The IL-33/ST2 Axis Protects the Hippocampus from LPS-Induced Inflammation and Damage by Modulating Microglial Phenotype

Abstract

Background/Objectives: Systemic inflammation is a known driver of neurodegenerative processes, with amyloid accumulation and neuronal loss. The Interleukin-33 (IL-33)/Suppression of Tumorigenicity 2 (ST2) signaling pathway has emerged as a critical immune regulator with dual roles in maintaining brain health. However, its role in pathological alterations in the central nervous system, and more specifically in the hippocampus during endotoxemia, is not fully elucidated. The aim of this research was to determine the role of the IL-33/ST2 axis in neurodegenerative processes in mice caused by systemic inflammation. Methods: BALB/c wild-type (WT) and ST2-deficient (ST2^−/−) mice were challenged with systemic lipopolysaccharide (LPS) for 7 days. One subgroup of WT mice also received exogenous IL-33. Expression of Iba1, myelin, and amyloid was detected by immunohistochemistry, the TUNEL assay was used for detection of apoptosis, flow cytometry was used to assess microglial phenotype, and RT PCR was used to detect the expression of cytokines. Results: LPS administration induced demyelination and amyloid deposition in the hippocampus. These pathological changes were the most pronounced in ST2^−/− mice, which exhibited an aggressive microglial phenotype, excessive production of IL-1β and massive apoptosis in the hippocampus. Conversely, exogenous IL-33 treatment in WT mice exerted a profound neuroprotective effect. IL-33 induced phagocytic morphology of Iba1-positive cells, redirected microglia toward a restorative M2 phenotype, and significantly upregulated IL-10. This immunomodulation led to the preservation of myelin integrity, a reduction in amyloid load, and the near-complete prevention of hippocampal apoptosis in IL-33 treated mice. Conclusions: This study identifies the IL-33/ST2 axis as an important defense signaling pathway in neuroinflammation induced by systemic LPS administration. By promoting a regulatory microglial state and balancing the IL-10/IL-1β ratio, IL-33 prevents neuroinflammation and neurodegeneration. Our data highlight the pharmacological potential of the IL-33/ST2 axis in counteracting amyloid-related pathologies.

1. Introduction

Alzheimer’s disease (AD), one of the most common forms of dementia, is characterized by the extracellular deposition of amyloid-β (Aβ) and the formation of senile plaques in the central nervous system (CNS) [1]. The accumulation of Aβ is not merely a marker of the disease but a primary driver of proteotoxicity, leading to widespread neuronal death, synaptic disruption, and the progressive decline of cognitive and behavioral functions [2].

While neuroinflammation in Alzheimer’s disease was previously viewed as secondary to neuronal damage, a growing body of evidence suggests that systemic inflammation acts as a critical upstream initiator. Chronic systemic inflammation can trigger innate immune responses within the CNS, initiating and accelerating Aβ deposition. Furthermore, systemic mediators exacerbate existing pathologies by stimulating further amyloid accumulation and by promoting the formation of intracellular neurofibrillary tangles, thereby fueling a self-perpetuating cycle of neurodegeneration [3,4]. Experimental models have validated this link; for instance, the repeated intraperitoneal administration of lipopolysaccharide (LPS), a potent systemic inflammatory stimulus, is sufficient to induce microglial activation, Aβ plaque formation, and measurable cognitive deficits in mice [5,6]. Given that with age, the immune system gradually transits into a more pro-inflammatory state, known as “inflammaging” with continuous increase in systemic proinflammatory mediators which might lead to neuroinflammation and microglial dysfunction, and finally to cognitive impairment, the systemic administration of LPS provides a clinically relevant framework for studying the immunopathogenic mechanisms of AD [7,8]. Within this inflammatory landscape, identifying endogenous modulators of the immune response is paramount; thus, intervening before significant neurodegeneration occurs may provide novel approaches to delay or prevent AD onset.

Interleukin-33 (IL-33), a member of the IL-1 family, functions as an “alarmin” with complex immunomodulatory properties. Predominantly expressed by stromal and endothelial cells, as well as CNS-resident oligodendrocytes and astrocytes, IL-33 signals through its cognate receptor, Suppression of Tumorigenicity 2 (ST2) [9]. In the brain, the IL-33/ST2 axis is vital for maintaining tissue homeostasis. Upon injury, IL-33 is released to activate microglial phagocytosis and to facilitate the clearance of cellular debris [10]. However, the role of IL-33 in the CNS is notably dualistic, often exerting either neuroprotective or pro-inflammatory effects depending on the pathological context [11].

Clinical data underscore the relevance of the IL-33/ST2 signaling pathway in AD development: patients with AD and mild cognitive impairment exhibit reduced cerebral IL-33 expression, elevated levels of the decoy receptor soluble ST2 (sST2), and specific genetic polymorphisms within the IL33 and ST2 loci [12,13]. Animal studies further support a protective role for this axis; IL-33-deficient mice develop tauopathies and cognitive impairments with age due to significantly greater neuronal loss in the cortex and hippocampus [14], while exogenous IL-33 treatment in Amyloid Precursor Protein (APP)/Presenilin 1 (PSEN1) transgenic mice enhances Aβ clearance and shifts microglial polarization toward a restorative, anti-inflammatory phenotype [15]. Conversely, in acute models of neuroinflammation induced by intracerebroventricular LPS administration, IL-33 has been shown to exacerbate neutrophil infiltration and tissue damage [16].

Despite these insights, a significant void remains: the specific impact of the IL-33/ST2 axis on neuroinflammation and Aβ deposition specifically triggered by systemic inflammatory insults has not been elucidated. This study aims to fill that gap. By utilizing ST2-deficient (ST2^−/−) mice and exogenous IL-33 administration, we have investigated the role of this signaling pathway in modulating hippocampal inflammation and neural tissue damage in response to systemic LPS challenge. The hippocampus was selected as the primary region of interest due to its exceptional vulnerability to systemic inflammatory insults and its central role in the progression of amyloid-beta pathology. Furthermore, the high expression of the ST2 receptor within the hippocampal formation, as documented in previous neuroanatomical studies [17,18], makes it a key anatomical site for investigating the neuroprotective potential of the IL-33/ST2 signaling axis.

2. Materials and Methods

2.1. Animals and LPS and IL-33 Treatments

BALB/c male mice, wild-type (WT) and mice deficient for ST2 receptor (ST2^−/−), 10 to 12 weeks old and weighing 22 to 22 g were used in experiments. The mice were housed in the Animal Facility of Faculty of Medical Sciences, University of Kragujevac under standard laboratory conditions (12 h light/dark cycle, temperature 22 ± 2 °C) with ad libitum access to food and water. All experimental procedures were approved by the Ethics Committee of Faculty of Medical Sciences, University of Kragujevac (protocol number 01-8667) and were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

A total of 72 mice were divided into three groups: (1) BALB/c wild-type mice, which received intraperitoneal (i.p) injection of lipopolisaharide E. coli 055:B5;L2880, LPS, (Cat. Number L2880, Sigma Aldrich, St. Louis, MO, USA) (750 µg/kg), (2) BALB/c wild-type mice, which received i.p. LPS (750 µg/kg) + IL-33 (Recombinant Mouse IL-33 Protein, Cat, Number # 3626, R&D Systems, Minneapolis, MN, USA) (500 ng/mouse), (3) ST2^−/−, which received i.p. LPS (750 µg/kg). The i.p. administration of LPS and IL-33 was conducted for seven consecutive days. All analyses were done 24 h after the last dose of LPS. Mice were sacrificed by cervical dislocation, a method performed by highly trained personnel in strict accordance with the institutional ethical guidelines to ensure a rapid and painless procedure.

For immunohistochemical analysis of amyloid-β and MOG and TUNEL analysis, 18 mice (12 BALB/c WT and 6 ST2^−/− mice) were divided into three groups (n = 6 per group), while for IHC analysis of Iba1, 21 mice (14 BALB/c WT and 7 ST2^−/− mice) were divided into three groups (n = 7 per group). For flow cytometric experiments, 21 animals were assigned to three groups with 7 mice per group (14 BALB/c WT and 7 ST2^−/− mice), and for RT PCR, 12 animals were assigned to 3 groups with 4 mice per group (8 BALB/c WT and 4 ST2^−/− mice).

2.2. Immunohistochemistry and TUNEL

Brains were fixed in 4% buffered formalin fixative overnight. Paraffin wax-embedded sections (5 µm) were deparaffinized, hydrated through xylene and several graded ethanol solutions and after rinsed in distillated water. After that, tissue sections were incubated with anti-MOG antibody (clone, (CL2852), Cat. Number # MA5-24644, Thermo Fisher Scientific, Waltham, MA, USA), anti-Iba1 (clone [1022-5]. Cat. Number ab15690, Abcam, Cambridge, UK) antibody, and anti-amyloid beta 1–42 antibody (clone [mOC64] Cat. Number ab224275, Abcam, Cambridge, UK). Dilution for all antibodies was 1:500. Sections were visualized using a specific HRP/DAB detection IHC kit (ab64264, Abcam, Cambridge, UK) and Rabbit-specific HRP/AEC Detection IHC Kit (ab64260, Abcam, Cambridge, UK). The slides were counter-stained with hematoxylin, dehydrated, and mounted with mounting media. The slides were analyzed using a light microscope (Leica DM2500) equipped with a Leica Flexacam i5 digital camera. Quantification of MOG and amyloid beta 1–42 staining in stratum lacunosum-moleculare (SLM) (magnification 200) and the number of Iba1-positive cells per mm³ were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Death of cells in brains was determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling) staining of hippocampal sections. Paraffin-embedded brain tissue sections were stained with in situ Cell Death Detection Kit, POD (Cat. Number 11684817910, Roche, Basel, Switzerland). DAB (3,3′-diaminobenzidine) was added as a substrate for peroxidase in order to obtain the typical brown coloration of the nuclei. Slides were counterstained with hematoxylin and photomicrographed with a digital camera mounted on a light microscope. The TUNEL-positive nuclei (brown) were quantified under magnification 400× in ten fields of stratum lacunosum-moleculare, and the data were summarized as the mean number of positive cells.

2.3. Isolation of Mononuclear Cells from CNS and Flow Cytometry

The brain tissue was carefully isolated and homogenized by passing it through a syringe with an 18 G needle. The homogenized tissue was then passed through a cell-strainer and washed with 5 mL of medium. The suspension was centrifuged for 10 min at 400 G, and the precipitate was resuspended in 10 mL 30% Percoll overlaid onto 5 mL of 70% Percoll and centrifuged at 390 G for 20 min. After the incubation was completed, the myelin layer was removed from the top, and the mononuclear cells that were in the interphase were collected with a pipette, washed twice in PBS, and resuspended in medium. Mononuclear cells isolated from CNS were resuspended in FACS buffer (PBS with 5 mM EDTA and 0.2% BSA) incubated with fluorochrome-conjugated anti-mouse CD45, CD11b, CD86, CD40, CD206, ST2 antibodies or their respective isotype controls. For intracellular staining, cells were incubated for 4 h at 37 °C in the presence of 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (P8139, Sigma-Aldrich, St. Louis, MO, USA), 1 μg/mL ionomycin (I0634, Sigma-Aldrich, St. Louis, MO, USA) and Golgi Stop (Cat. Number 554724, BD Biosciences, San Jose, CA, USA). After incubation with PMA and ionomycin, cells were fixed and permeabilized using a BD Cytofix/Cytoperm kit (Cat. Number 554714, BD Biosciences, San Jose, CA, USA). After fixation and permeabilization, cells were incubated with anti-mouse IL-1β and IL-10 antibodies. Isotype controls were included to set gates. Expression of cell surface and intracellular antigens was analyzed using a FACSCalibur Flow Cytometer (BD Biosciences). Flow cytometric analysis was conducted with FlowJo Software V9 (Tree Star, Phoenix, AZ, USA).

2.4. RNA Extraction and qRT-PCR

Total RNA from mouse hippocampal tissue was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA (2 μg) was reverse-transcribed to cDNA using a RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania). qRT-PCR was performed using Luminaris Color HiGreen qPCR Master Mix (Thermo Fisher Scientific) and miRNA-specific primers (sense, 5′-CCAGCTTCAAATCTCACAGCAG-3′ antisense, 5′-CTTCTTTGGGTATTGCTTGGGATC-3′) in a Mastercycler ep realplex (Eppendorf, Hamburg, Germany). Relative expression of genes was calculated according to the formula 2−(Ct−CtGAPDH), where Ct is the cycle threshold of the gene of interest and CtGAPDH is the cycle threshold value of the housekeeping gene (GAPDH).

2.5. Statistical Analysis

The data were statistically processed using IBM SPSS Statistics 22.0 for Windows. The normality of the distribution of continuous variables was initially assessed through the Shapiro–Wilk test. Continuous variables were presented as mean values ± standard deviations or as median (IQR), depending on data normality. For comparisons among the three groups, a one-way analysis of variance (ANOVA) was applied when the assumptions of normality and homogeneity of variances were satisfied. Homogeneity of variances was evaluated using Levene’s test. In cases where a statistically significant overall difference was detected, post hoc pairwise comparisons were conducted using Tukey’s honestly significant difference (HSD) test to identify specific group differences. When the data did not meet the assumptions for parametric testing, the Kruskal–Wallis test was used as a nonparametric alternative. If the Kruskal–Wallis test indicated a significant difference, post hoc analysis was performed using Dunn’s test with Bonferroni correction to control for multiple comparisons. A p-value of less than 0.05 was considered statistically significant.

3. Results

3.1. The IL-33/ST2 Axis Attenuates Damage of Hippocampal Tissue Following Systemic LPS Administration

To study the role of the IL-33/ST2 axis on the development of neurodegenerative changes following chronic systemic inflammation in mice, we compared histological markers of CNS tissue damage in BALB/c WT and ST2^−/− BALB/c mice treated with LPS intraperitoneally and BALB/c WT mice treated with LPS and IL-33 for seven days. MOG staining was the lowest in the hippocampi of ST2^−/− mice (Figure 1A), with the lowest staining intensity and stained area. On the other hand, the highest intensity of staining, higher in comparison to both BALB/c WT and ST2^−/− mice treated with LPS, was detected in mice that were systemically administered with IL-33 besides LPS over a 7-day period (Figure 1A). The highest MOG staining was detected in stratum lacunosum moleculare of hippocampus, and these differences between the groups are noticed in this area (Figure 1A). Further, IL-33 treatment of mice together with systemic application of LPS resulted in a significant increase in myelinated areas in the hippocampus compared to ST2^−/− and WT mice treated with LPS only (Figure 1B). The percentage of myelinated area was the lowest in ST2^−/− mice treated with LPS (Figure 1B). These findings suggest an important role of IL-33 in preserving myelin integrity during chronic systemic inflammation.

Since it is known that seven days of systemic inflammatory stimuli increases amyloidogenesis in the CNS through activation of β- and γ-secretases with concomitant inhibition of α-secretase resulting in elevated levels of Aβ1–42 levels in mice [6], we next analyzed the expression of Aβ1–42 in the hippocampi of WT, ST2^−/− and IL-33-treated WT mice exposed to inflammatory stimuli for seven days. Aβ1–42 depositions were detected in the hippocampi of WT mice treated with LPS, mostly in the stratum lacunosum moleculare (Figure 2A). Higher intensity of Aβ1–42 staining was noticed in ST2^−/− mice with almost the same spatial pattern of expression (Figure 2A), while the weakest immunoreactivity was noticed in the hippocampi of IL-33-treated mice (Figure 2A, medium panel). Figure 2B shows that IL-33 treatment significantly reduced the percentage of hippocampal tissue affected by amyloid deposition in comparison with WT mice exposed to inflammatory stimuli, while the highest percentage of affected tissue was detected in mice without the IL-33/ST2 signaling axis (ST2^−/− mice).

Aβ peptides trigger neurotoxic events, which activate caspase-dependent apoptotic pathways in neurons and induce apoptosis of neurons in vitro and in vivo [19,20]. In order to explore the effects of IL-33/ST2 signaling on neuronal cell death as a consequence of chronic systemic inflammation, the TUNEL assay was performed. As shown in Figure 3A, IL-33 administration reduced programmed cell death in WT mice, while in the brains of ST2^−/− mice, massive increases in TUNEL-positive cells were detected. The number of TUNEL-positive cells was the highest in ST2^−/− mice and the lowest in IL-33-treated WT mice, significantly lower in comparison with ST2^−/− (p < 0.005) and also with WT mice treated only with LPS (p < 0.001) (Figure 3B). Apoptotic cells were detected mostly in the stratum lacunosum moleculare of the hippocampus (Figure 3A).

3.2. IL-33/ST2 Axis Modulates Microglial Response to Systemic LPS Administration

Overall, these results suggest that IL-33/ST2 signaling exerts a neuroprotective effect in mice exposed to systemic inflammatory challenge for seven days. The ST2 molecule is expressed in microglial cells; phagocytic activity of microglia is important for the control of nervous tissue damage, and we next analyzed the characteristics of these cells.

Immunohistochemical staining for Iba1 shows the strongest staining in IL-33-treated WT mice and the lowest in ST2^−/− mice after systemic inflammatory stimuli (Figure 4A). Also, in Figure 4A, differences in microglial morphology can be observed. In the hippocampi of WT mice, both activated (bushy) and ameboid microglia can be noticed; in ST2^−/− mice, almost all are ameboid microglia; while in IL-33-treated WT mice, there is almost no ameboid type of microglia. The number of Iba1-positive cells per mm³ in hippocampal tissue was significantly lower in ST2^−/− mice compared with both WT and IL-33-treated WT mice (Figure 4B). The highest number of Iba1-positive cells was detected in IL-33-treated mice but did not reach statistical significance when compared with WT mice (Figure 4B). Flow cytometric analysis of mononuclear cells isolated from the CNS shows the highest percentage of ST2+ microglia (CD45lowCD11b+ cells) in IL-33-treated mice; significantly higher in comparison with WT mice exposed to inflammatory stimuli, and also in comparison to ST2^−/− mice after systemic inflammatory stimulation in which ST2-expressing microglia were not detected (Figure 4C). The highest percentage of microglia expressing markers of activation CD40 and CD86 was noticed in IL-33-treated WT mice injected with LPS, but with statistical significance only in comparison with ST2^−/− mice, not in comparison with WT mice (Figure 4C). However, the percentage of microglia expressing the marker of alternative activation CD206 was significantly higher in the groups of IL-33-treated and LPS-injected WT mice in comparison with both WT (p < 0.05) and ST2^−/− (p < 0.005), LPS-injected mice (Figure 4C). Also, the expression of pro- and anti- inflammatory cytokines in CD45lowCD11b+ cells was analyzed via flow cytometric analysis. The highest percentage of CD45lowCD11b+ cells containing IL-1β was found in the ST2^−/− BALB/c group of mice, significantly higher compared to WT and IL-33-treated WT mice (Figure 5A).

The percentage of IL-10-expressing CD45lowCD11b+ cells was higher in both WT and IL-33-treated WT mice in comparison with ST2^−/− mice (Figure 5A). Further, the percentage of IL-10-expressing microglia was the highest in IL-33-treated WT mice, significantly higher in comparison with WT mice (Figure 5A). The expression of IL-1β at the mRNA level in hippocampal tissue was the lowest in IL-33-treated WT mice, significantly lower in comparison with ST2^−/− mice, where the highest expression of IL-1β was detected, but more importantly in comparison to WT mice (Figure 5B).

4. Discussion

Our study suggests the IL-33/ST2 axis as one of the key checkpoints in the systemic-to-central nervous system inflammatory cascade. We demonstrate that IL-33 signaling is required for balancing the phenotype of microglia, resulting in preserving hippocampal integrity and limiting amyloid deposits induced by chronic peripheral inflammatory insults.

Our results indicate the critical role of IL-33 signaling in maintaining myelin homeostasis. It is known that the IL-33/ST2 signaling pathway plays a role in the myelination process during CNS development, and also in the repair phase in demyelinating diseases [11]. We found a significant reduction in MOG staining in ST2^−/− mice (Figure 1B). LPS activates microglia and astrocytes that release pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and free radicals, which together can induce degradation of myelin sheaths [21]. While we focused on microglial reprogramming, the potential involvement of other glial populations cannot be excluded. Recent literature suggests that IL-33 also acts on astrocytes, promoting an A2-like protective phenotype that supports neuronal survival and blood–brain barrier integrity [10]. Our results suggests that, in the absence of the IL-33/ST2 signaling axis, systemic inflammation triggers an uncontrolled neurotoxic environment that prioritizes white matter degradation. Damage to myelin is located particularly within the SLM, part of the hippocampus that is highly vulnerable in early stages of AD-like pathology [22]. Early signs of myelin damage pathways within this part of the hippocampus have been reported early in AD, leading to atrophy in the SLM and cognitive impairment [23]. Conversely, exogenous IL-33 administration significantly expanded the myelinated area in the hippocampus (Figure 1B). This finding is in line with previous reports that in vivo administration of IL-33 increases the myelin content in the spinal cord following spinal cord trauma [24] and stroke [25]. There are several mechanisms by which IL-33 promotes recovery of myelin sheets. It could be skewing the microglial cells, which are known mediators of inflammatory injury toward M2 phenotype [26]. Also, by creating a restorative microenvironment, IL-33 can contribute to oligodendrocyte health, differentiation of OPCs and their integration, leading to myelin repair [27]. It can also directly induce the expression of myelin-related genes in oligodendrocyte precursor cells [28]. Since we have also found reduced expression of IL-1β in the brains of IL-33-treated mice (Figure 4 and Figure 5), it can be assumed that IL-33 attenuates inflammation induced by systemic administration of LPS and thus attenuates myelin damage. Reduction in neuronal apoptosis in mice treated with IL-33 (Figure 3) supports the protective role of IL-33 and its significance in facilitating a restorative microenvironment.

It is known that the systemic inflammatory response induced by LPS shifts the balance of Aβ metabolism toward production and induces deposition of Aβ in mouse hippocampi and cortices [6]. Our results show that while LPS induces Aβ1−42 deposition in WT mice, the absence of ST2 signaling leads to significant exacerbation of amyloid burden (Figure 2). Most importantly, we show that exogenous IL-33 treatment can effectively reduce amyloid deposition induced by systemic inflammation. Since Aβ peptides can induce the creation of a neurotoxic environment that trigger apoptosis of neurons [19,20,29], the observed reduction in Aβ load in IL-33-treated mice can be the primary driver behind the observed decrease in apoptosis of neural tissue cells. The other possibility that does not exclude the contribution of decreased Aβ deposition is that IL-33 directly affects the expression of genes responsible for apoptosis in neural cells, since it has been published that IL-33 attenuates neurobehavioral disorders, learning and memory deficits partially by reducing apoptosis [30]. In summary, previous findings suggest that IL-33 probably interrupts amyloid deposition and neurodegeneration at its inception. It is possible that IL-33 initially attenuates neuroinflammation, the release of inflammatory factors known to induce myelin damage, apoptosis of neural cells, and also stimulates the formation of Aβ. This protective efficacy, however, appears to be highly sensitive to the temporal dynamics of the inflammatory insult. The literature suggests that the timing of IL-33 administration is critical, as its ability to shift microglial polarization is most effective when synchronized with the peak of the systemic inflammatory response [31]. Furthermore, evidence from similar neurodegenerative models suggests a dose-dependent relationship, where optimized concentrations of IL-33 are required to achieve maximal restoration of hippocampal homeostasis without triggering off-target immune activation [11,32].

The neuroprotective effects observed appear to be mediated by a profound modulation of the immune microenvironment in the CNS. Given that IL-33 was administered systemically, it is important to consider whether the observed hippocampal changes result from direct CNS action or peripheral immune modulation. While systemic IL-33 can attenuate peripheral inflammation, thereby reducing the surge of pro-inflammatory cytokines into the brain, its effects likely involve a direct interaction with resident microglia, which constitutively express high levels of the ST2 receptor [11,24,29]. Thus, the neuroprotection observed in our model likely reflects a synergistic mechanism, where IL-33 simultaneously dampens the peripheral inflammatory trigger and directly reprograms microglia toward a restorative phenotype. Interleukin-33 plays an important role in the regulation of the inflammatory response and innate immunity and microglia, which simultaneously express high levels of ST2 in several CNS regions, including the hippocampus as its main target cells [11,24,33]. Microglia are a double-edged sword in neuroinflammation and neurodegeneration: while they are necessary for Aβ clearance, chronic activation often leads to a neurotoxic, pro-inflammatory state that promotes neurodegeneration. Possible mechanisms by which IL-33 could modulate microglial activity and contribute to decreased neural damage after LPS administration include: (a) promotion of phagocytic activity of microglia by IL-33 [15], (b) stimulation of microglia migration toward Aβ plaques [15], and (c) modulation of microglial inflammatory response [31]. However, since IL-33 as an alarmin plays a role in the control of early inflammatory immune responses, it seems the most likely that IL-33 attenuates neural tissue damage mainly by regulating the initial activation of microglia induced by systemic inflammation and its phenotype. Our flow cytometric and morphological data reveal that IL-33/ST2 signaling dictates this microglial “choice”. In the absence of ST2, microglia adopted a predominantly ameboid, pro-inflammatory phenotype, characterized by high expression of the potent neurotoxin IL−1β and a lack of restorative markers (Figure 4 and Figure 5), while under IL-33 stimulation, there was a significant shift toward alternative activation (CD206+), and increased production of the anti-inflammatory cytokine IL-10 was observed (Figure 4 and Figure 5). Interestingly, while IL-33 increased the total number of Iba1+ cells (Figure 4B) and the expression of activation markers like CD40 and CD86 (Figure 4C), these cells were not neurotoxic. Instead, they likely represent a damage-associated microglia (DAM) phenotype [34] that is geared toward phagocytosis and tissue repair rather than bystander damage. The significant reduction in IL−1β at both the protein and mRNA levels in IL-33-treated mice (Figure 5) underscores the potency of this axis in silencing the “inflammasome-like” response typically triggered by systemic LPS. Importantly, IL-33 administration significantly increased expression of ST2 on microglial cells after systemic inflammatory challenge (Figure 4B). This is in line with previous reports that IL-33 induces increased expression of ST2 on microglia, driving microglia toward an IL-10-producing anti-inflammatory phenotype but with enhanced phagocytic capacity, which is crucial for tissue repair and survival of neurons [35,36]. Since treatment with IL-33 of LPS-exposed mice enhances ST2 expression in microglia and activates them, this led to a break in excessive neuroinflammation and limited damage of neuronal tissue.

While our study provides strong evidence that the IL-33/ST2 axis plays a neuroprotective role by modulating microglial phenotypes, certain limitations should be acknowledged. First, although we observed significant histopathological improvements, including reduced amyloid deposition and neuronal apoptosis, our study did not include behavioral or cognitive assessments. However, given that hippocampal integrity and reduced neuroinflammation are closely linked to cognitive performance in models of systemic inflammation, it is highly probable that the observed cellular changes correlate with improved functional outcomes [15]. Additionally, although the direct phosphorylation of ST2-associated signaling mediators, such as MAPK or AKT, was not quantified in this study, the observed shift toward an anti-inflammatory microglial phenotype and the reduction in neuronal apoptosis are hallmarks of the activation of these intracellular pathways [9,32]. Further molecular studies, including Western blotting for phosphorylated signaling mediators and behavioral testing, will be necessary to fully map the intracellular events triggered by IL-33 in this specific model.

5. Conclusions

In conclusion, our data suggest that the IL-33/ST2 axis serves as an endogenous controller of central inflammation to the neuroinflammation cascade. ST2^−/− mice exhibited a “perfect storm” that contributes to neurodegeneration: demyelination, high amyloid load, massive neuronal apoptosis, and a pro-inflammatory microglial profile. By restoring or enhancing the IL-33/ST2 axis, it was possible to reverse these pathological hallmarks. These findings provide a strong rationale for exploring IL-33-based therapies as a preventative strategy in patients with chronic systemic inflammatory conditions, potentially halting the transition from peripheral inflammation to neuroinflammation and irreversible neurodegenerative diseases like Alzheimer’s.