class I molecules (MHC1) functionality
and exhibiting antiviral properties via mechanisms thatare not yet fully understood [80]. In addition, He et al.
have shown that administration of itaconate and mesaconate, which are endogenously synthesized by IRG1, canincrease the levels of IFN-β and CXCL10, highlighting
the antiviral effects of these metabolites [19].
However, Wu et al. pointed out that the actions of IRG1
not only protect against inflammation but can also exacerbate tissue damage under certain conditions [78]. Thus,
increased Irg1 mRNA levels in macrophages infected with
V. Leishmania facilitated parasite growth and survival[81], and Irg1 expression was associated with lung damage during respiratory syncytial virus infection [82]. Furthermore, IRG1-driven itaconate production promotes
vesicular stomatitis virus replication, with Irg1−/− mice
exhibiting reduced infiltration of inflammatory cells in
the lung [83]. In addition, IRG1 has been associated withtumorigenesis, and knocking down Irg1 can reduce tumor
growth [90]. These results emphasize the complex role of
IRG1 in promoting and inhibiting disease processes. Theysuggest that the absence of IRG1 does not consistently
lead to worsened outcomes and emphasize the need forfurther research into its diverse biological effects.
Overall, our study focused on the acute phase of infection after LPS injection in order to determine how TCA
metabolites might dampen the initiation of inflammation. Not within the scope of our study, was whether
exogenously administered itaconate and mesaconate
can attenuate the long-term effects of inflammatory processes induced by LPS, such as memory deficits and thenegative neuronal effects triggered by LPS [6, 84, 85].
These could include behavioral assessments in the experimental groups used in this work to investigate how thesecompounds affect memory formation impaired by neuroinflammatory events as well as long-term morphologicalchanges in neurons.

Ohm et al. Journal of Neuroinflammation(2024) 21:207

Considering that microglia play a crucial role in maintaining the balance and proper function of the CNS
during development and adulthood, there is growing evidence that hyperreactivity and dysfunction of microglia
in conjunction with their excessive release of inflammatory mediators can lead to neurotoxic outcomes in various neurological and neurodegenerative diseases [86].
Therefore, modulation of microglia reactivity is a potential therapeutic strategy to treat a number of neurologicaland neurodegenerative diseases characterized by neuroinflammation. As endogenous compounds, as the TCA
metabolites itaconate and mesaconate are particularlyinteresting due to their biocompatibility and tolerability, which could also make them suitable for long-term
treatment. However, to verify these advantages, thoroughlong-term studies are needed to confirm that prolonged
administration has no detrimental effects. In addition,
research into less invasive delivery methods than injections, such as oral administration via drinking water orfood, could make these compounds suitable for oral therapies. A remarkable point in this study is the possibility
that the observed effect of itaconate and mesaconate onneuroinflammation may be an indirect effect of them in
influencing peripheral inflammation rather than a direct
effect inside the CNS. Although the study cannot definitively rule out this possibility, the finding that itaconate
levels in the brain remained elevated fifty-one hours after
the last injection suggests the ability of itaconate to crossthe blood–brain barrier (BBB) and be taken up by CNS
cells. This highlights further its therapeutic potential forthe treatment of neurological diseases, since this indicated a possible direct effect on microglia cells. However,the pharmacokinetic details of itaconate and mesaconate
need to be investigated in future studies to understandthe role of the two metabolites in modulating the immune
response. However, it is important to note that despite
the unknown and perhaps short half-life, itaconate andmesaconate appear to induce significant intracellularsignaling cascades leading to their immunoregulatory
functions, which need to be discovered by future studies.
Moreover, the metabolites here were administered prior
to LPS injection, mainly to demonstrate their preventive
importance. It would be of interest to investigate whether
post-treatment with itaconate and mesaconate could also
attenuate the pre-existing neuroinflammatory consequences. However, to clarify this hypothesis, the timingof administration of the metabolite is very important, as
the acute phase of immune responses triggered by LPS isvery short and prompt. In addition, future studies need toclarify whether the findings related to LPS can be extrapolated to sterile causes of disease, as the pathogenic
neuroinflammatory responses differ considerably fromthose caused by sterile insults such as proteinopathies

Page 27 of 30

or trauma. The path from laboratory research to clinicaltranslation is complex and multi-layered. However, thiswork lays an important foundation and highlights theneed for further research to fully exploit the therapeuticpotential of itaconate and mesaconate in the fight against
neuroinflammatory brain diseases.

Conclusion
This study investigated the effects of itaconate and mesaconate, derived from the metabolites of the TCA cycle,
on neuroinflammation in a mouse model of septic shock
induced by i.p. injection of LPS. The results of this study
showed that pretreatment with itaconate or mesaconate
significantly reduced the levels of key pro-inflammatory
cytokines in the brain. Importantly, these metabolites
significantly attenuated the excessive microglial reactivity induced by LPS. Moreover, the protective effects ofthese metabolites on neuroinflammation-induced damage were able to reverse the impairment of synaptic plasticity and maintain the density of dendritic spines after
LPS exposure. Overall, this study highlights the potential therapeutic value of itaconate and mesaconate in thetreatment of neuroinflammatory diseases and emphasizes their important role in protecting neuronal structure and function. Due to their endogenous origin andproduction, which generally leads to high tolerance, thesemetabolites could be of great importance as therapeuticstrategies in neuroinflammatory brain diseases.
Abbreviations
4-OI	4-Octyl itaconate
ACOD1	Aconitate decarboxylase 1
ACSF	Artificial cerebrospinal fluid
AD	Alzheimer’s disease
AMPA	α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid
BBB	Blood–brain barrier
BDNF	Brain-derived neurotrophic factor
C1q	Complement component 1q
CA	Cornu Ammonis
CD68	Cluster of differentiation 68
CNS	Central nervous system
CXCL10	C-X-C motif chemokine ligand 10
DG	Dentate gyrus
DMI	Dimethyl itaconate
ELISA	Enzyme-linked immune sorbent assa
FACS	Fluorescence-activated cell sorting
fEPSPs	Field excitatory postsynaptic potentials
GC–MS	Gas chromatography–mass spectrometry
GFAP	Glial fibrillary acidic protein
i.p.	Intraperitoneal
IBA1	Ionized calcium binding adaptor molecule-1
IL	Interleukin
IFN	Interferon
IRG1	Immune-responsive gene 1
Irg1-/-	Irg1-deficient mice
LPS	Lipopolysaccharide
LTP	Long-term potentiation
NMDA	N-methyl-d-aspartateNO	Nitric oxide
NRF2	Nuclear factor erythroid-2-related factor 2

Ohm et al. Journal of Neuroinflammation(2024) 21:207

PBS	Phosphate buffered saline
PD	Parkinson’s disease
PFA	Paraformaldehyde
PGE2	Prostaglandin E2
PPF	Paired pulse facilitation
ROS	Reactive oxygen speciesTBS	Theta-burst stimulation
TCA​	Tricarboxylic acid cycle
WT	Wild-type

Page 28 of 30

4.
5.
6.
7.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03188-3.

8.

Supplementary Material 1.
9.
Author contributions
MO and SH conducted the research and analysed the data. MO, SH, WH, TM,
KH and MK conceived and designed the research. OG and EM kindly provided
the Irg1−/− mice. SH, MK and MO wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL. DFG, SPP2395
to SH and MK.
Availability of data and materials
Data is provided within the manuscript or supplementary information files.
The datasets used during the current study are available from the corresponding author upon reasonable request.

10.
11.
12.
13.
14.

Declarations
Ethics approval and consent to participate
The animal study was reviewed and approved by the animal welfare representative of the TU Braunschweig and the LAVES (Oldenburg, Germany, Az. §4(02.05) TSchB TU BS and Az. 33.19–42502-04- 21–3734).

15.
16.

Consent for publication
Not applicable.

17.

Competing interests
The authors declare no competing interests.

18.

Author details
1
Department of Cellular Neurobiology, Zoological Institute, TU Braunschweig,
38106 Braunschweig, Germany. 2 Neuroinflammation and Neurodegeneration Group, Helmholtz Centre for Infection Research, 38124 Braunschweig,
Germany. 3 Department of Bioinformatics and Biochemistry, Braunschweig
Integrated Centre of Systems Biology (BRICS), TU Braunschweig, 38106 Braunschweig, Germany. 4 Infection Immunology Research Group, Helmholtz Centre
for Infection Research, 38124 Braunschweig, Germany.
Received: 3 June 2024 Accepted: 26 July 2024

19.
20.
21.
22.

23.
References
1. Carson MJ, Doose JM, Melchior B, Schmid CD, Ploix CC. CNS immune
privilege: hiding in plain sight. Immunol Rev. 2006;213:48–65.
2. Perry VH, Cunningham C, Holmes C. Systemic infections and inflammation affect chronic neurodegeneration. Nat Rev Immunol.
2007;7(2):161–7.
3. Biesmans S, Meert TF, Bouwknecht JA, Acton PD, Davoodi N, De Haes P,
et al. Systemic immune activation leads to neuroinflammation and sickness behavior in mice. Med Inflamm. 2013;2013:271359.

24.

25.

Catorce MN, Gevorkian G. LPS-induced murine neuroinflammation
model: main features and suitability for pre-clinical assessment of nutraceuticals. Curr Neuropharmacol. 2016;14(2):155–64.
Zhao J, Bi W, Xiao S, Lan X, Cheng X, Zhang J, et al. Neuroinflammationinduced by lipopolysaccharide causes cognitive impairment in mice. Sci
Rep. 2019;9(1):5790.
Beyer MMS, Lonnemann N, Remus A, Latz E, Heneka MT, Korte M. Enduring changes in neuronal function upon systemic inflammation are NLRP3
inflammasome dependent. J Neurosci. 2020;40(28):5480–94.
Lonnemann N, Hosseini S, Ohm M, Geffers R, Hiller K, Dinarello CA, et al.
IL-37 expression reduces acute and chronic neuroinflammation andrescues cognitive impairment in an Alzheimer’s disease mouse model.
Elife. 2022. https://​doi.​org/​10.​7554/​eLife.​75889.
Skrzypczak-Wiercioch A, Salat K. Lipopolysaccharide-induced model ofneuroinflammation: mechanisms of action, research application andfuture directions for its use. Molecules. 2022. https://​doi.​org/​10.​3390/​
molec​ules2​71754​81.
Wang L, Lin F, Ren M, Liu X, Xie W, Zhang A, et al. The PICK1/TLR4 complex
on microglia is involved in the regulation of LPS-induced sepsis-associated encephalopathy. Int Immunopharmacol. 2021;100:108116.
Deng I, Corrigan F, Zhai G, Zhou XF, Bobrovskaya L. Lipopolysaccharide
animal models of Parkinson’s disease: recent progress and relevance toclinical disease. Brain Behav Immun Health. 2020;4:100060.
Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer
L, et al. Neurotoxic reactive astrocytes are induced by activated microglia.
Nature. 2017;541(7638):481–7.
Sofroniew MV. Multiple roles for astrocytes as effectors of cytokines andinflammatory mediators. Neuroscientist. 2014;20(2):160–72.
Colombo E, Farina C. Astrocytes: key regulators of neuroinflammation.
Trends Immunol. 2016;37(9):608–20.
Stolp HB, Liddelow SA, Sa-Pereira I, Dziegielewska KM, Saunders NR.
Immune responses at brain barriers and implications for brain development and neurological function in later life. Front Integr Neurosci.
2013;7:61.
Kempuraj D, Thangavel R, Natteru PA, Selvakumar GP, Saeed D, Zahoor H,
et al. Neuroinflammation induces neurodegeneration. J Neurol Neurosurg Spine. 2016;1(1):1003.
Rauf A, Badoni H, Abu-Izneid T, Olatunde A, Rahman MM, Painuli S, et al.
Neuroinflammatory markers: key indicators in the pathology of neurodegenerative diseases. Molecules. 2022. https://​doi.​org/​10.​3390/​molec​ules2​
71031​94.
Heneka MT, Carson MJ, El Khoury J, Landreth GE, Brosseron F, FeinsteinDL, et al. Neuroinflammation in Alzheimer’s disease. Lancet Neurol.
2015;14(4):388–405.
Holmes C. Review: systemic inflammation and Alzheimer’s disease. Neuropathol Appl Neurobiol. 2013;39(1):51–68.
He W, Henne A, Lauterbach M, Geissmar E, Nikolka F, Kho C, et al. Mesaconate is synthesized from itaconate and exerts immunomodulatory effects
in macrophages. Nat Metab. 2022;4(5):524–33.
Peace CG, O’Neill LA. The role of itaconate in host defense and inflammation. J Clin Invest. 2022. https://​doi.​org/​10.​1172/​JCI14​8548.
Mills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylationof KEAP1. Nature. 2018;556(7699):113–7.
Michelucci A, Cordes T, Ghelfi J, Pailot A, Reiling N, Goldmann O,
et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc Natl Acad Sci USA.
2013;110(19):7820–5.
Nemeth B, Doczi J, Csete D, Kacso G, Ravasz D, Adams D, et al. Abolition of
mitochondrial substrate-level phosphorylation by itaconic acid produced
by LPS-induced Irg1 expression in cells of murine macrophage lineage.
FASEB J. 2016;30(1):286–300.
Lampropoulou V, Sergushichev A, Bambouskova M, Nair S, Vincent EE,
Loginicheva E, et al. Itaconate links inhibition of succinate dehydrogenase
with macrophage metabolic remodeling and regulation of inflammation.
Cell Metab. 2016;24(1):158–66.
Swain A, Bambouskova M, Kim H, Andhey PS, Duncan D, Auclair K, et al.
Comparative evaluation of itaconate and its derivatives reveals divergent
inflammasome and type I interferon regulation in macrophages. Nat
Metab. 2020;2(7):594–602.

Ohm et al. Journal of Neuroinflammation(2024) 21:207

26. Sun G, Zhang R, Liu C, Meng W, Pang Q. Itaconate attenuates neuroinflammation and exerts dopamine neuroprotection in Parkinson’s disease
through inhibiting NLRP3 inflammasome. Brain Sci. 2022. https://​doi.​org/​
10.​3390/​brain​sci12​091255.
27. Cordes T, Lucas A, Divakaruni AS, Murphy AN, Cabrales P, Metallo CM.
Itaconate modulates tricarboxylic acid and redox metabolism to mitigate
reperfusion injury. Mol Metab. 2020;32:122–35.
28. Demuth L, Ohm M, Michaelsen-Preusse K, Schulze K, Riese P, Guzman CA,
et al. Influenza vaccine is able to prevent neuroinflammation triggered by
H7N7 IAV infection. Front Pharmacol. 2023;14:1142639.
29. Hosseini S, Wilk E, Michaelsen-Preusse K, Gerhauser I, Baumgartner W,
Geffers R, et al. Long-term neuroinflammation induced by influenza A
virus infection and the impact on hippocampal neuron morphology andfunction. J Neurosci. 2018;38(12):3060–80.
30. Hosseini S, Michaelsen-Preusse K, Schughart K, Korte M. Long-term
consequence of non-neurotropic H3N2 influenza A virus infection forthe progression of Alzheimer’s disease symptoms. Front Cell Neurosci.
2021;15:643650.
31. Lonnemann N, Hosseini S, Marchetti C, Skouras DB, Stefanoni D,
D’Alessandro A, et al. The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer’s disease. Proc
Natl Acad Sci USA. 2020;117(50):32145–54.
32. Sapcariu SC, Kanashova T, Weindl D, Ghelfi J, Dittmar G, Hiller K. Simultaneous extraction of proteins and metabolites from cells in culture.
MethodsX. 2014;1:74–80.
33. Meiser J, Kramer L, Sapcariu SC, Battello N, Ghelfi J, D’Herouel AF, et al.
Pro-inflammatory macrophages sustain pyruvate oxidation through pyruvate dehydrogenase for the synthesis of itaconate and to enable cytokine
expression. J Biol Chem. 2016;291(8):3932–46.
34. More TH, Mozafari B, Martens A, Herr C, Lepper PM, Danziger G, et al.
Plasma metabolome alterations discriminate between COVID-19 andnon-COVID-19 pneumonia. Metabolites. 2022. https://​doi.​org/​10.​3390/​
metab​o1211​1058.
35. Jager C, Hiller K, Buttini M. Metabolic profiling and quantification of neurotransmitters in mouse brain by gas chromatography-mass spectrometry. Curr Protoc Mouse Biol. 2016;6(3):333–42.
36. Hiller K, Hangebrauk J, Jager C, Spura J, Schreiber K, Schomburg
D. MetaboliteDetector: comprehensive analysis tool for targeted
and nontargeted GC/MS based metabolome analysis. Anal Chem.
2009;81(9):3429–39.
37. Wendeln AC, Degenhardt K, Kaurani L, Gertig M, Ulas T, Jain G, et al.
Innate immune memory in the brain shapes neurological disease hallmarks. Nature. 2018;556(7701):332–8.
38. Miranda M, Morici JF, Zanoni MB, Bekinschtein P. Brain-derived neurotrophic factor: a key molecule for memory in the healthy and thepathological brain. Front Cell Neurosci. 2019;13:363.
39. Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells arehighly dynamic surveillants of brain parenchyma in vivo. Science.
2005;308(5726):1314–8.
40. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. Physiology of microglia. Physiol Rev. 2011;91(2):461–553.
41. Qin L, Liu Y, Hong JS, Crews FT. NADPH oxidase and aging drive microglial
activation, oxidative stress, and dopaminergic neurodegeneration following systemic LPS administration. Glia. 2013;61(6):855–68.
42. Norden DM, Trojanowski PJ, Villanueva E, Navarro E, Godbout JP. Sequential activation of microglia and astrocyte cytokine expression precedes
increased Iba-1 or GFAP immunoreactivity following systemic immune
challenge. Glia. 2016;64(2):300–16.
43. Chistiakov DA, Killingsworth MC, Myasoedova VA, Orekhov AN, Bobryshev
YV. CD68/macrosialin: not just a histochemical marker. Lab Invest.
2017;97(1):4–13.
44. Pekny M, Pekna M. Astrocyte reactivity and reactive astrogliosis: costs andbenefits. Physiol Rev. 2014;94(4):1077–98.
45. Sofroniew MV. Astrogliosis. Cold Spring Harb Perspect Biol.
2014;7(2):a020420.
46. Vereker E, Campbell V, Roche E, McEntee E, Lynch MA. Lipopolysaccharide
inhibits long term potentiation in the rat dentate gyrus by activating
caspase-1. J Biol Chem. 2000;275(34):26252–8.
47. Ogger PP, Albers GJ, Hewitt RJ, O’Sullivan BJ, Powell JE, Calamita E, et al.
Itaconate controls the severity of pulmonary fibrosis. Sci Immunol. 2020.
https://​doi.​org/​10.​1126/​sciim​munol.​abc18​84.

Page 29 of 30

48. Yi Z, Deng M, Scott MJ, Fu G, Loughran PA, Lei Z, et al. Immune-responsive gene 1/itaconate activates nuclear factor erythroid 2-related factor
2 in hepatocytes to protect against liver ischemia-reperfusion injury.
Hepatology. 2020;72(4):1394–411.
49. Zhu D, Zhao Y, Luo Y, Qian X, Zhang Z, Jiang G, et al. Irg1-itaconate axis
protects against acute kidney injury via activation of Nrf2. Am J Transl
Res. 2021;13(3):1155–69.
50. Kim HW, Yu AR, Lee JW, Yoon HS, Lee BS, Park HW, et al. Aconitate decarboxylase 1 deficiency exacerbates mouse colitis induced by dextran
sodium sulfate. Int J Mol Sci. 2022. https://​doi.​org/​10.​3390/​ijms2​30843​92.
51. Sohail A, Iqbal AA, Sahini N, Chen F, Tantawy M, Waqas SFH, et al. Itaconate and derivatives reduce interferon responses and inflammation in
influenza A virus infection. PLoS Pathog. 2022;18(1):e1010219.
52. Yang W, Wang Y, Huang Y, Wang T, Li C, Zhang P, et al. Immune response
gene-1 [IRG1]/itaconate protect against multi-organ injury via inhibiting
gasdermin D-mediated pyroptosis and inflammatory response. Inflammopharmacology. 2023. https://​doi.​org/​10.​1007/​s10787-​023-​01278-x.
53. Kuo PC, Weng WT, Scofield BA, Furnas D, Paraiso HC, Yu IC, et al. Immunoresponsive gene 1 modulates the severity of brain injury in cerebral
ischaemia. Brain Commun. 2021;3(3):187.
54. Jurgens HA, Amancherla K, Johnson RW. Influenza infection induces
neuroinflammation, alters hippocampal neuron morphology, and impairscognition in adult mice. J Neurosci. 2012;32(12):3958–68.
55. Vitkovic L, Konsman JP, Bockaert J, Dantzer R, Homburger V, Jacque
C. Cytokine signals propagate through the brain. Mol Psychiatry.
2000;5(6):604–15.
56. Lynch MA. Interleukin-1 beta exerts a myriad of effects in the brain and inparticular in the hippocampus: analysis of some of these actions. Vitam
Horm. 2002;64:185–219.
57. Nwaubani P, Cercignani M, Colasanti A. In vivo quantitative imaging of
hippocampal inflammation in autoimmune neuroinflammatory conditions: a systematic review. Clin Exp Immunol. 2022;210(1):24–38.
58. Kempuraj D, Thangavel R, Selvakumar GP, Zaheer S, Ahmed ME, Raikwar
SP, et al. Brain and peripheral atypical inflammatory mediators potentiate neuroinflammation and neurodegeneration. Front Cell Neurosci.
2017;11:216.
59. O’Connor JC, Lawson MA, Andre C, Moreau M, Lestage J, Castanon N,
et al. Lipopolysaccharide-induced depressive-like behavior is mediatedby indoleamine 2,3-dioxygenase activation in mice. Mol Psychiatry.
2009;14(5):511–22.
60. Chen WW, Zhang X, Huang WJ. Role of neuroinflammation in neurodegenerative diseases (review). Mol Med Rep. 2016;13(4):3391–6.
61. Batista CRA, Gomes GF, Candelario-Jalil E, Fiebich BL, de Oliveira ACP.
Lipopolysaccharide-induced neuroinflammation as a bridge to understand neurodegeneration. Int J Mol Sci. 2019. https://​doi.​org/​10.​3390/​
ijms2​00922​93.
62. Hanada T, Yoshimura A. Regulation of cytokine signaling and inflammation. Cytokine Growth Factor Rev. 2002;13(4–5):413–21.
63. Zhang JM, An J. Cytokines, inflammation, and pain. Int Anesthesiol Clin.
2007;45(2):27–37.
64. Kany S, Vollrath JT, Relja B. Cytokines in inflammatory disease. Int J Mol
Sci. 2019. https://​doi.​org/​10.​3390/​ijms2​02360​08.
65. Mendiola AS, Cardona AE. The IL-1beta phenomena in neuroinflammatory diseases. J Neural Transm. 2018;125(5):781–95.
66. Bambouskova M, Gorvel L, Lampropoulou V, Sergushichev A, Loginicheva E, Johnson K, et al. Electrophilic properties of itaconate andderivatives regulate the IkappaBzeta-ATF3 inflammatory axis. Nature.
2018;556(7702):501–4.
67. Zhao C, Jiang P, He Z, Yuan X, Guo J, Li Y, et al. Dimethyl itaconate protects
against lippolysacchride-induced mastitis in mice by activating MAPKs
and Nrf2 and inhibiting NF-κB signaling pathways. Microb Pathog.
2019;133:103541.
68. Chen F, Elgaher WAM, Winterhoff M, Bussow K, Waqas FH, Graner E, et al.
Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses
and oxidative stress, and modulates inflammation and cell metabolism.
Nat Metab. 2022;4(5):534–46.
69. Iyer SS, Cheng G. Role of interleukin 10 transcriptional regulation in inflammation and autoimmune disease. Crit Rev Immunol. 2012;32(1):23–63.
70. Wickstead ES, Elliott BT, Pokorny S, Biggs C, Getting SJ, McArthur S. Stimulation of the pro-resolving receptor Fpr2 reverses inflammatory microglial

Ohm et al. Journal of Neuroinflammation

71.
72.

73.
74.
75.
76.
77.

78.
79.
80.
81.

82.
83.
84.

85.

86.
87.
88.
89.
90.

(2024) 21:207

activity by suppressing NFκB activity. Int J Mol Sci. 2023. https://​doi.​org/​
10.​3390/​ijms2​42115​996.
Tao W, Zhang G, Liu C, Jin L, Li X, Yang S. Low-dose LPS alleviates earlybrain injury after SAH by modulating microglial M1/M2 polarization via
USP19/FOXO1/IL-10/IL-10R1 signaling. Redox Biol. 2023;66:102863.
Go M, Kou J, Lim JE, Yang J, Fukuchi KI. Microglial response to LPS
increases in wild-type mice during aging but diminishes in an Alzheimer’s mouse model: implication of TLR4 signaling in disease progression.
Biochem Biophys Res Commun. 2016;479(2):331–7.
Runtsch MC, Angiari S, Hooftman A, Wadhwa R, Zhang Y, Zheng Y, et al.
Itaconate and itaconate derivatives target JAK1 to suppress alternative
activation of macrophages. Cell Metab. 2022;34(3):487–501.
Li Z, Zheng W, Kong W, Zeng T. Itaconate: a potent macrophage immunomodulator. Inflammation. 2023;46(4):1177–91.
Paolicelli RC, Sierra A, Stevens B, Tremblay ME, Aguzzi A, Ajami B, et al.
Microglia states and nomenclature: a field at its crossroads. Neuron.
2022;110(21):3458–83.
Jurga AM, Paleczna M, Kuter KZ. Overview of general and discriminating markers of differential microglia phenotypes. Front Cell Neurosci.
2020;14:198.
Swanson MEV, Mrkela M, Murray HC, Cao MC, Turner C, Curtis MA, et al.
Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain. Acta
Neuropathol Commun. 2023;11(1):69.
Kang JB, Park DJ, Shah MA, Kim MO, Koh PO. Lipopolysaccharide induces
neuroglia activation and NF-kappaB activation in cerebral cortex of adultmice. Lab Anim Res. 2019;35(1):19.
Commins S, O’Neill LA, O’Mara SM. The effects of the bacterial endotoxin
lipopolysaccharide on synaptic transmission and plasticity in the CA1subiculum pathway in vivo. Neuroscience. 2001;102(2):273–80.
Hennigan A, Trotter C, Kelly AM. Lipopolysaccharide impairs long-term
potentiation and recognition memory and increases p75NTR expression
in the rat dentate gyrus. Brain Res. 2007;1130(1):158–66.
Di Filippo M, Sarchielli P, Picconi B, Calabresi P. Neuroinflammationand synaptic plasticity: theoretical basis for a novel, immune-centred,
therapeutic approach to neurological disorders. Trends Pharmacol Sci.
2008;29(8):402–12.
Camacho-Arroyo I, Lopez-Griego L, Morales-Montor J. The role ofcytokines in the regulation of neurotransmission. NeuroImmunoModulation. 2009;16(1):1–12.
Dantzer R. Neuroimmune interactions: from the brain to the immune
system and vice versa. Physiol Rev. 2018;98(1):477–504.
Avital A, Goshen I, Kamsler A, Segal M, Iverfeldt K, Richter-Levin G,
et al. Impaired interleukin-1 signaling is associated with deficits inhippocampal memory processes and neural plasticity. Hippocampus.
2003;13(7):826–34.
Kelly A, Vereker E, Nolan Y, Brady M, Barry C, Loscher CE, et al. Activation ofp38 plays a pivotal role in the inhibitory effect of lipopolysaccharide andinterleukin-1 beta on long term potentiation in rat dentate gyrus. J Biol
Chem. 2003;278(21):19453–62.
Barry CE, Nolan Y, Clarke RM, Lynch A, Lynch MA. Activation of c-JunN-terminal kinase is critical in mediating lipopolysaccharide-inducedchanges in the rat hippocampus. J Neurochem. 2005;93(1):221–31.
Coogan A, O’Connor JJ. Inhibition of NMDA receptor-mediated synaptictransmission in the rat dentate gyrus in vitro by IL-1 beta. NeuroReport.
1997;8(9–10):2107–10.
Lai AY, Swayze RD, El-Husseini A, Song C. Interleukin-1 beta modulates
AMPA receptor expression and phosphorylation in hippocampal neurons.
J Neuroimmunol. 2006;175(1–2):97–106.
Daniels BP, Kofman SB, Smith JR, Norris GT, Snyder AG, Kolb JP, et al. The
nucleotide sensor ZBP1 and kinase RIPK3 induce the enzyme IRG1 to promote an antiviral metabolic state in neurons. Immunity. 2019;50(1):64–76.
Chen YJ, Li GN, Li XJ, Wei LX, Fu MJ, Cheng ZL, et al. Targeting IRG1
reverses the immunosuppressive function of tumor-associatedmacrophages and enhances cancer immunotherapy. Sci Adv.
2023;9(17):0654.

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